Final

Question 1

What is size of nucleus?

Answer 1

• Largest organelle
• Nuclear size variation is influenced by several factors, and can differe significantly between cells within the same organism and diff
• Nuclear size varies from cell to cell and between organisms usually determined by cell size (ie cytoplasmic volume more cytoplasm more nucleus)
• increases during development and cancer cells-used for cancer diagnosis/prognosis

Question 2

Do cancer cells have larger nuclei?

Answer 2

YES

Question 3

What is the primary diff between pro and euk nucleus?

Answer 3

• Eukaryotes have membrane bound nucleus
• Euk: larger, structurally and functionally more complex interiors (possess single and double membrane bound organelles)
• Cellular “compartmentalization” allows for larger size and segregation and organization of specific cellular functions
• Each organelle contains both unique and common factors for functioning (eg metabolism) and their biogenesis (formation), maintenance, and turnover
• Pro have region bound nucleiod where chs located; less DNA packaging, and limited/no RNA processing

Question 4

What is the nucleus 2 main functions?

Answer 4

1. Compartmentalization of the cellular genome and its activites
   eg site of DNAreplication, transcription and RNA processing
   eg site where translation components (ribosomes, mRNAs, tRNAs) are made
2. Coordination of cellular activites
   eg control of metabolism, protein synthesis, reproduction (cell division etc)

Question 5

What are more functions of the nucleus?

Answer 5

• Nucleus seperates DNA from the rest of thr cell
• Allows for better timing and control over which genes are turned on and when (seperation of the cytoplasm from genome)
• Since pro have no nucleus, they dont seperate DNA from rest of cell, so mRNA is used right away while its still bring made (mRNA translated while transcription progress right away)
• In euk since mRNA is first edited (like splicing) in the nucelus
• Then sent out to be used in cytopladm or to ER to make proteins
• The nuclear envelope (membrane) acts like a security gate
• It controls whihc proteins (transcription factors) can reach the DNA from cytoplasm to genome in Euk

Question 6

What is the nucloeplasm?

Answer 6

• Gel-like inside of the nucleus
• Its very organized, not just empty space
• There are 30+ special regions inside the nucleus (called subdomians)
• Subdomains do specific jobs but arent surrounded by membranes)

Question 7

What is an example of a subdomain?

Answer 7

Nucleolus
- Most visible subdomain looks dense and grainy
- Cells can have 1-5 nucleoli, depending on how active the cell is
- More active cells=more/larger nucleoli
Main function: makes ribosomes
- Transcribes ribosomal DNA (rDNA)
- Processes rRNA
- Begins ribsome assembly (combining rRNA+protein)
- Final assembly happens in the cytoplasm

Question 8

What happens in the nucelus during interphase?

Answer 8

• Chromosomes during interphase (when the cell isnt dividing) are organized into seperate areas in the nucleus, called subdomains
• Genes locaiton in the nuclues affects how active it is
• Genes that are being used (active) are found near the edges of these CHS areas (periphery of chromsomal subdomains)
• Interchromsomal channels are spaces between chromsomal areas
• Help prevent unwanted interactions between different DNA regions or proteins

Question 9

What are chromosomal subdomains and what do they do?

Answer 9

• They are internally organized regions
• Active genes (from euchromatin) can reach outside their own zones (subdomains) into special areas called transcription factories
• Factories where transcription factors gather to help make RNA
• Interchromosomal interactions= when genes from different CHS connect
• Sometimes called kissing CHS
• A gene from one CHS ca turn on a gene on another CHS

Question 10

What do nuclea speckles do?

Answer 10

• Small spots inside the nucleus that look like bright dots under microscope
• These are subdomains where mRNA splicing factors gather (splicing= editing pre-mRNA before becomes mature mRNA)
• Often found near transcription factories (where RNA is made)
• SPeckles are very active and flexible
• Can move, grow, shrink, or increase/decrease in number depending on what the cell needs

Question 11

What is the nuclear matrix?

Answer 11

• Like a support net inside the nucleus
• Made of fibrous proteins that create a mesh-like structure throughout the nucleoplasm
• Similiar to cytoskeleton in cytoplasm, which helps give cells their shape and organize their contents
• Made of 3 main parts
• Microfilaments
• Microtbules
• Intermiadte filaments

Question 12

What does the nuclear matrix do?

Answer 12

• Serves as a structural role:maintains overall shape of nucleus
• Serves a scaffold: responsible for organizing nuclear subdomains and anchoring protein factors (eg proteins involved in DNA replication, transcription, RNA processing, etc)
• Very little known about composition and assembly/disassembly of nuclear matrix) (still dont know alot about what its made of or how its built and taken apart)

Question 13

What is the nuclear envelope and what does it do?

Answer 13

• Seperates the contents (eg genome) from surrounding cytoplasm
• Serves as a barrier (requires regilated passage of molecules; eg RNA and proteins) between nucleus and cytoplasm
• Establishes unique composition of nucleus (comapred to cytoplasm) and spatially regulates gene expression
• provides structural framework for nucelus
• Made of 3 parts:
• Nuclear membranes
• Nuclear lamina
• Nuclear pore complexes

SImplified:
- Protects and seperates the nucelus from the rest of the cell
- Acts like a gate:
- Controls what goes in and out (like RNA and proteins)
- keeps the inside of the nucelus different from the cytoplasm, which helps control gene activity
- Nuclear membranes: double layered wall
- Nuclear lamina: support mesh just inside the membrane
- Nucelar pore-comlexes: little holes that tegualte traffic in and out

Question 14

How are the nuclear membranes arranged?

Answer 14

Has 2 layers:
1. Inner membrane
2. Outer membrane

• Both are made of phospholipid bilayers (like cell membrane) and sit parallel to each other
• These 2 membranes are seperated by a small space called the nuclear evelope lumen (10-50 nm wide)
• Membrane acts as barriers to control what moves in and out of nucelus- like ions, solutes, and large molecules (eg proteins, RNA)

Question 15

Where is the outer and inner mebrane connected to?

Answer 15

Outer membrane: connected to rough ER
- It even has ribsomomes attached to it, just like the RER
- Space between the inner and outer membranes (nucelar evelope lumen) is continouus with the inside of thr ER
- Inner nuclear membrane has a different protein makep then outer one, so it does different jobs
- Both membranes come togehter at nuclear pores, which are curved oepning that allow things to move in and out nucleus

Question 16

What is a nuclear lamina and what does it do?

Answer 16

• Located on inner surface (nucleoplasmic side) of nuclear inner membrane
• Network (mesh) of long, filament-like proteins
• ABC nuclear lamins: related to proteins that form intermidate filaments in cytoskeleteon network
• provides mechanical support to nuclear envelope (binds to nuclear inner membrane integral proteins)
• Serves as scaffold for attachment of chromatin and nucelar matrix to nuclear envelopoe

Question 17

What happens if there are mutations in nuclear lamina?

Answer 17

• Mutations in LAMIN genes responsible for several human diseases
• Eg Hutchinson-Gilford Progeria syndrome
• Rare, characterized by premature aging in children, hair loss, wrinkles, artery damage, death by early adolescence
• Due to a point mutation (sporadic: occurs during in embryo development) in LAMIN A gene leading to truncated lamin protein
• Results in destabilization/breakdown of nuclear lamina (causes abberant changes in nuclear (envelope) morphology and function
• Recently promising advances using CRISPR/Cas9 genome editing-based (gene) therapy in mice

Question 18

What is the nuclear pore complex?

Answer 18

• Channel (doorways) in nuclear envelope
• Responsible for regulated trafficking (import and export) of all substances between nucleus and cytoplasm
• Small, polar molecules (eg nucleotides for DNA/RNA synthesis) RNAs-mRNA, tRNA, rRNA
  proteins- eg transcription factors, RNA-binding proteins, ribosomal (subunit) proteins, and cyclins
• typically 3000-4000 per nucleus: number of NPC related to nuclear activity

Question 19

What is the structure of NPC?

Answer 19

What is it?
- Large, complex structure in the nuclear envelope
- Much bigger then a ribosome (about 30x the size)

Made of:
- About 40 proteins called nuceloproins (Nups)
- Found in all eukaryotes
- Found in both inner and outer membranes of the nucleus (use diff types of proteins that are either inserted into or stuck onto nuclear mebranes to build structure)
- Some nups are simialr to those in the ER (relted to COPII involced in vessivle formation), suggesting they share a common origin
- Both nups and copii proteins finction to deform (curve membranes)
- Proteins can bend membranes, helping shape them

Structure:
- NPCs have a symmetrical 8-sided shape
- Built around a central channel that allows molecules (like RNA and proteins) to pass through

Question 20

What is nuclear pore complex made of?

Answer 20

Several parts:

Central scaffold:
- Made of membrane-bound proteins called nucleoporins
- Gives structure to pore

Anchoring:
- NPC is attached to point where the inner and outer nuclear membranes meet

Central channel:
- Water-filled tunnel in the middle (about 20-40 nm wide)
- This is where molecules pass through

Question 21

What are FG Nuceloporins?

Answer 21

FG Nuceloporins (filament-like proteins in the NPC)
- Line the inside of the central channel
- Called FG nuceloporins because they have FG (phenylalanine-glycine repeats)

Key features:
- Unusual amino acids: mix water-loving (hydrophilic) and water hating (hydrophobic parts)
- FG domains are disordered and flexible, filling the cetral channel like spaghetti

What do FG domains do?
- Form a mesh-like gel that acts like a sieve
- FG part hydrophobic
- Limit what can pass through, only allowing certain molecules to get through the nuclear pore
- Form secondary structure (how they fold; flexible unstructured

Question 22

What do nucleoporins let in?

Answer 22

• FG domains stick into the central channel and form a gel-like mesh
• Mesh blocks large molecules from diffusing through
  What can pass through:
• Small molecules (such as nucleotides)
• Can move freely in and out in both directions
• Larger molecules greater then 40 kDa or about 39 nm wide cant pass through on their own
• Need speical trasnport proteins to be moved actively

Question 23

What determined the size-exclusion limit of NPC?

Answer 23

Microinjection of nuclear protein coated gold particles into mammalian culture cells

Question 24

What are the y-complexes??

Answer 24

• Includes the cytoplasmic ring and nuclear ring
• made of structural nucleoporins (Nups)
  Located on:
• Cytoplasmic side (outer part)
• Nuceloplasmic side (inner part)

Connected to:
- Central scaffold
- Cytoplasmic filaments or nuclear basket

Question 25

What are cytoplasmic filaments?

Answer 25

- Long, thread like proteins sticking out into the cytoplasm - Help recognize and import cargo proteins from cytoplasm into nucleus

Question 26

What is nucelar basket?

Answer 26

- Looks like a basket on nucleoplasmic side (inside the nucleus) - Made of structural nuceleoporins too - Connected to y-complex nuclear ring - Helps with import and export of cargo proteins

Question 27

What is the nucleocytoplasmic transport via NPC?

Answer 27

- One of the busiest transport routes in the cell - Transport goes in both directions: Cytoplasm to nucleus (import) Nucelus to cytoplasm (export) - Many types of cargo moves through... - Proteins - RNAs - Ribosomal subunits

Question 28

What goes into the nucleus from cytoplasm?

Answer 28

Proteins needed for: - DNA replication - Transcription - RNA splicing - RIbosome assembly - Chromatin packing (like histones) - Nuclear matrix structure (lamins, etc)

Question 29

What foes out of the nucleus to cytoplasm?

Answer 29

- All RNA types: mRNA, tRNA, rRNA - Partially assembled ribosomes (that are needed for protein synthesis in cytoplasm) - Some proteins

Question 30

How much does an animal cell transport?

Answer 30

imports about 300,000 histones, and 560,000 ribosomal proteins/min Exports 14,000-20,000 ribosmoems per minute About 3,000 NPCs handle all this transport About 100 histones, 180 ribosmomal proteins, and 6 ribomsomes per NPC per minute

Question 31

What does nucelocytoplasmic transport via NPC require?

Answer 31

- Well understood need energy, specific protein receptors, and unique targetting signals

Question 32

How do proteins get into the nucleus (cytoplasm to nucleus)?

Answer 32

- Most proteins such as transcription factors, histones, lamins cyclins etc that go into the nucleus have a special tag called a nuclear localization signal - NLS is like a zip code a short amino acid sequence that tells the cell hey send this protein into the nucleus - There are different types of NLS depending on the proteins amino acid sequence - The NLS sequence is recongized by nuclear receptor proteins

Question 33

What are NLS (nuclear localization signals)?

Answer 33

Classic NLS- most common - Short stretch of positvely charged amino acids (like lysine and arginine) - Example KKQRKK- in simian virus 40 large T antigen protein (found one of the first known nuclear localization signals) Bipartite NLS: - Two short stretches of basic amino acids, with a spacer (7-10 amio acids) in between eg KR(PAATKAGQA)KKKK in nucleoplasmin - protein can have more then one NLS and nuclear export signal (NES) - NLS identified in proteins based on mutational analyses

Question 34

What is the definition of Nuclear Localization Signals (NLS)?

Answer 34

NLS: an amino acid sequence that is both neccessary and suffieicent for cytoplasm-to-nuclear targetting (neccessary must be present and have enough on its own to send a protein into the nucleus) What does that mean? Neccessary: - If you remove or mutate the NLS, the protein wont go into the nucelus anymore - Need to pass for the specific one or wont get in Sufficient: - If you attach NLS to proteint aht usually stays in cytoplasm, it will now go into the nucelus - If given to random enough to get in different protein Pyruvate kinase alone stays in the cytoplasm When NLS is added to it, it moves into the nucleus

Question 35

How do you find an NLS in ARC1?

Answer 35

- ARC1: protein that helps with plant pollination (specifically, recongizing incompatible pollen) - ARC1 moves between the nucelus and cytoplasm How does ARC1 get into nucleis - Has a classic NLS - Found at amino acids 261-266 - Also has a nuclear export signal (NES) to leave the nucleus How do scientists test this? - Normal ARC1 (goes into the nucleus) - Mutated ARC1 (NLS removed): doesnt go into nucleus - CAT protein alone: stays in cytoplasm - CAT+ NLS added moves into the nucelus

Question 36

What are the 2 experiments that determined if the NLS is neccessary?

Answer 36

Experiment 1: is the NLS neccessary? - Scientists mutated amino acids (261-266) the suspected NLS - Result ARC1 stayed in the cytoplasm, meaning it couldnt go into nucleus - Conclusion: NLS is neccessary for nuclear import Experminet 2: is the NLS sufficient? - Attached the same 261-266 sequence to a protein that usually stays in the cytoplsm (CAT) Result: CAT moved into nucelus Conclusion: the NLS is sufficient to direct proteins into the nucleus Proves that those 6 amino acids (261-266) in ARC1 are the NLS

Question 37

What are the Myc and the CAT?

Answer 37

Myc= tag to detect protein CAT= a test protein that normally stays in cytoplasm

Question 38

What did studying NLS help with?

Answer 38

- Studying NLS helped scientists discover the proteins needed to import other proteins into the nucleus - These helper proteins are called transport receptors (recognize and carry out function): - Act as shuttles or ferries that carry cargo (proteins) through the nuclear pore - What are karyopherins? - A big family of transport receptors Two main types: - Importins: bring into nucleus - Exportins: out of nucleus

Question 39

What are the steps of cytoplasm-to-nucleus transport?

Answer 39

- Protein import into nucelus is a multi-step process - 5 main steps... Step 1: - newly made protein (called "cargo" has a NLS tag on it - Importin has 2 parts: - Importin alpha-> sticks to NLS on the cargo - Importin beta-> helps to guide the whole complex toward the nucleus Step 2: - The cargo and importin alpha/beta complex moves toward the nucelus through the cytoplasm - It travels using the cytoskeleton, which acts like a highway inside the cell (movement of proteins, RNA, organelles etc) - Once the complex reaches the nuclear pore, the importin beta subunit helps it attach to cytoplasmic filaments at the pore - First contact before the complex moves through the pore into the nucleus Step 3: - The cargo/importin complex goes through the central channel of the nuclear pore complex (NPC) - We dont fully understand how this works, but current idea is... - Complex passes by interacting with special proteins inside the channel called FG-nucleoproins - These interactions help untangle the channel so the cargo can pass through Step 4: - Once inside the nucelus, the cargo-importin complex reaches the nuclear basket (beta binds with it) - There it binds to molecule called Ran-GTP which... - Causes the complex to break apart, and the cargo is released into the nucleus (nucleoplasm) - NLS tag still attached to protein (not cut off) so protein can be imported again later if needed What is Ran? - Ran is a small protein called a GTPase (binds and breaks down GTP) - Helps control nucelar transport by switching two forms: Ran-GTP (active form with GTP) Ran-GDP (inactive form with GDP) Where is Ran-GTP found? - Lot of Ran-GTP in the nucleus - Very little in cytoplasm (so theres a gradient) - Gradient helps as importins known when to release cargo; direction of transport (import vs export) stays organized What maintains the Ran-GTP gradient? - GEF (in the nucleus) turns ran-gdp to gtp - Keeps high ran-gtp levels in nucleus - GAP (in cytoplasm) - Turns ran-gtp to gdp - Keeps low ran-gap levels in cyotplasm - Ran-gtp gradient controls direction of transport - Breakiing down GTP (hydrolysis) provdes the energy needed for moving stuff through nuclear pore Step 5: - Importin beta and ran-gtp retuns to cytoplasm - Happens because the Ran-GTP is more concentrated in nucelus (naturally flows out) - In cytoplasm, GAp turns Ran-GTP to ran-gap (breaks down GTP) - Importin beta now free to be reused for more cargo import - Ran-GDP moves back into the nucleus (since ran-gdp is low inside) - IN the nucleus, GEF turns it back into Ran-GTP

Question 40

What is the fate of importin alpha in the nucleus?

Answer 40

- After importin alpha finishes brining cargo into the nucleus; it needs to go back into the cytoplasm to do its job again - Similairy some proteins (like ribosmoal proteins, RNA-binding proteins, and cyclins) must be exported from the nucleus to the cytoplasm after their work is done - Importin binds to exportin (a transport protein) to leave nucleus - Trasnport is handled by karyopherin proteins (family of transport proteins) - Once importin alpha delivers its cargo in the nucleus, a signal called NES (nuclear export signal) is exposed on it - This nES tells the cell its time to export importin alpha out of the nucleus and back to cytoplasm - Exportin binds to other proteins that need to leave the nucleus - These proteins have a nucelar export signal- a spceicifc amino acid sequence that acts like a zip code telling exportin to move them out of the nucleus - Most NESs have leucine rich sequences like (LxxLxxL) x=any amino acid residue (leave bye L) - Importin alpha (or cargo with NES) binds to exportin and ran-gtp in the nucleus - Forms a stable complex - The complex moves through the nuclear pore into the cytoplasm (because of the Ran-GTP gradient) - in cytoplasm: - Ran-GTP is hydrolyzed (broken down by GAP to ran-gdp - This causes a release of thr cargo and importin alpha from exportin Then: - Importin alpha is reused for nuclear import - Ran-GDP goes back into nucelis and becomes Ran-GTP agai (via GEF) - Exportin also returns to nucelus; using its own import signal)

Question 41

What is piggyback nuclear protein import?

Answer 41

- Some proteins that go into the nucelus dont have a NLS (nuclear localization signal) - Instead use a piggyback method - These proteins bind to another protein that does have a NLS - NLS containing protein brings them into the nucleus together - Import still uses imprtins and follows the normal steps (1-5) for nuclear import

Question 42

What happens to proteins that move in and out of nucleus and cytoplasm (shuttling)?

Answer 42

- Some proteins move back and forth between the nucleus and cytoplasm - These proteins usually have both: - NLS (to go into the nucleus) - NES (to go out to the cytoplasm) - Where the protein ends up most of the time depends on which signal is stronger (NLS vs NES) - The strength of the signals can be changed by modifications like phoshorylation (adding a phopshate group) NLS= xKKQRKK - K and R posotively charged amino acids (lysine and arginine) - Basic residues that importins recognize x= any amino acid, but in this case, often S,T or Y - Can be phosphorylated (can turn signal on or off) NES= LxxLxxL - Leucine rich sequences X can be any amino acid just a placeholder

Question 43

What happens during nucelocytoplasmic transport of ARC1 using the shuttle analysis?

Answer 43

- ARC1 moves between nucleus and cytoplasm - It has both NLS and NES, meaning it can be imported and exported Before pollination: Before pollination NLS>NES means ARC1 is in nucleus During self pollination: - NLS is disrupted by phosphorylation (adds phosphate groups to nearby amino acids) - Now NES>NLS and ARC1 is now in cytoplasm Why important? - When ARC1 is in cytoplasm, helps degrade proteins using the proteasome (protein shredder) - Prevents self-pollination in plants What are the experiments on ARC1 Transport? Experiment 1: - If you phosphorylate residues near NLS, ARC1 cant enter the nucleus it stays in cytoplasm - Mimicked by replacing residues with aspartic acid (D), which behaves like phospohrlyated S/T/Y - Induced phosphomimetic mutations Experiment number 2: - If you mutate the NES, ARC1 cant exit the nucleus, stays in nucleus

Question 44

What does in vitro co-immunopreciptiation assay to assess cargo protein importin binding do?

Answer 44

What is the goal? - To find out if a nuclear-localized protein (cargo) can bind to importin (the transporter) What is the method? - Co-immunoprecipitation (Co-IP) Two things you need? - Bait= a tagged nucelar protein (eg Myc-ARC1 or Myc-ARC1 with a mutated NLS) Prey= importin alpha and beta proteins 1. Mix bait and prey proteins together 2. Add antibody-coated beads that stick to tagged bait 3. Spin (centrifuge) to isolate beads and anything stuck to bait (including prey if it binds) 4. Run on a gel (SDS-PAGE) and stain to see if importin bound - What are we testing? - If the nucelar protein has a working NLS, it should bind importin - If the NLS is mutated, it wont bind importin Result: - Importin only binds to Myc-ARC1 with a working NLS - No binding seen with the NLS mutatn, showing NLS is needed for importin interaction What is you used only importin beta? - Might not see binding; importin alpha usually recognizes the NLS and is needed for thr full complex to form How to test if Ran-GTP is involved? - Try Co-IP with and without Ran-GTP; if Ran-GTP affectd ARC-1 importin interaction you will see a difference How to test if exportin is involved? - Do Co-IP with ARC1 and see if it binds to exportin in the presence of Ran-GTP (which is needed for nuclear export)

Question 45

What are cyclins?

Answer 45

- Cyclins are proteins that help control the cell cycle (when 2 cells grow and divide) - They are made and destroyed each cycle to keep things on tract

Question 46

What are the cell cycle phases?

Answer 46

1. Interphase (when the cell isnt dividing) - G1 (Gap 1): cell does its regular job and responds to signals s (synthesis): DNA is copied, and proteins like histones are made G2 (Gap 2): cell prepares for division (mitosis) 2. M phase (Mitosis): - Cell divides Go (Gap 0): resting phase: the cell stops dividing (most body cells are here)

Question 47

What is the M phase?

Answer 47

M phase: mitosis - consists of prophase, metaphase, anaphase and telophase Prophase: chs condense, spindle forms, and the nuclear evelope breaks down Metaphase: chs line up in the middle Anaphase: CHS get pulled apart Telophase: two new nuclei form Cytokinesis happens after mitosis: cell splits into two daughter cells Important notes: - During prophase, the nucleus breaks down temporarily (envelope, lamina, nuclear pores) - Allows CHS to move and seperate

Question 48

Why is proper control of the cell cycle important?

Answer 48

- Proper control of the cell cycle prevents uncontrolled cell growth, like in cancer Cell cycle is controlled at checkpoints (think of them as quality control stops): - Checkpoints make sure everything is working properly before moving to next stage - If there is something wrong (like DNA damage), the cell can: - Fix it - Pause the cycle - Die (apoptosis) or go into a permnanent rest state (senescence) Main checkpoints: 1. Mid-G1 checkpoint (aka start or restriction point): cell decides whether to start DNA replication and organelle duplication 2. End of G2 checkpoint: cell decides whether to enter mitosis 3. End of metaphase: cell decides whether to seperate the CHS and finish dividing

Question 49

Who are the key players in cell cycle maintenance?

Answer 49

- Cell cycle is guided by positive controls: special mobile proteins that help the cell move from one phase to the next Who are key players? 1. Cyclins: proteins that rise and fall in amount during the cell cycle (why they are called cyclins) - They control when the cell progresses through stages like G2 to M 2. Cdks (cyclin-dependent kinases) - Enzymes found in nucleus - They need to bind cyclins to become active - Once active they phosphorylate (add phosphate groups too) target proteins to turn things off or on for cell cycle progression How it works together: - Cyclins bind Cdks and cdks get activated then cdks phosphorylate target proteins and the cell moves onto the next stage Nucleocytoplasmic (mobile) factors: responsible for mediating transitions from one phase to next Cdks: cell-cycle specific kinase enzymes located in nucleus: phosphorylate various 'target' (nuclear) proteins: turn on or off Cyclins: nucelocytoplasmic portines: bind Cdks and regulate their activity during specific stages of the cell cyle eg mitotis cyclins during G2-to-M transition Cyclical= they cycle with the cell

Question 50

How do mitotic cyclin levels change throughout the cell cycle?

Answer 50

Early G1: - low cyclins low cdk activity End of G2/Start of M phase -high cyclins means a high cdk activity - triggers the start of mitosis by activating cdks What do cdks do (once activated)? - phosphorylate (add a phospahte to) important nucelar proteins to prep for mitosis: Histones and condensins: - help pack dna into CHS Lamins: - Cause nuclear envelope to break down (phosphrlyation causes) - Disassembly of nucelar lamina Nups (nucleoporins): - Cuase the nuclear pore complex (NPCs) to break apart - Prepares the nucleus for mitosis by condensind DNA and breaking down nuclear strcture - Phoshorlyation causes

Question 51

What is open mitosis? and what happens to the nucleus during mitosis?

Answer 51

Early in mitosis (prophase to metaphase) - Nucleus breaks down completely - Nuclear membrane disappears - Nuclear lamina and nuclear pores (NPCs) fall apart - Soluble nuclear proteins (like ones with NLS) are released into the ER or cytoplasm What happens after mitosis (telophase)? - Two new nuclei are built in the daugher cells - Cyclin levels drop means cdk activity goes down - This causes: - Dephosporylation (removal of phosphates) - Reassembly of: - Nuclear lamina - Nuclear envelope - Nuclear pores - Nuclear proeins (with NLS) can now be re-imported back into nucleus - Nucleus completely disassembled by metaphase - Prophase: outer and inner membranes break down, lamina and NPCs disassemble, membrane-bound and soluble nuclear proteins released into ER membrance and cyotplasm, respectively

Question 52

How is the cell cycle regulated?

Answer 52

1. Cyclin levels go up and down (oscillate) during the cell cycle - Caused by how fast cyclins are made vs how fast they are destroyed 2. After mitosis begins, cyclin levels drop because: - New cyclins stop being made and old cyclin s are destroyed by the proteasome (cells protein shredder) 3. Cdks get turned off; this happens through phosporylation by other kinases 4. After mitosis starts, leftover cyclins are also kept out of the nucleus - So they cant turn cdks back on

Question 53

How does nucleocytoplasmic transport of cyclins happen? eg Cyclin B1?

Answer 53

Cyclins move between the nucleus and cytoplasm (they both have NLS and NES) - Where the cyclin is found depends on which signal is stronger (NLS or NES): - Up to G2:cyclin shuttles back and forth, but stays mostly in the cytoplasm (NES stronger) - At the end of G2/start of M phase NES gets phosphorlayed and turned off, NLS becomes stronger: cyclin accumulates in nucelus, activates cdks - After M phase starts: - NES gets dephosphorlyated and is strong again - Cyclin moves back to cytoplasm Why this matters: - cyclin b1 needs to be in nucelus at right time to activate cdks and start mitosis, but after that, ots kicked out again to shut down

Question 54

What is a standard brightfield microscope?

Answer 54

Main components: light source, condenser lens, stage (holding specimen), objective and ocular (projection) lenses, and "detector" eye - light diffracted by specimen and undiffracted light (eg field of view) focused by objective lens - Image usually captured by video camera - more sensitive to low light intensities: living cells can be viewed with limited photo (light) damage - record image as digital file: diff light intensities converted into 2D array of numbers (quantified) - easily manipulate digital images using various computer software programs eg deconvolution: designed to remove background and out of focus light (yields and increases contrast and clarity) - cameras can see better in lower light (helpful for viewing living cells without damaging them with too much light) - how it works: - camera captures light intensity (brightness) - turns this into numbers in a 2d image: so data can be analyzed or adjusted Image editing: - software allows you to edit the image - one tool is called deconvolution - removes background blur and sharpens the image - makes it clearer and easier to understand

Question 55

What is the purpose of brightfield microscopy?

Answer 55

- Primary purpose of microscopy to generate magnified, high-quality view of specimen overall magnification=objective lensxoccular lens

Question 56

What about the quality of the image? Does continuining to enlarge image provide more details/info?

Answer 56

No, it is "empty" magnification

Question 57

What is resolution?

Answer 57

- most important aspect of todays microscope - minimum distance that can seperate two points that still remian idetifiable as seperate points, ie ability to distunguish two close objects as seperate entities - Ability to show two things as seperate even if they are close together

Question 58

Resolving of microscope depends on two main factors what are they?

Answer 58

1. wavelgenth of illumination light 2. numerical aperture (NA) (light gathering qualities of objective lens and specimen mounting medium) Resolution (distance in nm)=(0.61xwavelegnth)/NA - higher/better resolution has a lower number

Question 59

How is resolution of a microscope maximized?

Answer 59

- use shorter wavelength of illuminating light eg. red light 0.61x700nm/1 air= 427 nm eg. blue light 0.61x400 nm/1 air=244 nm - Increase NA- after mounting medium (eg air to oil) - eg. blue light 0.61x400nm/1.4 (oil)=174 nm Two main factors affect microscope resolution... 1. Wavelength - shorter wavelength (like blue light) gives better resolution - Longer wavelengths like red give worse resolution 2. Numerical aperture - measures how much light the lens can gather - Higher NA=better resolution - Increase NA by changing the medium (eg using oil instead of air between the slide and lens) - standard light microscopes cant resolve things smaller then 200 nm - so you can see large organelles like nuceli, mitochondria, cholorplasts but not smaller structures

Question 60

What are the limits of resolution?

Answer 60

Human eye about 0.1 mm (cant see cell or tiny structures) Standard microscope (CLSM): about 200 nm (500x) (can see larger organelles like nuclei) Super-resolution CLSM: about 20 nm (5,000x) (can see smaller strctures like some vessicles) Electron microscopes (EM): about 0.2 nm (500,000x) can see tiny molecules like proteins EM uses electrons (wavelength about 0.0045nm) rather than photos- lower wavelgenth yields higher resolution

Question 61

What is brightfield microscopy?

Answer 61

- Basic type of light microscopy where light passes through the sample - Main problem: the image often has poor contrast: hard to see details unless the sample is specially treated

Question 62

How samples are prepared in brightfield microscopy?

Answer 62

1. Fixation - use chemicals like formadehyde to "freeze" the strcture - looks like proteins/nucelic acids in place by cross-linking them 2. Embedding - Sample is put into wax or plastic to hold its shape 3. Sectioning - Thin slices are cut using a microtome (cutting tool) 4. Staining - Dyes are added to highlight specific molecules (like proteins or DNA) so they are visible under the microscope What are the downsides of this process? - Sample is dead (fixation kills cells) - Artifacts (fake-looking structures) can appear due to the preperation steps - Staining is limited to a few molecule types

Question 63

What is fluorescence microscopy? How does it work?

Answer 63

- Technique to see glowing (fluorescent) molecules in living or fixed cells -Lets you watch specific strctures or processes using special glowing tags How does it work? it uses: - Natural fluorescence in cells (autofluorescence) - FLuorescent dyes or antibodies bind to specific molecules (immunofluoresnce) - Fluorescent proteins like GFP that glow in diff colours Pros: - High contrast=easier to see details - Can show 3D strctures and track live cell activity Cons: - Fluorescne out-of-focus areas can cause the image to look blury, especially in thick samples

Question 64

How does fluorescence work?

Answer 64

1. Light hits a fluorescent molecue (like GFP) - absorbs a photon of light (usually high energy like blue, which has a short wavelgenth) 2. Electrons get excited - absorbed energy makes electron jump to a higher energy level 3. Excied state is unstable - electrong quickly falls back down to normal state (ground state) 4. Energy is released: - when the electron falls back down, it gives off light-but with less energy (since some is lost as heat) - so light that comes out hasa longer wavelength (like red or green) KEY POINTS: - absorbed light=high energy/shprt wavelength (blue) - emitted= lower energy/longer wavelength (green/red for ex)

Question 65

What is confocal lasr-scanning microscopy (CLSM)?

Answer 65

- type of fluorescence microscopy - uses lasers and advanced features to get very clear and detailed images How is it diff? - Similar to a regular brightfield, but better: - Uses one or more lasers to shine wavelengths of light - laders excited fluoresncet molecules in sample - Microscope is set up to focus , so only light from sharpest plane is captured - Gives clear, high resolution imsges, even in thick samples

Question 66

What makes CLSM special?

Answer 66

- usually with living speciments - no need to kill/fix the cells - lets you see real-time cell activity - can see the dynamic procoesses like organelle movement in living cells Uses: - natural fluoresncece - stains/dyes - genetically added fluorescent proteins (eg GFP fusion proteins) - lasers go deep into thick samples beter then standard light microscopes for thick tissue

Question 67

How does CLSM work?

Answer 67

1. Laser scans the sample point by point with specific light to excite 2. Only fluorescent light from one sharp layer (focal plane) is allowed through a pinhole 3. Light from above or below that layer is blocked, so: - image is not blurry - you get a clear and focused view of one thin slice at a time Key features: Pinhole: acts likr a filter: only lets in in-focus light Scanning system: moves the laser across the sample Computer: collects the light info and builds a sharp image - This setup allows one to build a detailed 3D images layer by layer

Question 68

What does the CLSM give you?

Answer 68

- Clear, thin 2D images of sample called z-sections or optical slices - Slices are taken one layer at a time, so image is less blurry than a regular fluorescence microscope Why useful? - by collecting many thin slices (like pages in a book) can: - see fine details at diff depths - combine into 3d if needed - form z-stack and give 3d image

Question 69

What are the limitations to CLSM?

Answer 69

1. too slow for fast cell activity - it scans one point at time, so cant keep up with very fast changes in live cells 2. Photobleaching - laser can burn out fluorescnet molecules, so stop glowing 3. phototoxicity - laser can harm or kill living cells py producing chemicals (free radicals) 4. Not great for thick samples - hard to see into thick tissues 5. resolution limited - can only see about 200 nm, which is good not the best

Question 70

What is the endomembrane system?

Answer 70

- The endomembrane system is a group of connected organelles inside a cell that work together to: - Move materials like proteins and lipids around the cell - Do this by using small transport vesicles (little bubbles that carry stuff) What is included? Mitochondria and cholorplasts NOT PART - Endoplasmic reticulum (ER): makes and moves proteins/lipids Golgi apparatus: - modifies, sorts, and ships proteins/lipids Endosomes: - help sort and transport materials coming Lysosomes/vacuoles: break down waste or store things Secretory granules: store materials to be released from the cell Plasma membrane: outer layer of the cell, where materials enter/exit ER-derived organelles: lipid body and peroxisomes and lipid bodies - Large amounts of material (eg proteins, lipids, etc) exchange (trafficked) between each organelles/strcuture via small, membrane-bound transport vesicles

Question 71

How does vessicle transport happens?

Answer 71

Step 1: Vesicle formation (budding from donor) - move materials (like proteins or lipids) from one organelle to another using transport vesicles Step 1: Budding - vessicle forms by budding off the membrane of a donor organelle (eg ER or golgi) - vessicle carries "cargo", which includes: - soluble proteins (float inside the vesicle) - membrane proteins, lipids, or carbs (stuck in the vessicle membrane) How does the vesicle know what to carry? - coat proteins (special helper) does two things: - select cargo from the donor organelle - helps shape the vesicle so it buds off properly Key terms: - Donor compartment=where the vesicle starts (eg ER) - Receptor proteins= help load the correct cargo into the vessicle - coat proteins=help select cargo and form vesicle Step 2: nascent vessicle transported through cytoplasm to "acceptor" membrane compartment - vessicle receptor (coat) proteins regulate intracellular trafficking of vesicle to proper acceptor membrane - also involves molecular motors and cytoskeleton highways - motor proteins direct vesicle movement by linking to vesicle surface and cytoskeleton element Coat proteins on vesicle help guide the vessicle to the correct location - vessicle uses the cells cytoskeleton (like train tracks) to move through the cell - Motor proteins (like little engines) pull the vessicle along the cytoskeleton Step 3: vesicle "fuses" with proper aceptor membrane compartment - receptors proteins also regulate vesicle-acceptor membrane fusion - vesicle (donor) membrane and lumenal cargo proteins incorporated into acceptor compartment Step 4: process of budding and fusion repeated and can occur in reverse direction - other receptor proteins regulate recycling of proteins that "escape" to acceptor membrane compartment back to donor membrane compartment - budding and fusion can happen in reverse; some proteins accidently go to the wrong place- so they get sent back to the orginal organelle (the donor compartment) - Special receptor proteins help idetify and recycle these scaped proteins

Question 72

What is the biosyntetic pathway (main transport route)?

Answer 72

- materials (like proteins for lysosomes) are: 1. Made in ER (endoplasmic reticulum) 2. Sent to Golgi 3. Then moved to endosome 4. And finally to lysosomes (for vacuole in plants)

Question 73

How does HIV work?

Answer 73

- they can go from endosome to the plasma membrane and out of the cell into extracellular space, using exosomes (small vessicles released from cells)

Question 74

What is the secretory pathway?

Answer 74

two types of secretion? 1. Constitutive secretion - materials transported from golgi to plasma membrane and/or released (via exocytosis) outside of cell (ie extracellular space) in secretory vessicle - secretory vessicle membrane components incorporated into pm and lumenal "cargo" released into extracellular space - exocytosis=vessilce trafficking to and fuson with plaama membrane, release of contents 2. Regulated secretion - occurs only in specialized cells - er-derived materials from golgi stored in secretory granules - in response to cellular signal, secretory signal, secretory granule fuse with pm and release (via exocytosis) lumenal cargo into extraceullular space eg regulated release (secretion) of neurotransmitters by nerve cells into synaptic gap - secretory granule membrane components incorporated into pm - only happens with specific cells - proteins from er go to golgi and are packed into secretory granules - these granules wait inside the cell until a signal tells them to release content - qhen signal comes like nerve imppulse granules - fuse with plasma membrane, and relase their cargo out of cell granules wait; grannies

Question 75

What is the endocytic pathway?

Answer 75

- operates in opp direction of secretory pathways (materials move into cell) - materials from pm (eg receptor proteins destine for degredation or bound to ligand) and/or extracellular space incorported into cell (via endocytosis) and then trasnported to endosomes and lyssoomes (vacuole)

Question 76

What pathway is used to study protein traffickig in cells?

Answer 76

secretory, where proteins are made, processed and transported out of cell

Question 77

Do all cells secrete the same amount of materials?

Answer 77

No; amount of secretion varies

Question 78

Name three types of cell types with high levels of secretion?

Answer 78

1. Yeast and plant cells: secrete wall materials 2. Panreatic acinar cells: secrete digestive enzymes 3. Intestinal epithelial cells: secrete mucus - pancreatic and intestine epithelial cells highly polarized

Question 79

What does it mean when a cell is highly polarized?

Answer 79

Its oganelles organized into specific regions, each with unique fincton

Question 80

Where are the nucelus and rough er found in secretory cells

Answer 80

basal end of cell

Question 81

Where are golgi and lysosomes found in polarized cells?

Answer 81

in central region of cell

Question 82

Where are the secretory granules found in polarized cells?

Answer 82

At the apical end (facing the duct or lumen, read to release enzymes or mucus)

Question 83

Where are secretede proteins made, and how do they reach the surface?

Answer 83

- made in rough er - processed in golgi - packaged in vesicles - released at plasma membrane (apical end)

Question 84

What is the purpose of a pulse chase experiment?

Answer 84

track how proteins move through secretory patheay over time

Question 85

What happens during the pulse step?

Answer 85

cells are given radioactive amino acidsthat get built in new proteins

Question 86

What happens during the chase step?

Answer 86

cells are then given non-radioactive amino acids, so researcers can follow whew the labeled proteins go

Question 87

How do scientists see protein movement in these experiments?

Answer 87

usong autoradiography: exposing cells to x-ray film to see where radioactive proteins are

Question 88

What did pulse chase experiemtns prove about protein transport?

Answer 88

proteins move through organelles using vesicles, not by drifting in cytoplasm

Question 89

In pulse chase experiment where owud toy first find radioactive proteins?

Answer 89

In the rough er, then later in golgi, vessicle and plasma membrane

Question 90

in a pulse chase experiment, where are radioactive proteins found after a short chase about 3 min?

Answer 90

in rough er, where protewins first made

Question 91

Where are radioactive proteins lcoated after an intermeidate chase about 20 min

Answer 91

in golgi where peoteinhs modfified

Question 92

After loing chase about 120 min whwre do proteins go?

Answer 92

secretory vessicles and some released out of cell

Question 93

What pathway did the pulse chase experiemnt help to confirm?

Answer 93

secretory pathway: er to golgi to vessicles to plasma membrane

Question 94

What did pulsechase experiments revelaed in non-secretory (non poarized cells)?

Answer 94

portiens trafficking between golgi, endoasomes and lysosomes

Question 95

What is the purpose of using autofluorescent proteins like GFP and in live-cell imaging?

Answer 95

to track protein location and movement in living cells

Question 96

How are fluorescent proteins like GFP used in molecular bio?

Answer 96

their gene is fused to gene of interest, creating a glowing versio of the target protein

Question 97

What technique is used to visualize fluorescent proteins insdie living cells?

Answer 97

Fluoresence microscopy, such as CLSM (confocal laser scannong microscopy)

Question 98

What is a recombinant gene fusion?

Answer 98

lab created gene that combies a fluorescnet protein gene with a protein of interest gene

Question 99

What kinds of organizmz or cells can be used?

Answer 99

Yeast, plants, mice and many others

Question 100

What are aome colours of fluorsence proteins used in imaging?

Answer 100

GFP, RFP, YFP, BFP, tangerine, cherry etc

Question 101

What does VSVG-GFP stand for?

Answer 101

viral glyocprotein fused to green fluoresenct protein (GFP)

Question 102

What happens o vsvg-gfp at 40 degress celsius

Answer 102

it misfilds and gets traooed in er (cant be transported)

Question 103

What happens to vsvg-gfp at 32 degrres?

Answer 103

it folds correctly ad moves through secretory

Question 104

Whar is purpose of using a temp senstive protein like vsvg?

Answer 104

control and track when and how proteins move through cell

Question 105

What technique is used to watch vsvg-gfp?

Answer 105

fluoresnce microscopy like clsm

Question 106

Aftering shifting to 32 degrres where would VSVG-GFP go over time?

Answer 106

er to golgi and then to secretory plasma membrane

Question 107

How does vsvg-gfp relate to pulse chase?

Answer 107

similar results of how proteins go thorugh endomembrne system

Question 108

What is subcellular fractionation?

Answer 108

a method to seperate organelles based on their size ir density using centrifugation

Question 109

What does subcellular fractionation help scietnists study?

Answer 109

structure and function of specific organelles

Question 110

What organelle is often ilsoated to study protein syntethsis?

Answer 110

rough er

Question 111

What are free vs attached ribosmomes?

Answer 111

free ribosmes float in cytosol attached ribosmes are bound to er me,rane

Question 112

What kinds of prcoesses can be studied using osolated rough er?

Answer 112

protein synthesis cotranslocation (proteins entering the er) vessivle trafficking

Question 113

What is the first step in subcellular franctionation?

Answer 113

homogenization: gently breaking open cells while keeping organelles intact

Question 114

What is a homogenate?

Answer 114

the mix of cell contents after homogenizatio (contains organelles, proteins, etc(

Question 115

What does differntial centrifugation do?

Answer 115

seperates organelles based on size and density using diff spin speeds

Question 116

What organelle is found in the pellet at 600gx10 min?

Answer 116

nuclei

Question 117

What organelles pellet at 100,000 gx60 min?

Answer 117

Plasma membrane and ER fragments

Question 118

What are microsomes?

Answer 118

Vesicle-like fragments of ER or plasma membrane formed during centrifugation

Question 119

What is the difference between pellet and supernatant?

Answer 119

Pellet: solid at bottom (heavier stuff) Supernatant= liquid on top (lighter stuff)

Question 120

what is 15Kgx5 min?

Answer 120

mix of mitochondria, lysosomes, etc

Question 121

What does equilibrium density-gradient centrifugation seperate organelles based on?

Answer 121

their density

Question 122

What solution is used to create the density gradient in this method?

Answer 122

surcrose solution with increasing density from top to bottom

Question 123

what happens to organelles during centrifugation in a sucrose gradient?

Answer 123

they move to the point where the surcose density matches their own (equilibriun density)

Question 124

What are the aprox densites of lysosomes, mitochondria and peroxisomes?

Answer 124

lysosomes= least dense 1.12 mitochondria= middle 1.18 peroxisomes= most dense

Question 125

What is the main purpose of using this centrifugation method?

Answer 125

to purify and study specific organelles and their contents (eg protein, lipids)

Question 126

What can the seperated organelles be used for?

Answer 126

studying strcture and function - running in vitro assays for protein import or vessicle trafficking

Question 127

What is the goal of using cell free systems in endomembrane studies?

Answer 127

study the function of specific proteins from the endomembrane system outside of living cells (in vitro)

Question 128

What decade did cell free systems become a common appraoch in studying the endomembrane system?

Answer 128

1990s "biochem approach"

Question 129

What are liposomes?

Answer 129

artifical spherical vessilces made ffrom phospohlipid bilayers, used to mimic membranes in lab experiments

Question 130

How are purified endomembrane proteins studied in cell free systems?

Answer 130

insterted into liposomes to study their functino and interaction

Question 131

What is the strcuture of a liposome similar to?

Answer 131

a cell membrane, with a phospholipid bilayer and a hydrophobic interior

Question 132

Why are liposomes useful for studying membrane proteins?

Answer 132

they provide a controlled, simplified system to test how proteins behave without using whole cells

Question 133

What kind of protein actiivty can be obsrved using liposome systems?

Answer 133

Proteins involved in vesicle formation and budding (ie transport proteins)

Question 134

What are liposomes mixed with?

Answer 134

purified proteins: allows for study of proteins in vitro natural lipid environment; eg proteins involved in formation of trasnport vesivles cause liposome membrane budding

Question 135

What is the goal of mutant phenotype analysis?

Answer 135

identify genes or proteins involved in vessicle trafficking by studying mutant cells with defects

Question 136

What type of approach is mutant genotype analysis?

Answer 136

gentics approach (used from 1990s to 2000s)

Question 137

How are mutant phenotypes found?

Answer 137

by screening cells for changes when certain genes are mutated

Question 138

What does it mean that vessicle trafficking is evolutionary con served

Answer 138

similar across sprcies from yeast to humsnd

Question 139

What kind of organism used in this research?

Answer 139

Yeast, since it secretory pathway is essential and easy to studt

Question 140

What are secretory "sec" mutants

Answer 140

mutant yeast strains thst can secrete proteins at low temp, but not at high restrctive temp; mutations in secretory patheay grnes that cause proteins to gedt stuck at diff steps of the endomembrane system

Question 141

What do sec mutants help scientsirts study?

Answer 141

where and how protines normally move through the secretory patheay and what happens when specific genes dont work

Question 142

What happens to secreted proteins in sec mutants?

Answer 142

they accunulate in the cell at the step where the secretory pathway is blocked

Question 143

What is a class A sec mutant?

Answer 143

proteins accumulate in cytosol due to defect in protien translocation into the ER

Question 144

what is class b mutant?

Answer 144

proteins accumulate in er due to defect in er vesicle formation example sec12 mutant shows enalrged er

Question 145

What are classes c d and e

Answer 145

class c: accumulation er-to-golgi vessicles class d: accumulatoin in golgi class e: accumulstion in secretory vessilces cant reach cell surface

Question 146

What can double sec yeast mutants tell us?

Answer 146

the order of the steps in the secretory pathway ex if both b and d mutants block diff steps, comparing shows that er vessicle budding happens before golgi vessicle budding

Question 147

How can organelle morphology changes in mutants help?

Answer 147

they show which proteins are invoved in organelle fomration or maintenance ex golgi fragmentation means affects golgi fission/fusion

Question 148

What happens when scietnists clone sec genes?

Answer 148

they can priduce and studt the normal (wild-type) versions of the proteins to see how they work

Question 149

What do sec mutants help us to understand overlall?

Answer 149

mechsnidmd of prtoein and vessicle trafficking in endomembrane system

Question 150

What is the endoplasmic reticulum?

Answer 150

highly complex network of membrane enclosed, rod-like tubles and sheet-like cisternae (ie flattened sacs) organlles with largest surface area

Question 151

What is the lumen of er?

Answer 151

the liquid filled space inside the er tubles and cistenae

Question 152

What are the 2 shapes the ER can take?

Answer 152

tubules (thin tubes) and cistenae (flat sacs)

Question 153

What protein shapes the ER membrane?

Answer 153

Reticulons (endorplasmic reticulum)

Question 154

What is special about reticulons?

Answer 154

they have a v-shaped (hairpin) structure that helps bend the membrane

Question 155

What do reticulons do for the ER?

Answer 155

control the curvature and overall shape of the ER

Question 156

What does it mean that the ER is a "highly dynamic network"?

Answer 156

Means that the ER is constantly changing- bending, growing, shrinking, fusing and splitting

Question 157

What are the two main parts of the ER that change shape?

Answer 157

Tubules (tube like structures) and cisternae (sheet-like structures)

Question 158

What types of changes happen in the ER?

Answer 158

Bending, gorwing, shrinking, fusion (joining), and fission (splitting)

Question 159

What is the ER made of?

Answer 159

ER has multiple subdomains: diff regions with unique shapes and functions

Question 160

What are the two main types of er subdomains?

Answer 160

rought er and smooth er

Question 161

What does rough er do?

Answer 161

rer has ribosomes, makes proteins and phospholipids for membranes

Question 162

What does smooth er do?

Answer 162

ser has no ribosomes, makes hormones, and stores calcium (Ca2+)

Question 163

What do RER and SER look like?

Answer 163

rer has flat sacks with ribsomoes (cisternae); SER has curved tubles without ribosomes

Question 164

How many ER subdomins are there?

Answer 164

Over 20 ER subdomains, each with unique proteins and lipids for diff functions

Question 165

What is the nuclear envelope in the context of the ER?

Answer 165

The outer nuclear membrane is continuous with the rough ER, and contains nuclear pore proteins (Nups) and ribomsomes - outer layer of the nucleus is connected to rough er, has holes (called nucelar pores) and tiny machines (ribosomes) that help make and move proteins

Question 166

What are MAM and PAM in the ER?

Answer 166

MAM (mitochondria associated membranes): er parts that contact mitochondria, involved in lipid and protein exchange PAM (plasma membrane associated membranes): er parts that contact the plasma membrane, also for lipid/protein exchange

Question 167

What are the ER exit sites (ERES)?

Answer 167

eres are parts of the ER where transport vessicles form and leave to golgi

Question 168

Why are theres ER subdomains important?

Answer 168

they allow the ER to multitask-helping with protein/lipid exchange, organelle interaction, and directing vessicle traffic

Question 169

What are the two main sites for prtoein synthesis in the cell?

Answer 169

Rough ER and free ribosomes in the cytoplasm

Question 170

Where are free ribosomes located?

Answer 170

in the cytoplasm (not attached to er)

Question 171

What happens to proteins made by free ribosomes?

Answer 171

either stay in cytoplasm (eg glycolytic enzymes) or go to other organelles like the nucleus, mitochondria, or chloroplasts

Question 172

What does "post-translational targeting" mean?

Answer 172

the protein is sent to the correct part of the cell after is it fully made in the cytoplasm

Question 173

What is one example of a protein staying in the cytoplasm?

Answer 173

glycolytic enzymes

Question 174

Where are the membrane-bound ribosomes found?

Answer 174

attached to rough ER

Question 175

What is the fate of proteins made by membrane-bound ribosomes

Answer 175

They either stay in the RER, move to another er subdomain, go to other ER-derived organelles, or are sent to other organelles via vessicles

Question 176

What does it mean when a protein stays in the RER or localizes to another ER subdomain?

Answer 176

The protein either remains in place or moves through the ER to regions like the nuclear envelope

Question 177

What is an example of an ER-dervied organelles a protein might bind to?

Answer 177

peroxisome (which bud off the er)

Question 178

How do proteins get from er to other organelles like the golgi or plasma membrane?

Answer 178

They are transported in vessicles as part of the endomembrane system

Question 179

What does "co-translational translocation" mean?

Answer 179

its the process where a protein is made (translated) and inserted into the ER at the same time

Question 180

What is the signal that directs the ribosome to the ER?

Answer 180

a signal sequence at the begginning (n-terminus) of the protein

Question 181

What binds to the signal sequence during translation?

Answer 181

the signal recognition particle (SRP)

Question 182

What happens when SRP binds the signal sequence?

Answer 182

translation puases, and the complex (ribosome+mRNA+SRP) moes to ER membrane

Question 183

What receptor does the SRP binds to at the ER membrane?

Answer 183

the SRP receptor

Question 184

What is the translocon?

Answer 184

channel in the ER membrane that allows the growing protein to pass into the ER lumen

Question 185

What happens when the translocon opens?

Answer 185

ribosome resumes translation, pushing the protein through the translocon in the ER

Question 186

What enzyme removes the signal sequence inside the ER?

Answer 186

Signal peptidase

Question 187

What happens to the protein after it enters the ER lumen?

Answer 187

it folds into its correct shape

Question 188

where does translation begin for proteins destine for the ER?

Answer 188

on free ribsomes in the cytoplasm

Question 189

What is found at the N-terminus of a growing protein that is destine for the ER?

Answer 189

A signal sequence, which is a stretch of 8-15 hydrophobic amino acids

Question 190

What is the purpose of the signal sequence?

Answer 190

it acts as a "mailing label" that directs the ribosome to the rough er

Question 191

What is the SRP made of?

Answer 191

ribonucleoprotein complex: 6 proteins+ 1 small RNA

Question 192

What happens when SRP binds to the signal sequence?

Answer 192

it binds to ribsosome and puases translation

Question 193

What does the SRP do after it binds to the ribosome and signal sequence?

Answer 193

it targets the complex (ribosome, stalled nascent polypeptide, mRNA) to the surface of the rough ER

Question 194

Where does the SRP bind on the ER membrane?

Answer 194

to SRP receptor, which is an ER integral membrane protein complex

Question 195

What part of the SRP receptor serves as the docking site?

Answer 195

Cytoplasmic-facing domain on the SRP receptor

Question 196

What strengthens the interaction between SRP and its receptor?

Answer 196

Both SRP and SRP receptor bind GTP, which stabilizes their interaction

Question 197

What happens when GTP is hydrolyzed during protein translocation?

Answer 197

SRP and SRP recrptor are relased, so they can be reused in more protein import rounds

Question 198

What happens to ribosme and new polypeptide after SRP is released?

Answer 198

They are trasnferred to cytoplasmic side of Sec61 translocon

Question 199

What is the sec61 translocon?

Answer 199

multi protein complex that helps move proteins into the ER

Question 200

What makes up the sec61 translocon?

Answer 200

contains seceral ER membrane proteins (ie sec61alpha, beta and gamma) that form an hourglass shaped channel

Question 201

What is the function of the sec61 translocon?

Answer 201

forms an aqueous channel that allows polypeptide to enter the er of the lumen

Question 202

What happens after the polypeptide ribosome is trasnferred to sec61 translocon?

Answer 202

n-term of growing polypetide is interted into the opening of the translocon channel

Question 203

What resumes after the polypeptide is inserted into the sec-61 translocon?

Answer 203

translation resumes, and the elongating polypeptide is pused through the translocon into the ER lumen

Question 204

What drives the movement of the growing polypetpide through the translocon?

Answer 204

translation itseld (called co-translatioal translocation)

Question 205

What is the name of the translcoon used to move proteins into the ER lumen?

Answer 205

Sec61 translocon

Question 206

What is the shape of the sec61 translocon channel?

Answer 206

hour-glass shape

Question 207

What is the "pore ring" in the sec61 translocon made of?

Answer 207

ring of 6 hydrophobic amino acids located at the narrowest part of this channel

Question 208

What is the function of the pore ring in sec61?

Answer 208

acts as a gate to seal the channel and prevent ion/small molecule leakage, maintiaingin the er compartmentalization

Question 209

What is the second gatekeeping feature in the sec61 translocon?

Answer 209

a short alpha-helix "plug" that blocks the pore ring

Question 210

What happens to the alpha helix plug during protein translocation?

Answer 210

the growing polypeptide pushes the plug out of the way to open the channel

Question 211

What happens to the N-terminal sequence once it enters the er lumen?

Answer 211

it is cleaved off and degraded by signal peptidase

Question 212

What is signal peptidase?

Answer 212

an er iegral membrane protease (enzyme) that removes the signal sequence from the polypeptide

Question 213

Where is the catalytic domain of singal peptidase located?

Answer 213

in faces the ER lumen (does the actual cutting)

Question 214

How does signal peptidase know where to cleave?

Answer 214

it recognizes a cleavage sequence motif just downstream (c-terminal) of the signal sequence

Question 215

Does protein translocation stop after cleavage of the signal seqauence?

Answer 215

No, co-transloational translocation continues untl the full polypeptide is in the ER lumen

Question 216

What happens in step 7 of co-translational tranlocation into the rer lumen?

Answer 216

translation finishes and the ribomse is released from the transclocon

Question 217

What happens in step 8 of co-translcatinoal translocation?

Answer 217

the rest of the protein enters the er lumen, and the translcon closes as the pore pluh moves back in to block the channel

Question 218

What happens to the protein after it enters the ER lumen?

Answer 218

gets glyocylated (sugars added) and preoprly folded by reticuloplasmins

Question 219

What are reticuloplasmins and name a few examples?

Answer 219

they are ER chaperon proteins that help with proper protein foldig, examples include BiP, calnexin and calreticulin

Question 220

What do ER molecular chaperones do?

Answer 220

they bind to new proteins to help fold them properly, stop clumping (aggregation) and help them form complexes

Question 221

What is glyosylation?

Answer 221

Addition of sugars to protein

Question 222

Where are most membrane prtoeins made?

Answer 222

on membane-bound ribosomes on the rough er

Question 223

What type of membrane proteins are made at RER?

Answer 223

resident membrane proteins of the er and proteins for all other post-er compartments (eg golgi, lysomes, plasma membrane, nucelar envelope)

Question 224

What is the exception to membrane protein synthesis at the RER?

Answer 224

membrane proteins destined for mitochondria and chloropasts are sytnetsize in the cytoplasm

Question 225

What does co-transloational insertion mean?

Answer 225

the membrane protein is inserted into the er membrane while it is still beig made by the ribosomes

Question 226

Why are the ribomosmes important for inserting membrane proteins into the ER?

Answer 226

ribosmoes translates mrna into protein, and signal sequence directs the growing protein into or onto the er membrane

Question 227

How does does the insertion of integral membrane proteins differ from soluble proteins?

Answer 227

ingetgral membrane proteins are integrated into the er membrane and require proper orientation or topology, unlike soluble proteind which are fully translocated into thr ER lumen

Question 228

What is membrane protein topology?

Answer 228

the number of membrane spanning domains and the oritentation of the protein in the membrane

Question 229

What is a transmembrane domain?

Answer 229

an alpha helix of about 16-25 hydrohpobic amino acids that speans the membrane, interacting with hydropohibc core of the phospholipid bilayer

Question 230

Where are most inteegral membrane proteins synthesized?

Answer 230

on membrane-bound ribsomes at the rough er

Question 231

What are the main types of single-pass membrane proteins?

Answer 231

type I: n-terminal in er lumen, 1 TMD, cleaved signal sequence type II: n-terminal in cytosol, 1 TMD, no signal sequence type III: n-term in er lumen, 1 TMD, no singal sequwnce

Question 232

What is a tail anchored protein?

Answer 232

A membrane protein with 1 c-terminal tmd and n-term in cytosol

Question 233

What is a type iv membrane protein?

Answer 233

multi-pass membrane protein with 2 or more TMDs and varied topologies, with no cleaved signal sequence

Question 234

What role does the transmembrane doamin play?

Answer 234

acts a stop-transfer anchor (STA) sequence, stopping further translocation

Question 235

What happens to stop-transfer anchor sequence after entering the translocon

Answer 235

STa moves or exits laterally out of translcocon

Question 236

What continues afrer the STA stops further translocatin?

Answer 236

translation continues, and rest of polypeptide chain elongates into the cytosol

Question 237

Do type II membrane proteins have an N-terminal signal sequence?

Answer 237

no, they have an internal signal anchor (SA) sequence instead

Question 238

What does the interal signal-anchor (SA) sequence do in type II membrane proteins?

Answer 238

it acts as both the signal sequence for SRP binding and as the membrane anchor

Question 239

What happens to the signal anchor sequence when it enters the translocon?

Answer 239

it gets flipped positioning the n-terminus of the protein in the cytosol

Question 240

What determines the orientation of the protein across the membrane in type II membrane proteins?

Answer 240

severely postivetly charged amino acids just upstream (N-terminal) of SA sequence

Question 241

What is the positive outside rule?

Answer 241

positively charged regions of the protein tend to face they cytosol (outside of er), helping orient the protein in the membrane

Question 242

During type II membrane protein insertion, what happwns in step 2?

Answer 242

translation continues, and the c-term of the polypeptide extends into the ER lumen through the translocon

Question 243

What determines the orientation of type III membrane proteins?

Answer 243

positively charged amino acids located downstream (c-terminal) of the SA sequence prevent flipping, keeping the SA in place

Question 244

What are multi-spanning (type Iv) er membrane proteins?

Answer 244

er membrane proteins with multiple transmembrane domains (TMDs) and no N-terminal signal sequence

Question 245

What two internal sequences do type IV membrane proteins contain?

Answer 245

internal signal anchor (SA) sequences and stop transfer anchor sequences

Question 246

What types of sequences do type i and 4 have vs 2 and 3?

Answer 246

i and 4: i has sta and 4 has sta and sa 2 and 3: just sa

Question 247

Do membranes form from scratch (de novo) in cells?

Answer 247

No, all membranes come from pre-existing membranes

Question 248

Where are most membrane proteins and lipids made in the cell?

Answer 248

at the er

Question 249

What are the exceptions to er synthesis of membrane components?

Answer 249

glycolipids are made in golgi, and some proteins and lipids are made in chlorplasts and mitochondria

Question 250

What happens to newly made er membrane proteins and lipids?

Answer 250

they move to other membraned in the cell (er subdomains or other organelles)

Question 251

How do ER components move to other parts of the cell?

Answer 251

by lateral diffusion or by tranport vessicles

Question 252

What does it mean that ER membrane proteins and lipids are distributed asymetrically?

Answer 252

Not evenly distributed between the two side (leaflets) of the lipid bilayer-diff types face diff directions

Question 253

What are integral membrane proteins and how are they distributed in the ER membrane?

Answer 253

integral membrane proteins span the membrane and have diff regions exposed on either the cytoplasmic side or exoplasmic side (facing the ER lumen). Orientation is specific and determine during protein synthesis

Question 254

What are peripheral membrane proteins?

Answer 254

do not span the membrane. they are attached to either the cytoplasmic or lumenal side of the er membrane through weak interactions

Question 255

How are membrane phospholipids distributed in the ER membrane?

Answer 255

they are distributed unequally between the cytoplsmic and exoplasmic leaflets, contributing to mebrane asymmetry

Question 256

Where is membrane protein and lipid asymmetry established?

Answer 256

at the er; this assymetry is maintained through the entire endomembrane system

Question 257

What happens to the cytoplasmic and exoplasmic faces of membranes as vesicles move through the endomembrane system?

Answer 257

- they are conserved - the cytoplasmic face always stays facing the cytosol - the exoplasmic face always stays facing the lumen of organelles or the extracellular space after fusion with the plasma membrane

Question 258

What does it mean that the exoplasmic face of the ER becomes the extracellular face?

Answer 258

protein facing the er lumen wull face the lumen of vessicles and golgi, and eventually outadie the cell when the vesicle fuses with the plasma membrane

Question 259

What are the final steps in co-translational translocation at the ER?

Answer 259

the final steps involve processing of the newly synthesize proteins in the ER lumen 1. signal sequence cleavage 2. inital glycosylation 3. protein folding and assembly 4. protein quality control (any misfolded or improperly assembled proteins recognized and degraded)

Question 260

What is the signal sequence cleavage in the ER?

Answer 260

the removal of the N-terminal signal seqeuence by an enzyme called signal peptidase, once the protein enters the ER lumen

Question 261

What happens during the intial stages of glycosylation in the ER?

Answer 261

covalent addition of carbs chains to specific amino acids of the protein. this helps in folding, stability, and protein-protein interactions

Question 262

What is the role of protein folding and assembly in the er?

Answer 262

proteins fold into their correct 3d shape and may form oligomers with help from molecular chaperones like reticuloplasms

Question 263

Why is the ER ideal for prcoesing and quality control of new proteins?

Answer 263

first compartment iof the endomembrane system, where biosynthetic and secretory proteins are initially process and checked for proper folding

Question 264

What is the function of sugar groups in glycoproteins?

Answer 264

sugar group helps with: - proper protein folding - serves as binding sites for other macromolecules (eg receptors, chaperones)

Question 265

What is the most common type of glycosylation in the er?

Answer 265

N-linked glycosylation: sugar groups are attched to the nitrogen of the side chain of asparagine (Ask) residues

Question 266

What are the two stages of N-linked glycosylation?

Answer 266

Core glycosylation: addition of basic sugar chain in er core modification: futher processing, which often continues in golgi

Question 267

What is added to asparagine during N-linked glycosylation?

Answer 267

short chain of sugar monomers, assembled in a precise order to form an oligosaccharide

Question 268

What makes the core oligosaccharide in the ER?

Answer 268

ER membrane-bound glycosyltrasnferases build the core oligosaccharide, a branched surgar chain of 14 resudies, including mannose and glucose

Question 269

What is the functio of the core oligosacharide?

Answer 269

important for protein quality control and helps ensure proper folding

Question 270

What molecule acts as the anchor for building the core oligosacchardie

Answer 270

Dolichol phospahe, a membrane lipid that anchors and carries the growing sugar chain

Question 271

What is the first step of core glycosylation?

Answer 271

addition of the first sugar to dolichol phopshare on the cytosolic side of the er membrane

Question 272

What happens after the intial sugars are added to dolichol phosphate?

Answer 272

the sugar chain is flipped into the er lumen, where more sugars are added to complete the 14-sugar core oligosaccharide

Question 273

What does tunicamycin do?

Answer 273

tunicamycin blocks the first step of N-linked hlycosylation by inhibitng the glycosyltransferases, which prevens proper folding of newly synthesize ER proteind

Question 274

What types of sugars are found in the core oligosaccharide?

Answer 274

- N-acetlyglucosamine - Mannose - Glucose

Question 275

What is the final step of N-linked glycosylation in the ER?

Answer 275

transfer of the core oligosaccharie rom dolichol phopshate to a new soluble or membrane protein as it is being synthesize via the sec61 translocon

Question 276

What happens to dolichol phospahte after the sugar is transfered?

Answer 276

it is recycled and reusud for another round of core oligosaccharide synthesis

Question 277

Where on the protein is the oligsaccharide attached?

Answer 277

to asparagine residue in a specific amino acid sequence motif N-X-S/T where, N=asparagine X= any amino acid (except proline) S/T= serine or threonine

Question 278

What part of the protein recieves the core oligosaccharide?

Answer 278

the lumen-facing side of the protein that is being translated into the er during co-translational translocation

Question 279

What does the sec61 complex do during glycosyltation?

Answer 279

acts as the protein translocation channel through new polypetides enter the er lumen, allowing co-trnalsational glycolsylation to occur

Question 280

What is core modification in N-linked glycosylation?

Answer 280

the second stage of N-linked glycosylation wher the attached 14-sugar oligosaccharide is trimmed and modified after attachment to the protein

Question 281

Which enzyme trim the oligosaccharide during core modification?

Answer 281

ER luminal glucosidases, which remove two of the three glucose residues

Question 282

Why is thr last glucose removed and re-added during core modification?

Answer 282

the removal and re-additoin of the final glucose unit (steps 3 and 3a) are crucial for proper protein folding and quality control

Question 283

What is the purpose of trimming sugars from the oligosaccharide?

Answer 283

to help regulate protein folding, check folding accuracy, and prepare the protein for export from the ER

Question 284

What happens to protein after core modification in the ER?

Answer 284

goes to cis-golgi for further processing if properly folded

Question 285

What happens to new proteins during N-linked glycosylation and modification?

Answer 285

they are rapidly foleded inito theor proper 3d conformation, aided by er-resident proteins

Question 286

What is the function of protein disulfide isomerase (PDI)?

Answer 286

PDI catalyzes disulfide bond formation between cysteine resudes in proteins, promoting proper folding and stabilizing 3d structure

Question 287

How do chaperons like BiP and calnexin help proteins in the ER?

Answer 287

temporarily bind to new proteins to ensure proper folding, prevent aggregation and assist with quality control

Question 288

What do reticuloplasmins and PDI do during ER protein synthesis?

Answer 288

They bind to new glycoproteins (with one reminaing glucose) as they are synthesized through sec61, helping with protein folding, oligomer assembly and stability

Question 289

What is the role of the remaining glucose unit in glycoproteins?

Answer 289

acts as signal for chaperon binding (like BiP, calreticulin, calnexin) chaperones ensure proper folding before final gluocse is trimmed

Question 290

What enzyme removes the last lguocse unit from glyoprotein?

Answer 290

er lumen glucosiade, which trims the final gluocse of N-linked glcosylation

Question 291

What happens when the final glucose is removed?

Answer 291

new peotein released from reticuloplasms/chaperons and can go into the secretory pathway ie to golgi

Question 292

What happens if a protein is properly folded and assembled in the ER?

Answer 292

undergoes final trimming by ER lumen mannosidase, which removes one mannose unit

Question 293

What is the role of ER mannosidase in protein quality control

Answer 293

Removes a mannose reisude, which marks that the protein as correctly folded and reasy for export or functino

Question 294

Through what pathwat is a protein transported from the ER to rhe golgi?

Answer 294

via vessicle, which bud off the er and fuse with the ci-golgi allowing the protein to continue along the secretory pathway

Question 295

Where foes the N-linked glycosyaltio continie after the er?

Answer 295

in golgi apparatus, where the core oligosaccharide can be further trimmed or modified depending on the protiens final destination

Question 296

What happens if a protein is released from reticuloplasmins but is misfolded or misassembled?

Answer 296

Recognized by UGGT, a glucosyltransferase monitoring enzyme that detects improperly folded proteins by sensing exposed hydrophobic regions

Question 297

What does UGGT do to misfolded proteins?

Answer 297

Adds back one gljucose unito to core making it for re-binding to chaperons

Question 298

What does UGGT recognize to detect misfolding?

Answer 298

sneses hydrophobic amino acid residues that are buried insde correctly folded protines but exposed in misfolded ones

Question 299

After glucose is re-added by uggt, what happens?

Answer 299

protein re-binds to chaperones like calnexin, calreticulun, biP for another round of folding and assembly

Question 300

How long does the ER try to fold a misfolded protein before degredation?

Answer 300

er attemps folding, but if the protein remains misfolded for about 5-60 mins, it is targetted for degredation

Question 301

What is the ERAD pathway?

Answer 301

ER- associated degredation misfolded unfixable prteins are exported from the er and degraded in cytosol

Question 302

What enzyme pwoers the erad pathway?

Answer 302

AAA ATPase p97, which uses ATP hydrolysis to pull misfloded proteins from the er lumen into cytosol (process called retrotranslocation)

Question 303

How is cystic fibrosis related to erad pathway?

Answer 303

in most patients, cftr protein is misfolded due to inporoper gylcosylation. instead of reacing the plasms membrane, it is degraded via the erad pathway

Question 304

What happens to misfolded proteins after retrotranslocation into the cytoplasm?

Answer 304

oligosaccharide chains are removed, and they are tagged with poly-uniqutin chains mark them for degredation

Question 305

What is unniquitn?

Answer 305

small (76) amino acids regulatroy protiein that acts as a signal for protein degredation

Question 306

What does mono-ubiquinatino signal for?

Answer 306

signals membrane proteins for sorting into endosomes or multivesicular bodes

Question 307

What does polu-ubiquationatn signal for?

Answer 307

protein that is destine for degredatin, especially for misfolded er proteins and other cellular ptoeins undergoung turnover

Question 308

What degrades poly-ubiquinated proteins?

Answer 308

proteasome, a large protein complex that unfolds and digestes. tagged rpteins into peptides

Question 309

What is the proteasome?

Answer 309

barrel-shaped, multi-subunit machine found in te cytoplasm and nucels that breaks down proteins

Question 310

What happens first when a protein rwaches the proteosome?

Answer 310

ubiquianted protein binds to cap (lid) of the proteosome

Question 311

What happens to the poly-ubiquitin chain?

Answer 311

removed and recylced before the protein is degraded

Question 312

How does the proteosome degrade proteins?

Answer 312

threads the protein into its barrel and breals it down through proteolysis (protein cutting)

Question 313

What happwns when the ER is under stress ( cant get rid of misfolded proteins fast enough)?

Answer 313

activates unfolded protein response (UPR) set of signalling pathways that try to restore balance by improving folding or degrading faulty proteins

Question 314

What diseases are linked to er stress?

Answer 314

cystic fibrosis and alzheimers are assocaited with er stress due to buildup of misfolded proteins forming toxic aggregates

Question 315

What types of proteins detects er stress?

Answer 315

er membrane spanning proteins sensors such as ire1, perk, atf6 - detect unfolded proteins and activate UPR pathways

Question 316

What are PERK and ATF6?

Answer 316

they are er memrane bound proteins that act as stress sensorts and acitvate unfolded protein response pathways during ER stress

Question 317

What do PERK and ATF6 have on their lumenal side?

Answer 317

Both have stress-sensing domains that bind to chaperon BiP under normal conditions

Question 318

What keeps PERK and ATF6 inactive when theres no stress?

Answer 318

Bound by BiP, which prevents them from activating the UPR pathways

Question 319

What triggers PERK and ATF6

Answer 319

er stress- an accumulation of misfolded or misassebked proteins-causes BiP to release PERK and ATF6, which then beocmes active

Question 320

What happens after PERK is activated?

Answer 320

PERK phosphorlyates translation factors, which reduced overall protein syntehsis and helps cells deal with stress

Question 321

What is the overall goal of activating perk anf atf6?

Answer 321

reduce er load, increase protein folding capactiy, and alleivate stress by making stress-relief protein

Question 322

What causes perk to activate during er stress?

Answer 322

BiP is released from PERK to help fold misfolded proteins. without bip bound, perk dimerizes and becomes active

Question 323

What does PERK do?

Answer 323

phosphorylates elF2alpha, a translation factor, which inhibits protein synthesis

Question 324

What is elf2alpha and wht does perk target it?

Answer 324

is needed to start translation (protein sythnesis) oerk phosphroyayes it to slow down translatin, reudcing the number of new proteins entering the er

Question 325

What triggers ATF6 activation duing er stress?

Answer 325

biP is released from ATF6 to help fold misfolded proteins. without biP, ATP6 becomes active

Question 326

What happens to ATF6 after its activated?

Answer 326

ATF6 moves from er to golgi in transport vesicles

Question 327

What happens to atf6 at golgi?

Answer 327

a golgi-assocaited protease cleaves off its cytoplasmic transcription factor domain

Question 328

What does the cleaved ATF-6 tf do?

Answer 328

enters the nucleus (via an exposed NLS) and activate genes that help reduce ER stress, like chaperones and folding enzymes

Question 329

Why doesnt full-length ATF6 go to the nuceleus?

Answer 329

its NLS is hidden, the NLS (nuclear localization signal) is only exposed after cleavage at the golgi

Question 330

What does ATF6 do once it enters the nucleus?

Answer 330

ATF6 upregualtes (activates) genes that help with er protein quality control

Question 331

What types of genes does ATF-6 turn. on in nucleis

Answer 331

Reticuloplasmins-help fold proteins (ie BiP) er export components: help move properly folded proteins to golgi erad components- help degrade misfolded proteins (eg AAA ATPase p97)

Question 332

What happens to properly folded and glycosylated protein in the RER?

Answer 332

either retained er or exits to the golgi through er exit sites (ERES)

Question 333

What does it mean for a protein to be retained in the er?

Answer 333

either stays in the rer or moves sideways through the er membrane or lumen to another er subdomian (eg ser, nuclear envelope)

Question 334

What are ER Exit Sites (ERES)?

Answer 334

special er regions where vessicles form to carry proteins to golgi apparatus after snythesis and folding; regions are enriches with machinery needed for vessicle budding

Question 335

Where are er exit sites located?

Answer 335

usually located right next to cis face of the golgi complex

Question 336

What happens ar er exit sites?

Answer 336

membrae-bound transport vesicles are formed (budded off), carrying proteins from the er to the cis-golgi

Question 337

What molecular machinery is enriched at eres?

Answer 337

machinery responsible for vesicle fomraiton, such as COPII coat proteins, which help shape the vesicle and pinch it off the er membrane

Question 338

What is the role of eres machinery besides forming vesicles?

Answer 338

it ensures proper packing of vessicles with the correct lumenal and membrane cargo proteins (and lipids) destined for the golgi

Question 339

Are all er proteins sent to golgi in vessicles?

Answer 339

no, resident er proteins (like BiP) are usually prevented from entering golgi-bound vessicles

Question 340

Is protein trafficking in the endomembrane system random?

Answer 340

no- bulk flow, protein transport is selective and based on targetting signals and receptors

Question 341

What gives vessicles at eres their fuzzy appearance

Answer 341

layer of coat proteins (COPs) attached to cytoplasmic surface of vessicle membrane

Question 342

What are COPs and where do they assemble?

Answer 342

soluble proteins that assemble on the outside (cytoplasmic surface) of vessicles budding from er to eres

Question 343

What are the main two functions of the coat proteins (COPs)?

Answer 343

shape the membrane to form vessivles pick and pack the right cargo into vessicles (like proteins, lipids and machinery)

Question 344

What do COPs gelp vessicle carry?

Answer 344

soluble and membrane proteins/lipids targetting machiery like rabs and snares to guide vessicle to right location eg the golgi

Question 345

What are the coat proteins used for in endomembrane system?

Answer 345

they help form transport vessicles by shaping the membrane and selecting cargo for specofic destinatinos

Question 346

What direction do COPII-coated vessicles travel

Answer 346

foward (anterograde) transport: from er to golgi (especially from er exit sites)

Question 347

What direction do COPI-coated vessicle travel?

Answer 347

backward (retrograde) transport: from golgi back to er, and b etween golgi compartments

Question 348

What is the function of clathrin-coated vessicles?

Answer 348

helps vessicles go from: - golgi to endosomes or - from plasma membrane to endosomes (eg during endocytosis)

Question 349

What does the "coat" formed by COP and clathrin look like?

Answer 349

curved, cage-like lattice that surrounds the vessicle and help it bud off from the membrane

Question 350

What are two main roles of vesicle coat proteins

Answer 350

1. mediate membrane curvature and vessicle formation 2. select and concentrate proper cargo for vessicle

Question 351

What does clathrin look like when assembles into coat?

Answer 351

triskelion (three legged shape) that builds into a soccer ball like cage structure

Question 352

What is the function of SNARE proteins in vessicle trafficking?

Answer 352

snare proteins guide vessicles to correct membrane and help them fuse with the target (like cis-golgi)

Question 353

What is Sar1 and where is it recruited from?

Answer 353

Sar1 is a COPII GTPase recuirted from cytoplasm to ER membrane at the ERES

Question 354

What does Sar1 bind to at the ER membrane?

Answer 354

Sar1 binds to Sec12, an ER membrane protein that acts as a GEF (guanine nucleotide exhange factor)

Question 355

What happens when Sec12 activates Sar1?

Answer 355

Sec12 promotes the excahgne of GDP to GTP on Sar1, activating it and causing a shape change

Question 356

What does activated Sar1 do?

Answer 356

exposes a hydrophobic N-terminus, allowing Sar1-GTP to insert into the ER membrane like anchor

Question 357

Why is Sar1-GTP important in vesslce formation?

Answer 357

Sar1-GTP marks the future site of vessicle budding and starts recruiting other COPII components to form the vessicle coat

Question 358

What does Sar1-GTP do after embedding in the ER membrane?

Answer 358

it recruits copII coat proteins from cytosol to the ERES membrane surface

Question 359

Which two proteins does Sar-1 GTP recruit first?

Answer 359

Sec23 and Sec24

Question 360

What do Sec23 and Sec24 do?

Answer 360

they act as scaffolding and help bend the er membrane outward toward the cytosol, start of vessicle budding

Question 361

What kind of complex do Sar1, Sec23, and sec24 form?

Answer 361

a ternary complex on the er membrane surface that begins copII vessicle formation

Question 362

What role does Sec24 play besides scaffolding?

Answer 362

helps select vesicle cargo proteins by binding to cytoplasmic facing domains of er membrane proteins

Question 363

What are membrane cargo receptor proteins

Answer 363

bind to soluble proteins inside the er lumen and help carry them into vessicle for transport to golgi

Question 364

What are membrane trafficking proteins?

Answer 364

proteins like v-snares that help the vessicle dock and fuse with the correct target membrane (eg the golgi)

Question 365

What does sec24 recognize to select vessicle membrane proteins?

Answer 365

sec24 recognizes er export sorting signals, like di-acidic motif (Asp-x-glu) in cytoplasmic facing domains of the membrane proteins

Question 366

Where are the er export sorting signals located?

Answer 366

on cytoplasmic facing side of membrane proteins destined to exit the er

Question 367

Do ER resdient proteins have ER export sorting signals?

Answer 367

no, they lack export signals to prevent them from entering transport vesicles

Question 368

What happens to sec-24 bound proteins during copII vessicle assembly?

Answer 368

they become conentrated inside the growing copII vessicle

Question 369

What do sec23 and sec24 do during copII vessicle assembly?

Answer 369

they recruit more soluble copII proteins from the cytoplasm to the surface of the growing vessicles

Question 370

What is the role of sec13 and sec31 in copII vessicle assembly?

Answer 370

they self-assemble into an outer, cage-like lattice that acts as a structural outer scaffold for the vessicle bud

Question 371

What does the outer scaffolding formed by sec13/31 promote?

Answer 371

promotes additional outward bending of the eres membrane towards the cytosol

Question 372

What happens to copII vessicle after it finishes forming?

Answer 372

it is relased (scission) from the eres membrane into the cytosol

Question 373

What does sec23 do after the vessicle buds from the er?

Answer 373

sec23 promotes gtp hydrolysis in sar1-gtp

Question 374

What does the conversion of Sar1-GTP to Sar1-GDP trigger?

Answer 374

it causes disassembly of the copII coat from vessicle

Question 375

What happens after copII proteins after coat dissasembly

Answer 375

they are reasled into cytoplasm to be reused for building new vessicles

Question 376

What is the result of copII coat disassembly?

Answer 376

naked (uncoated) transport vessicle is formed, ready to fuse with golgi

Question 377

What do copII vessicles carry from the er?

Answer 377

Protein cargo and helper molecules like v-SNAREs and Rabs

Question 378

Where do COPII vesscles travel after leaving the ER?

Answer 378

travel to cis-golgi networn, entry point of the golgi

Question 379

What is the role of v-snares and rabs in copII vessicles?

Answer 379

adress labels and keys, helping the vessicle dock and fuse with the correct membrane

Question 380

What happens when vessicles reach the cis-golgi netwoek

Answer 380

fuse with each other to fomr the cgn and start processing their cargo through the golgi

Question 381

What is the cis-golgi network?

Answer 381

its the front door of the golgi where incoming vessicles from thed er unload their cargo

Question 382

What does the cgn consist of?

Answer 382

interconnected network of vessicles and tubules

Question 383

What is the role of the cgn in anterograde trasnport?

Answer 383

recieves vessicles transorted from the er exit sites (ERES)

Question 384

What is the role of microtubles in vessicle transport?

Answer 384

micrtubles act as tracks or highways that vessicle travel along using molecular motors

Question 385

What is the main goal of vessicle docking and fusion at the cis-golgi network?

Answer 385

ensure that a transport vessicle recognizes and fuses with the correct "acceptor" membrane or organelles

Question 386

How many steps in vesslcle docking and fusion?

Answer 386

4

Question 387

What proteins are involved in vessicle docking and fusion?

Answer 387

SNARE proteins (v-snares and t-snares), Rab-GTP, and effector proteins

Question 388

What form during the docking process

Answer 388

SNARE complex forms between the vessicle and target membrane

Question 389

What happens during the fusion step?

Answer 389

snare complex pulls the membrane together, allowing them to fuse

Question 390

How is the snare complex dissasebmked after fusion?

Answer 390

using NSF, alpha-snap, and atp hydroylsis (ATP to ADP+Pi)

Question 391

What is the first step in vessicle docking at the cis-Golgi network?

Answer 391

Vessicle docking with the acceptor (target) membrane

Question 392

What protein family mediates vesicle docking?

Answer 392

Rab proteins (GTP-binding proteins)

Question 393

What happens when rab ias activated (Rab-GTP)?

Answer 393

binds to specific rab effector rptoeins on the target membrane

Question 394

What is the function of rab effectors?

Answer 394

help vessicles recognize and bind to correct membrane and can interact with motor proteins for vessicle movement

Question 395

What ensures that vessicles dock to the correct target membrane?

Answer 395

Specific rab-rab effector interavtions (molecular bridge)

Question 396

What happens after a vessicle docks with the target membrane?

Answer 396

SNARE proteins interact to bring the membranes close together for fusion

Question 397

What are SNARE proteins?

Answer 397

SNAREs(Noluble NSF attachment protein REceptors) are integral membrane porteins on vessicles and target membranes that help with membrane fusion

Question 398

What do SNARES do uding fusino?

Answer 398

form snare complexes that tighlty pull the vessicle and target the membranes together, allowing them to fuse

Question 399

How do SNAREs help with vessicle targetting?

Answer 399

each SNARE is speicifc to certain membranes, helping ensure vessicles fuse with the correct target (along with rab proteins and rab effectors)

Question 400

What two main roles fo SNARES play?

Answer 400

1. vessicle targetting specific, fusion of vessicle with the target membrane

Question 401

what are the two main classes of SNARE proteins?

Answer 401

v-snares and t-snares

Question 402

Where are v-snares found?

Answer 402

on vessicle membranes

Question 403

Where are t-snares found?

Answer 403

on target or acceptor membranes (eg cis-golgi network)

Question 404

What is the role of v-snares during vessicle transport?

Answer 404

they built into vessicle membrane during budding and help veisslce recognize its correct target

Question 405

How are v-snares incorporated into vessicles?

Answer 405

interaction with sec24 and an er export signal in their cytoplasmic facing domain

Question 406

What do v-snares and t-snares do togetjher?

Answer 406

pair up to bring vessicle and tsrget membrane close together for fusion

Question 407

what do all snare proteins have in common?

Answer 407

all have snare motif

Question 408

What is SNARE motif?

Answer 408

a coiled-coil domain on the cytoplasmic sdide of v-snares and t-snares that helps them interact

Question 409

What does SNARE complex formation lead to?

Answer 409

membrane fusion

Question 410

Is the mechanism of membrane fusion fully understood?

Answer 410

No, biophysical mechanism is not well understood

Question 411

What happens after vessicle and target membranes fuse?

Answer 411

SNAREcomplexes and Rab/Rab effector proteins dissociate and are recylcled

Question 412

What proteins are responsible for disassembling SNARE complexes?

Answer 412

NSF and SNAP

Question 413

How do NSF and SNAP dissasemble SNARE complexes?

Answer 413

they bind to SNARES and unwind them using hydrolysis

Question 414

What happens to soluble cargo proteins after vessicle fusion at the cgn?

Answer 414

released into cgn lumen due to lower pH in the can compared to er lumen; releases from cargo receptor proteins

Question 415

What proteins remian in the CGN membrane after fusion?

Answer 415

membrane cargo proteins, cargo-receptor proteins, and membrane trafficking proteins (eg V-snares)

Question 416

What are examples of vessicle-specific proteins or proteins that escape from the ER?

Answer 416

v-snares unique to eres-derived vessicles and er resident proteins like biP or calnexin

Question 417

How are most ER residet proteins kept inthe ER?

Answer 417

excluded from budding copIItransport vessiles at eres by not having er export signa;s

Question 418

What happens to er resident proteins that are accidentally escape to the CGN?

Answer 418

retrograde transport back

Question 419

What do most resdient soluble er proteins like (bip have)?

Answer 419

have a c-terminal kdel sequence, which acts as an er retrieving sorting signal

Question 420

What recognizes escaped membrane er proteins in the cgn?

Answer 420

cytoplasmic facing domain o f cgn (with a dilysine sequence) is recognized by copI protein coat

Question 421

What condition causes the release of proteins from kdel receptors in the er?

Answer 421

the lower ph in the cgn compared to cgn causs release of kdel bound proteins

Question 422

What happens to the KDEL recepotr after releasing its cargo in the er?

Answer 422

the free kdel receptor returns to the cgn via copII coated eres vessicles

Question 423

What signal allows the KDEL recepotr to be included in COPII vessicles?

Answer 423

di-acidic er export sorting signal recognized by sec24

Question 424

What sequence do most residnet ER membrane proteins (like calnexin) contain for retrieval?

Answer 424

they contain C-terminal di-lysine (-KKxx) sequence; serves as er retrieval sorting signal allowing recognition by copI for retrograde transport

Question 425

What other proteins besdies resident er proteins use retrograde transport from cgn to er?

Answer 425

proteins like kdel recetor, membrane cargo receptor proteins, er membrane trafficking proteins (eg vnares)

Question 426

What is the golgi complex?

Answer 426

organelle discovered by camillo golgi in 1898; composed of stacked,, flattened membrane bound cisternae with associated tubles and vesicles. functions in modifying sorting, and packaging proteins and lipids

Question 427

How many golgi in mammalian cell?

Answer 427

typically contains one (large) golgi complex located near center of cell

Question 428

How many golgi in plant and yeast cells?

Answer 428

contaon several colgi complexes through cell

Question 429

What is the overall structure of the golgi complex?

Answer 429

made up of several subcompartments and appears as a stack of flattenned sacs (cisternae) with associated tubles ans vessicles

Question 430

what does polarity in the golig complex refer to?

Answer 430

polarity means that the golgi has structural and functinoal differences across its regions: specifically from the cis (entry) to trans (exit) sides

Question 431

How many membranes make up a golgi stack and what are the name?

Answer 431

3 or more; cis, medial and trans cisternae

Question 432

What are the functiond of the Golgi cisternae?

Answer 432

sites of golgi metabolism, like complex polysaccharide synthesis, glycosylation of proteins/lipids, and phosphorlyation of mannose on lysosomal protein

Question 433

Where is the trans-golgi network?

Answer 433

on the trans face of the golgi complex

Question 434

What role does tgn servie in the golgi?

Answer 434

sorting statio for forward trasnport from trans cisternae to endosomes

Question 435

What type of vessicle coating is assembled at tgn?

Answer 435

clathrin-coated vessicles for anterograde transport to endosomes

Question 436

What does TGN sort for anterograde transport?

Answer 436

secretrory vessicles and secretory granules going to the plasma membrane (PM) for secretion

Question 437

What does the TGN do in retrograde transport?

Answer 437

site of COPI vessicle assembly for trasnport back to trans-golgi cisternae

Question 438

WHat organizes the Golgi complex stack?

Answer 438

golgi matrix organizes the stack using peripheral and integral membrane proteins that interact via their cytoplasmic-facing domains

Question 439

What is the functions of GRASPs in the golgi?

Answer 439

golgi reassembly and stacking proteins act as tethering protens that link diff golgi subcompartments (eg cgn, cis, medial, trans, tgn) together to maintain the golgi structure

Question 440

What does the golgi matrix also do?

Answer 440

liks golgi complex to cytoskeleton

Question 441

What is the final step of N-glycosylation in the ER?

Answer 441

trimming of 1 mnaose sugar from the core oligosacchardie by the enzyme mannosidase (mannose signals it is folded and can move on)

Question 442

What happens after a protein is properly folded and processed in the ER?

Answer 442

packaged into copII vessicles and trasnprted to cis-golgi network (CGN)

Question 443

Where is N-linked glycosylation completed?

Answer 443

In the golgi complex

Question 444

What type of enzymes are foind in cis, medial, and trans golgi cisternae?

Answer 444

unique glycosyltrasnferase and glycosides enzymes

Question 445

What is the function of these enzymes in the golgi cisternae?

Answer 445

modify the n-linked core oligosaccharides on glycoproteins for peroper finction and targetting

Question 446

How do golgi cisternae act like an assembly line?

Answer 446

prtoeons are modified step-by-step by diff enzymes in each subcompartment

Question 447

What happwns in the cis cisternae during glycoprotein processing?

Answer 447

alpha mannosidase I removes 3 mannose sugars from glycoprotein

Question 448

What happens after modoficatio of the protein in the cis golgi?

Answer 448

glycoprotein moves to medial and then trans cisternae for further processing

Question 449

What is the role of the golgi complex in M6P sorting?

Answer 449

adds mannose-6-P (M6P) to lysosomal-bound proteins as as a sorting signal for delivery to lysosomes

Question 450

Where in the golgi does M6P targetting occur?

Answer 450

in cis-golgi cisternae

Question 451

What enzyme adds the M6P tag to lysosomal proteins?

Answer 451

N-acetylglucosamine phosphotransferase, which recognizes a signal patch on the lysosomal proteins (phosphorylate mannose)

Question 452

What does the enzyme N-acetylglucosamine phosphotransferase do?

Answer 452

transfers a phospahe group onto mannose sugars in the oligosaccharide chain of lysosomal proteins

Question 453

Why is m6P tag important?

Answer 453

it prevents the protein from undergoing more N-linked glycosylation and sends it to lysosome

Question 454

What happens to proteins that do not have M6P?

Answer 454

packahneged into secretory trasnport vessicles at the TGN and sent to plasms mebrane or remain in golgi

Question 455

What happens to proteins that do have M6P?

Answer 455

packaged into clathrin coated vessicles at tgn and send to endosmes and lysosomes

Question 456

What model is currently most accepted for protein movement through the golgi complex

Answer 456

cisternal progression/maturation model

Question 457

What does the cisternal progression model suggest?

Answer 457

golgi cisternae themsevles moves from cgn to tgn, maturing as they go, carrying proteins with them

Question 458

Is the movement of materials through the golgi complex a settled topic in cell bio?

Answer 458

no, heavily debated area with multiple model proposed

Question 459

are golgi subcompartments static or dynamic?

Answer 459

dynamic: move forward from the cis to trans slide of the golgi complex

Question 460

What mediates the movement of golgi subcompartmets?

Answer 460

golgi matrix proteins and the cytoskeleton (motor proteins)

Question 461

How does the golgi maintain its structure and function during this movement?

Answer 461

COPI transport veiscles return golgi-resident proteins b ack to the correct subcomparmtnet (retorgrade transport)

Question 462

What do COPII-coated vessicles carry?

Answer 462

newly-synthesized cargo proteins like lysosomal destined proteins and golgi resident enzymes

Question 463

What evidence supports this model??

Answer 463

cryo-electron tomography images show COPII vessicles arriving and fusing at the cgn

Question 464

What is the role of COPI-coated vessicles in the golgi complex?

Answer 464

they bud from the edges of cisternae and transport resident golgi enzymes backward (retrograde transport) through the complex from trans to cis

Question 465

Why are enzymes like alpha mannosidase I transported bsckwards?

Answer 465

because they function in the cis cisternae, so they are recycled back from the trans/medial cisternae

Question 466

What is the role of clathrin-coated vessicles from the TGN?

Answer 466

they deliver M6P-bearing protein cargo to endosomes (which become lysosomes)

Question 467

Where do scretory vessicles from the TGN go?

Answer 467

Target the plasma membrane and extracellular space

Question 468

What are secretory granules?

Answer 468

vessicles from the TGN that store and release proteins into the extracellular space upon signalling

Question 469

What is the main function of lysosomes?

Answer 469

lysosomes are digestive organelles that break down all types of macromolecules (lipids, sugars, proteins, nucelic acids)

Question 470

What cellular process involves the degredation of large components or organelles?

Answer 470

Autophagy-lyosomes help break down all types of macromolecules (lipids, sugars, proteins, nucleic acids)

Question 471

What enzymes do lysosomes contain, and when are they active?

Answer 471

lysosomes contain about 60 soluble acid hydrolase enzymes, active only at low pH (about 4.6)

Question 472

How are lysosomal membrane proteins protected from being degraded?

Answer 472

shielded by lumen-facing cargo groups added via N-glycosyltation in the ER and Golgi

Question 473

What happens to products of lyosomsal degredation?

Answer 473

Transported into the cytoplasm to be resued in biosynthetic or metabolic pathways

Question 474

How is the low pH in lysosomes maintained?

Answer 474

ATPase proton pumps move H+ ions into the lysosome from the cytoplasm

Question 475

Are lysosomes static or dynamic strcutres??

Answer 475

Highly dynamic: their shapes and sizes vary based on cell type, tissue, and organism

Question 476

Where are lysosomal proteins first made and modified?

Answer 476

in the rough ER (RER), where they are N-glycosylated

Question 477

How do lysosomal proteins get from ER to Golgi?

Answer 477

they are transported in COPII vessicles to the cis-Golgi network (CGN)

Question 478

What happens to lysosomal proteins in the cis-Golgi cisternae?

Answer 478

their mannose sugars are phosphorylated by N-acetlyglucosamine phosphotrasnferase, creating a M6P tag

Question 479

What is M6P and why is it important?

Answer 479

M6P (mannose-6-phospahte) is a lysosomal targetting signal that sends proteins to lysosome

Question 480

What happens to proteins without M6P tag at TGN?

Answer 480

They are sent to secretory vessicles/granules and secreted to plasma membrane or extracellular matrix

Question 481

What happens to proteins with an M6P tag at the TGN?

Answer 481

packaged into clathrin-coated vessicles, sent to late endosomes, and then to lysosome

Question 482

What enzyme adds the M6P tag?

Answer 482

N-acetylglucosamine phosphotransferase, in the cis-golgi

Question 483

Where do later parts of the biosynthetic pathway intersect with the endocytic pathway?

Answer 483

at late endosomes

Question 484

What is the endocytic pathway responsible for?

Answer 484

internalizing materials like: - hormones - low-density lipoproteins (LDL) - iron (fe3+) - then delivering them to endosomes/lysosomes for internalization or degredation

Question 485

What is the final destination for many soluble lysosomal proteins from the TGN?

Answer 485

lysosome, via the late endosome

Question 486

What is the role of TGN in lysosomal protein trafficking?

Answer 486

sorts soluble lysosomal proteins (tagged with M6P) into clathrin-coated vessicles, which fuse with late endosomes

Question 487

What happens after soluble lysosomal proteins reach the late endosome?

Answer 487

delievered to the lysosome for function

Question 488

What type of protein is the M6P receptor?

Answer 488

integral transmembrane protein

Question 489

What does the lumen-faing domain of M6P receptor do?

Answer 489

binds to m6p groups on soluble lysosomal proteins in the lumen of the tgn

Question 490

What happens after m6p recepotor binds to its cargo?

Answer 490

helps concentrate the cargo (lysosomal proteins) into clathrin-coated transport vessicles

Question 491

What kind of vessicle carry M6P-tagged proteins from the TGN to endosomes?

Answer 491

clathrin-coated vessicles

Question 492

What adapter proteins are involved in clathrin-coated vessicle formation at the TGN?

Answer 492

AP1 and GGA adapter proteins recruited by Arf1-GTP

Question 493

What is a similar mechanism to M6P receptor function that happens at the ER exit sites(ERES)?

Answer 493

membrane cargo receptors bind soluble proteins and help package them into COP-II coated vessicles for transport to the golgi

Question 494

What is the role of AP1/GGA in vessivle trafficking?

Answer 494

select cargo (like M6P-tagged proteins) for packaging into vessicles

Question 495

What is AP1/GGA's role similar to in COPII vessicle formation?

Answer 495

act like sec24 in copII coated vessicles at er exit sites (ERES)

Question 496

What additional role do AP1/GGA adaptor proteins serves in vessicle formation?

Answer 496

act as linkers for clathrin coat assembly, helping form clathrin-coated vessicles

Question 497

What is Arf1 similar to in COPII vessicle formation?

Answer 497

sar1, which recuirts coat proteins at the er exit sites (ERES)

Question 498

what does arf1 also help with besdies clathrin-coated vessicle formation?

Answer 498

initates copI vessicle assembly for retrograde transport within the golgi

Question 498

What does the conformational changed in Arf-1-GTP do?

Answer 498

exposes a lipid anchor, directing arf-1-gtp to outer leaflet of the tgn membrane and starts membrane bending

Question 499

what is one molecule of clathrin made of?

Answer 499

three light chains and three heavy chains

Question 500

What structure do clathrin molecules form?

Answer 500

three-legged strcture called a triskelion

Question 501

What forms the inner layer of the clathrin-coated vessicle?

Answer 501

AP complex (AP1/GGA) linked to Arf1 and M6P receptors that are bound to lysosomal cargo

Question 502

What is clathrin funtionally similar to in COPII vessicles

Answer 502

to sec13/31 which form the outer scaffold of copII vessicles at er

Question 503

What do individual clathrin triskelions do first during assembly?

Answer 503

self-assemble into hexagons that lie flat on the cytoplasmic surface on the membrane; turn into pentagons after help curve the membrne

Question 504

What protein mediates the release (scission) of clathrin coated vessicles from the tgn membrane?

Answer 504

dynamin, large soluble gtp-binding protein

Question 505

Where is dynamic recruited from and where does it go?

Answer 505

dynamin is recruited from the cytosol to the stalk between the clathrin-coated bud and the tgn membrane

Question 506

What drives the scission (cutting off) of the vessicle

Answer 506

GTP hydrolysis by dynamin

Question 507

What happens if a cell is treated with non-hydrolyzable GTP eg gamma gtp

Answer 507

dynamin ring keeps forming but scisison does not occur, leading to long, uncut stalk

Question 508

What happens to clathrin coat after the vessicle pinches off from the tgn?

Answer 508

clathrin coat disassembles

Question 509

What happens to Arf1-GTP after vessicle budding?

Answer 509

converted to arf1-gdp

Question 510

What happens to arf1-gdp, ap1/gga and clathrin after disassembly?

Answer 510

released into cytoplasm and recycled for new rounds of vessicle formation

Question 511

What does the uncoated vessicle with M6P-bound lysosomal cargo do next?

Answer 511

fuses with late endosome

Question 512

What mediates vessicle fusion with the late endosome?

Answer 512

rab/rab effector proteins and v-SNARE/t-SNARE pairing

Question 513

What is the trafficking of soluble lyosomsal ortoeins frin tgn to lysosomes similar to?

Answer 513

eres-to-golgi copII vessicle trafficking

Question 514

What is the pH inside the lumen of late endosomes?

Answer 514

around 5-5.5 (acidic)

Question 515

What is the pH of tgn and tgn-derived vessicles?

Answer 515

around 6.5 less acidic

Question 516

What does the acidic pH of late endosomes have on M6P receptors?

Answer 516

causes m6p receptors to release their bound lysosomal cargo (eg acid hydrolases)

Question 517

Why is phosphate removed from m6p groups on cargo proteins in the late endosome?

Answer 517

prevent the cargo from rebinding to m6p receptor

Question 518

What is the retromer complex?

Answer 518

recently discovered protein coat (like COPI, COPII, or clathrin) that helps with vessicle formation and cargo selection

Question 519

Where does retromer coat assemble?

Answer 519

on the cytoplasmic surface of the late endosome

Question 520

What are the main functions of the retromer coat?

Answer 520

1. mediates membrane curving and budding 2. selects cargo, like empty m6p receptors for recylong

Question 521

What happens to retromer coat after the vessicle forms?

Answer 521

disassembles just like other vessicle coats (COPII, COPI, clathrin etc)

Question 522

Where else can retromer vessicles (besides the tgn) deliver m6p receptors?

Answer 522

can also traffic to plasma membrane

Question 523

What can happen to some m6p tagged lysosmal proteins during secretion?

Answer 523

may escape to plasma membrane via the scretory pathway

Question 524

What happens to escaped m6p tagged lysosomal proteins at the plasma membrane?

Answer 524

recaptured by m6p receptors at the plasma membrane

Question 525

How are m6p receptor-cargo complexes retrieved from the plasma membrane?

Answer 525

through receptor-mediated endocytosis

Question 526

Where are m6p receptor-cargo complexes delivered after endocytosis?

Answer 526

back to late endosomes

Question 527

What is one of the last steps of lyosomal protein traffixking?

Answer 527

late endosome fuses with the lysosome

Question 528

What mediates the fusion of the late endsome with the lysosome?

Answer 528

rab/rab effector proteins and snares that are sepcpfic to each organelles

Question 529

What happens to the soluble lysosomal cargo proteins (like acid hydrolases) in the lyosome?

Answer 529

become acticated by the low ph (4.6) of the lyosome

Question 530

Why is te lysosomes low ph important?

Answer 530

ensures acid hydrolases are only active in the lysosome, protecting other parts of cell

Question 531

What is an example of a lysosomal membrane protein that move during fusion?

Answer 531

v-ATPase H+ pump, which helps maintain low pH inside the lysosome

Question 532

What else is deleiverd to the lysosome during late endosome fusion?

Answer 532

materials from endocytic pathway (eg receptors, ligands, nutrients) for degredation

Question 533

What does the biosytnhetic pathway deliver to lysosome?

Answer 533

newly made lysosomal membrane proteins from tgn

Question 534

What structures and systems are involved in the endocytic pathway?

Answer 534

early endosomes late endsomones/multivesicular bodies escrt machinery (sort and packages cargo)

Question 535

What does the constitutive secretion pathway do?

Answer 535

continuosly trasnports materials from the trans-Golgi network (TGN) to plasma mebrane (PM) via secretory vessicles

Question 536

How do vessicles release their cargo in the contitutive secretion pathway?

Answer 536

Vessicle fuse with the plasma membraine using Rab and SNARE proteins, releasing soluble cargo outisde the cell via exocytosis

Question 537

What is an exmaple of soluble cargo released through the constitutive secretion pathway?

Answer 537

soluble eznymes or meterial used for extracellular matrix or cell wall synthesis

Question 538

What is another name for the constitutive secretion pathway?

Answer 538

default pathway

Question 539

why is the constitiutve secretion pathway called the "default" pathway?

Answer 539

it is used for proteins that are not selectively sorted into other pathways, such as those targetted to ewndosomes/lysosomes or regulated secretion

Question 540

What types of proteins follow the constituve secretion pathway?

Answer 540

proteins that are not targetted to: - late endosomes - lysosomes - regulated secretory vessicles

Question 541

What are two alternatives to constitituve secretion pathway?

Answer 541

biosynthetic pathway (targetting to lysoomes via m6P) regulated secretionp pathways

Question 542

What is regulated secretion pathway?

Answer 542

secretion pathway where materials are stored in secretroy granules anf only released in repsonse to a cellular singal

Question 543

Where are materials packaged in the regualted secretion pathway?

Answer 543

into secretory granules at the gtrans golgi network

Question 544

How doe secretory granules release their cargo?

Answer 544

fuse with the plasma membrane (via rab and snare proteins) and release their contents by exocytosis

Question 545

What are examples of materials released by the regulated secretino pathway?

Answer 545

hormones from endocrine cells and neurotransmitters from nerve cells

Question 546

What happens to materials in the endocytic pathway?

Answer 546

large molecules (macromolecules) enter the cell by vesiculation of the plasma membrane

Question 547

What are the two main process for internalization in the endocytic pathway?

Answer 547

1. phagocytosis 2. receptor-mediated endocytrosis

Question 548

What are examples of phagocytosis?

Answer 548

ingestion of microorganisms (bacteria, yeast) by single celled ameobos for nutrtion or by wbcs

Question 549

How does the immune system recognize bacteria as foreign?

Answer 549

by generating antibodies againt bacrterial-cell surface componenets

Question 550

What part of the antibody binds bacterial proteins or sugars?

Answer 550

fab domain of antibody

Question 551

What is opsonization?

Answer 551

the process where antibodies coat bacteria, making them easier for immune cells ti recognize and engulf

Question 552

What do fc recepotrs on leukocytes?

Answer 552

they reconize the fc domain of antibodies bound to bacteria

Question 553

What happens after fc receptors bind to fc domain of antibodies?

Answer 553

signal reassembly of the actin microfilament network, changing the cells shape

Question 554

What do pseudopods do in phagocytosis?

Answer 554

they engulf the bacterium anf fuse around it to oform a phagosome

Question 555

What is a phagosome?

Answer 555

membrane bound compoartment inside the leukocyyte that contains the engulfed bacterium

Question 556

What happens after phagosome forms?

Answer 556

phagosome fuses with a lysosome

Question 557

What is the purpose of phaogsome lyosome fusion?

Answer 557

digest the bacterium and release its nutrients into the leukocytes cytoplasm

Question 558

What pathogen can block phagosome-lysosome fusion?

Answer 558

mycobacterium tuberculosis

Question 559

what are the two main types of endocytosis

Answer 559

1. Bulk phase endocytosis (pinocytosis) 2. receptor mediated endocytosis

Question 560

What happens during bulk-phase endocytosis? and how often does the plasma membrane recycle through the bulk phase

Answer 560

uptake of extracellular fluids, membrane proteins, and lipids into small vessicles - evety 20-90 mins

Question 561

What happens during receptor mediated endocytosis?

Answer 561

receptor on the plasma membrane binds to a ligand and the complex is internalized in a clathrin coated vessicle

Question 562

What are lysosomal proteins bound to m6P receptors an example of?

Answer 562

proteins that escaped from the tgn and are internaled by recepotr mediated endocytosis

Question 563

How is iron taken into cells through receptor-mediated endocytosis?

Answer 563

iron is bound to transferrin, which is recognized by trasnferrin receptor

Question 564

what happens after the receptor binds to ligand?

Answer 564

cytoplasmic domain of the recepotr binds to ap2 adaptor protein

Question 565

what does ap2 adapter protein bind to in vessicle formatin?

Answer 565

links the recwpotr ligand complex to clathrin

Question 566

What accumulates in a clathrin coated pit during endocytosis?

Answer 566

the receptor ligand ap2 complex

Question 567

what are clathrin coted pits?

Answer 567

specialized indentations in the plasma membrane where recepotr ligand complexes gather and endocytic vessicles begin to form

Question 568

What phosphlipid is enriched at clathrin coated pits?

Answer 568

phosphatidylinositol (4,5)-bispohsphare or PI(4,5)P2

Question 569

WHat ois the function of pi(4,5)p2?

Answer 569

helps recruit ap2 with the bound receptr-ligand complex into the coated pit

Question 570

What are the 3 main things that ap2 binds to?

Answer 570

1. PI(4,5)P2 (lipid in the inner plasma membrane) 2. cytoplasmic domain of receptor with cargo 3. clathrin

Question 571

What does ap2 reciurt from cytoplasm?

Answer 571

clathrin triskelions, qhich assemblw outer coat of vessicle

Question 572

What are new uncaoted endocytic vessicle referred to as?

Answer 572

early endosome

Question 573

What could happen to recepotrs in endocytosis instead of being recycled?

Answer 573

they might be degraded in lysosomes if not reused

Question 574

What are the two main fates of free receptors after recrpot mediated endocytosis?

Answer 574

1. recycled back to plasma membrane 2. deleivered to lysosome for degredation

Question 575

What type of recepotrs are commonly degraded in lyososmes?

Answer 575

cell surface recepotrs like insulin recepotrs or epidermal growth factor (EGF) receptors

Question 576

How are endocyotised membrane proteins felevered into the lysosome interior?

Answer 576

through inward budding of vessicles into the late endosome, forming a multivesucular body (MVB)

Question 577

What is a multivesuclar body?

Answer 577

late endomse thst contains many small internal vessicles each holding membrane proteins destined for degredation

Question 578

What are the two sources of membrane proteins are sorted ast the mvb?

Answer 578

1. lysosmal membrane protiens (from tgn via biosynthetic pathway) 2. endocytosed membrane proteins destine for degredation via endocytic pathway

Question 579

what is special about the strcture of an mvb?

Answer 579

contrains many intralumenal vessicles (tiny vessicles inside it)

Question 580

How is mvb vessucle budding diff from other transport vessicles like copI/copII?

Answer 580

mvb vessicles bud inward, away from cytoplasm (opposite direction ofr other)

Question 581

Wht machinery helps mvb vessicles form and select their cargo?

Answer 581

escrt machinery (endosomal sorting complex required for transport)

Question 582

What kind of proteins does escrt help degrade?

Answer 582

endocytosed membrane protens that are tagged with mono-ubiqutin

Question 583

What does escrt protein do?

Answer 583

recognizes the ubinquiation asnd recruits other escrt proteins to help bud the vessicle inward

Question 584

What powers the disssasembly of the escrt complex?

Answer 584

vps4 ATP ase which uses atp to break apart the complex so it can be reused

Question 585

What do retroviruses like hiv hijack for virus budding at the plasma mebrane?

Answer 585

escrt machinery

Question 586

what viral protein in hiv mimicks the escrt hrs protein?

Answer 586

hiv gag protein

Question 587

What does escrt do during virus budding?

Answer 587

helps pinch off the briud fgrom the plasma mbenrae so it ca exit the cell into tje extracelljlar spce

Question 588

What is the main role of mitochondria in eukaryotic cells?

Answer 588

energy production from carbs and lipid breakdwon via oxidative phopshorlylation (TCA cycle)

Question 589

What is the main role of chlorplasts in eukaryotic cells?

Answer 589

energy production and carb snthesis through photosynthesis

Question 590

What makes mitocondria and chloroplasts unique from other organelles?

Answer 590

they have their own DNA (genome) and code for 20-30% of the proteins theyneed

Question 591

What is organelle biogenesis?

Answer 591

the process of organelle formation including protein targetting, membrane assembly, replication, degredation, and inheritance during cell division

Question 592

Are mitochondria and chloroplasts fully independent?

Answer 592

no, they are semi autonomous replication is controlled by both nuclear and organelle genomes

Question 593

How do mitochondria and chloroplasts arise in the cell?

Answer 593

come from pre-existing organelles, not formed de novo (anew) like endocmmebrane organelles cannot form from scratch, whereas some other organlles like those in endomembrane sytem (eg er, golgi, lysosomes), can form from scrath from other membranes

Question 594

What kind of membrane structure do mitochondria have?

Answer 594

they are double membrane bound organelles (outer and inner membranes)

Question 595

What is the function of the mitcohondrial outer membrane>

Answer 595

permeable to ions and smal l molecules

Question 596

What proteins are found in the outer mitochondrial membrane?

Answer 596

porins: barrel shaped proteins that form large channels for small molecule trasnport

Question 597

What is found in the intermmebrane space of mitchondria?

Answer 597

high concentration of H+ (protons)

Question 598

What is the structure and function of inner mitochondiral membrane?

Answer 598

forms cristae (folds) that increase surface area -impermebale to most molecules - it maintains the H+ gradienta dnt eh sit of atp syntheiss

Question 599

How do outer and inner mitochondrial membranes differ?

Answer 599

they differ in function and protein/lipid composition

Question 600

What is the mitchondrial matrix?

Answer 600

aqueous interior of the mitochondrion, surrounded by inner membranr

Question 601

What important preocesses occurs in the mitochondrial matrix?

Answer 601

tca cycle (krebs cycle), which produced ATP via oxidative phosphorlyation

Question 602

What type of DNA is found in mitochondria?

Answer 602

circular mitochondrial DNA, called the mitochondrial genome

Question 603

What does the human mitochondrial genome encode?

Answer 603

- 13 proteins - 2 rRNAs -22 tRNAs

Question 604

Do mitochondria have ribsomes? If so, what for?

Answer 604

yes, mitochondria contain ribosomes to translate prtoeins encoded by the mitochondrial genome

Question 605

Where are most mitochondiral proteins made?

Answer 605

in the cyotosl, from nuclear genes

Question 606

How are these proteins sent to mitochondria?

Answer 606

they are targeted to the mitochondria after translation (post-transtionally)

Question 607

What is mitochondrial network?

Answer 607

highly branched, long and interconnected series of tubles

Question 608

Why are mitochondria often appear bean-shaped in electron microscopy (EM) images?

Answer 608

because em sections usuaully capture only a single tubule of the larger mitochodnrial netwro kduring sample preperation

Question 609

What is the function of the mitochondrial network?

Answer 609

cell-wdie coordination of mitochondrial functions (like energy productino) and biogenesis (formation of new mitochondria)

Question 610

What do mitochindrial tubules constantly undergo?

Answer 610

fusion (joining) anf fission (splitting)

Question 611

Why do mitcohdnira undergo fission and fusion?

Answer 611

- environemtal signals - developmenmtal chanes - cells energy needs

Question 612

When does mitochondiral fission commonly occurd?

Answer 612

end of g1 pahse of cell cycle (when all organelles replicate) - during cell death (apooptosis)

Question 613

What determines the number, size and connectivity of mitochondira?

Answer 613

balance between fission and fusoin

Question 614

What happens if mitochodnrial network baalcne is disrputed?

Answer 614

neurodegerative disorders like alzheimers and parkinsons

Question 615

How many steps are there in mitocondrial fission?

Answer 615

three steps

Question 616

What happens in step 1 of mitchondrial fisison?

Answer 616

er tublues wrap arounf the mitochondira at future fission site, chanigng shape to help beging constrictionl happens at a special er subdomain called MAM (mitochondria-associated membrane)

Question 617

What happens in step 2 of mitochidra fission?

Answer 617

protein called drp1 is recuitted from cytoplasm to constriction site

Question 618

What does drp1 do during fission?

Answer 618

it forms a ring-like strucutre (Drp1) ring asrounf the outer membrane to help cut it

Question 619

What lind of protein is drp1

Answer 619

dyniamin related gtp binding protien, simiolar to tjhosr thst help pinch off vessicles in other memrbanes (eg clathrin coated vessicles)

Question 620

What helps recriut drp1 and er tubluels to fission site?

Answer 620

lipid microdomains on outer mitcocondrial membrane

Question 621

What specifc lipid is enriched in these microdomains?

Answer 621

cardiolipin: a mitochondrial specifc phosphlipid

Question 622

Where is cardiolipin normally found?

Answer 622

in the inner mitochondrial membrane, but it gets recuited to the oter membrane at the constriction site during fission

Question 623

What happens in step 3 of mitcohdrial fission?

Answer 623

drp1 hydolyzes gtp, chaniging shape and tightening its ring, which constricts the membrane and leads to fission

Question 624

When does mitochondrial fusion often occur?

Answer 624

in response to cell stress, to increase coordination of mitochondrial function

Question 625

What is required for mitcohdonrial fusion?

Answer 625

- mitcohdnrial membrane proteins - enegy (GTP) - remodelling of membrane proteins

Question 626

What is the first step of mitochondrial fusion?

Answer 626

tethering of the outer membranes of adjacent mitochondria

Question 627

What GTPases mediate outer membrane tethering during mitochodnrial fusion?

Answer 627

mitofusins mfn1 and mfn2

Question 628

What type of proteins are mfn1 and mfn2?

Answer 628

integral outer membrane proteins with: - gtpase domain - coiled-coil, protein proteins interaction domain

Question 629

What regulates proper mfn1/2 binding and prevetns self-bdining?

Answer 629

other mitochondrial outer membrane proteins, especailly bak and bax (prevention of slef binding)

Question 630

What is step 2 of mitochondiral fusion?

Answer 630

Fusion of the outer membrane of adjacent mitochindria

Question 631

What forms at the outer membranr fusion sites?

Answer 631

lipid microdomains at mfn1/2 tethering sites

Question 632

What enzyme is involved in converting membrane lipids during this step?

Answer 632

phosphlipase D (PLD)

Question 633

What does pld do?

Answer 633

converts cardiolipin (moved from inner to outer membrane) into phosphatidic acid

Question 634

what is phosphatidic acid and whats its role?

Answer 634

cone-shaped lipid that promotes the bending/curvature of the outer membrane, aididng fusion

Question 635

What is the protein that mediates inner membrane fusion?

Answer 635

OPA1 (optic atrophy 1)

Question 636

What type of protein is OPA1?

Answer 636

inner membrane bound mitofusin that contsins a gtpas domain facing the intermembrane space

Question 637

What regulates OPA1 binding and activity?

Answer 637

other inner mmebrane proteins such as prohibitn (prevents self fusion)

Question 638

Where are most mitochondrial proteins encoded?

Answer 638

in the nucleus (not mitochondrial DNA)

Question 639

Where are mitochindrial proteins made?

Answer 639

on free ribsomoes in the cytoplasm

Question 640

How many mitochondrial proteins are there

Answer 640

around 1,000 proteins including soluble and membrane bound ones

Question 641

What is the challene for the cell in mitochondirial protein targetting?

Answer 641

1. correct organelle (mitochondria) 2. the correct subcompartment (eg matrix, inner memrbane, intermembrane sapce)

Question 642

Is mitochodnrial protein targetting efficent?

Answer 642

yes, its a highly efficinet and regulated process

Question 643

What do all nucelar-encoded mitochodnrial proteins have?

Answer 643

unique targetting sequences-short amino acid sequences acting like zip codes

Question 644

What do mitochondrial targetting sequences do?

Answer 644

1. from cytoplasm to the mitochondrion, and 2. correct mitochondiral subcompartment

Question 645

What are the 4 possible final destinatins for mitochondrial proteins?

Answer 645

1. outer membrane 2. intermembrane space 3. inner mebrane 4. matrix

Question 646

What does each targetting pathway depend on?

Answer 646

- specific sub-mitochondrial targetting signal - and shared or unique import achinery (like tom, tim, sam, mia etc)

Question 647

what mitochondrial protein import pathway is best understood?

Answer 647

targetting and import of matrix proteins

Question 648

What sequence do matrix-destine proteins have?

Answer 648

20-50 amino acid-long matrix targettting sequence

Question 649

where are the matrix targetting sequence located on the protein?

Answer 649

n-termnus of the nascent protein

Question 650

Whst is the structure of the matrix targetting sequwnce?

Answer 650

ampihpathic alpha helic (one side positively charged, other one polar); on posoticve side argininge and lysine

Question 651

What is the role of the matrix targetting sequence?

Answer 651

targets the orotein to the mitchodnrial surface and allows it to be translocatedf throggh the oputer and inner membranes into the matrix

Question 652

what recognizes matrix-destined proteins in the cytoplasm?

Answer 652

cytoplasmic molecular chaperones, such as hsp70 (heat shock protein 70 kDa)

Question 653

what is the role of hsp70 in matrix protein import?

Answer 653

keeps the protein in a partially unfolded, import-compentent state so it can pass through the mitochidnral membranes

Question 654

Why do matrix-bound proteins tend to accumulate near the mitochondrial surface?

Answer 654

because of diffusion and mrna localization near the mitochondria

Question 655

where are mrnas encoding mitochondrial proteins often found?

Answer 655

in cytoplasm near mitochindria, forming a "mitochondrial RNA cloud"

Question 656

What mediates mitochondrial localization?

Answer 656

rna-binding proteins located on the mitochondrila outer membrane

Question 657

How do these rna-binding proteins recognize mrna?

Answer 657

they bind to specific sequences the mrnas utrs (untrasnalted regions) that act as anchors

Question 658

What is the result of mitochdonrial mrna localization?

Answer 658

protein synthesis (translation) happens right next to mitochondira improving targetting efficiency

Question 659

Why is mrna localization important?

Answer 659

allows site-sepcific (spatial) control of mitcohdonrial gene expresison, ensuring proteins are madewhere they are needed

Question 660

What happens in step 2 of matrix protein import?

Answer 660

proteins matrix-targetting is recognized by the import receptor complex on the mitochondiral surface

Question 661

What are the two main proteins in the im port receptor complex?

Answer 661

tom20 and tom22 (integral outer membrne proteins)

Question 662

What does tom stand for?

Answer 662

translocase of the outer membrane

Question 663

What are the molecular weights of tom20 and tom22?

Answer 663

about 20 kda and 22 kda

Question 664

What is the general import pore in the mitochondrial outer membrane?

Answer 664

tom 40, which allows the protein to pass into the intermembrane space

Question 665

What does tom40 do?

Answer 665

main entry point for almost all mitochondrial proteins-both matrix and membrane-bound

Question 666

What happens after the protein passes through the general import pre (tom40)?

Answer 666

it enters the inner merbane channel to continue translcoation into the matrix

Question 667

What proteins make up the inner mmebrane channel?

Answer 667

tim17, tim23, tim44 integral inner membrane proteins

Question 668

What do tim proteins stand for?

Answer 668

translocase of the inner mmebrane

Question 669

What are the molecular weights of tim17, tim23, tim44?

Answer 669

17, 23 and 44 kda

Question 670

What are mitochondrial contact sites?

Answer 670

areas where outer and inner mmebranes are closely aliged, allowing the e tom40 and tim channels to work together

Question 671

How are contact sites stabilized?

Answer 671

by interactions between tom40 and intermembrane space facing domains of tim23/17

Question 672

What happens as the protein exits the inner memrbne channel into the matrix?

Answer 672

its n-terminal matrix-targetting sequence is cleaved off

Question 673

What enzyme removes the matrix-targetting sequence?

Answer 673

matrix processsing protease

Question 674

What binds to the emergind precursor protein in the matrix?

Answer 674

matrix hsp70, a chaperon protein

Question 675

Where is matrix hsp70 located?

Answer 675

on the matrix side of the inner mmebrane channel, bound to tim44

Question 676

What is the function of matrix hsp70?

Answer 676

acts as a molecular motor (ratchet) to pull the protein into the matrix and prevent it from sliding back out

Question 677

How does hsp70 pull the protein into the matrix

Answer 677

uses atp hydrolysis pull protein in

Question 678

Does matrix protein import require energy?

Answer 678

yes, several steps require atp and a h+ gradient

Question 679

What additional energy source helps drive protein import?

Answer 679

the H+ electrochemical gradient across the inner membrne

Question 680

where is H+ concentration higher during import?

Answer 680

concentration is higher in the intermembrane soace than in the matrix

Question 681

how does the H+ graident help import?

Answer 681

positive charges in the proteins targetting sequence are pulled into the less positively charged matrix (electrochemical attraction)

Question 682

What drives directional movement of the protein into the matrix?

Answer 682

1. h+ gradient 2. Atp- powered hsp70 motor in the matrix

Question 683

What happens in step 6 of matrix protein import?

Answer 683

the imported, cleaved protein folds into its final active shape

Question 684

What happens in step 7 if folding doesnt occur on its own?

Answer 684

matrix localized chaperson assist the protein in folding, using atp

Question 685

What does step 7 represent in terms of energy use?

Answer 685

4th point where enrgy inout atp hydrolysis is needed in matrix protein imports

Question 686

What are the total energy requiring steps in mitochondrial matrix protein import?

Answer 686

1. cytosolic hsp70 2. matrix hsp70 3. h gradient 4. matrix chaperon-assited folding

Question 687

What are chloroplasta and what is their origin?

Answer 687

chloroplasts are semi-autonomus plant cell organelles, derived from photosynthetic cyanobacteria

Question 688

What process occurs in chloroplasts?

Answer 688

site of photosytnhesis and are involved inother metabolic proceses such as: - fatty acid biosythensis - amino acid biosythesis - nitrogen and sulfur assimilatio

Question 689

are chloroplasts stationary in the cell?

Answer 689

higly mobile and move along cytoskeleton elemetns ausing molecular motors

Question 690

What is the morpholgoy of chloroplasts?

Answer 690

chloroplasts are double-membrane bound organelles, with outer membrane and inner membrane that encloses the intermembrane spcae

Question 691

What does the chloroplast envelop consist of?

Answer 691

envelope consists of the outer membrane and inner mmebrane of chloroplasts (just thick and thin layer)

Question 692

What is the function of the outer membrane of chlorplasts?

Answer 692

the outer membrane contains proins (proteins) that allow small molecules to pass through and maintain the integrity of the of the chloroplasts outer surface

Question 693

What does the inner membrane of the chloroplast do?

Answer 693

the inner membrane is higly impermeable and contains trasnporters that regulare what enters or exits the chlorplast

Question 694

What is the role of the intermembrane space in chloroplasts?

Answer 694

intermembrane space lies between the outer and inner memrbanes and is involved in the mboement of moleucles and ions between these two membranes

Question 695

Where are thylakoids and where are they located?

Answer 695

thylakoids are flattenned, membranous discs arranged in stacks called grana (or between stacks as stroma thylakoids part of the third (internal) membrane system of the chloroplast

Question 696

What is the role of thylakoid membranes?

Answer 696

site of atp synthesis and help maintain the H+ gradient inside the thylakoid lumen

Question 697

Does thylskoid lumen have a higher H+?

Answer 697

yes

Question 698

What ois the stroma?

Answer 698

aqueous interior of chlorplast, found insdie the envelope and outside the thylakoids

Question 699

What is the plastid genome and what does it encode?

Answer 699

plastid genoe is cricular, varying in size and gene copy number between plant species; encodes ribosomal proteins, some photosyntheiss (Ps) proteins, trnas, rrnas and some rna polyermase subunits

Question 700

Where are the majority of plastid proteins encoded?

Answer 700

about 3000 plastid proteins encoded by nuclear genome and must be imported in plastid

Question 701

How are chloroplasts conneced?

Answer 701

via stromules

Question 702

What are stromules?

Answer 702

long stroma filled membrane tubules; dyanimc pradily extend and contract; allows for efficient metabolite transfer, communication etc between chloroplast and/or other organelle (eg er, mitochondria, etc)

Question 703

how are chlorplasts multiplied?

Answer 703

fission occurs end of g1: organelle duplication prior to s phase

Question 704

what sets or protein involved in chloroplast division?

Answer 704

FtsZ and PD; fprm ring like streuctires either on outside or inside of chlorplast envelope

Question 705

is ftz ring internal?

Answer 705

yes

Question 706

PD ring?

Answer 706

internal and externa

Question 707

What is FtsZ?

Answer 707

is a filamentous temperature sensitve protein (Z) involved in chloroplast division; homolog of bacterial division proteins and is located on the stromal side of the inner membrane of chloroplasts

Question 708

What are ftsz1 and ftsz2?

Answer 708

are two forms of ftsz proteins involved in chloroplast division - they assemble into long, filamentous polymers at the midway point (equator) of the inner membrane during division

Question 709

What proteins mediate the positioning of ftsz during chloroplast division?

Answer 709

positoning of ftsz proteins at the equaotr is mediated by several stromal proteins: arc3, minD and minE

Question 710

How is the FtsZ-ring linked to inner membrane at the chloroplast division site?

Answer 710

ftz-ring is linked at inner membrane at the chloroplast division site by arc6, which help stabilize the fts-z ring during division

Question 711

What role does arc6 play in chloroplasy divison?

Answer 711

arc6 is an integral inner membrane spanning protein localized to the equator of divinding chlorplasts; links and stabilizes the ftsZ-ring at the division site

Question 712

How is arc6 seen?

Answer 712

using confoca; laser scanning microscopy (CLSM) shows it at the equator of dividing chloroplasts

Question 713

How does arc6 interact with other proteins in chloroplast division?

Answer 713

ims-facing domain of arc6 binds to pladtid divding (PD) machinery in the chloroplast inner membrane space (IMS)

Question 714

What are pdv1 and pdv2??

Answer 714

plastid division 1 and 2 are hetero-dimeric, integral outer membrane proteins, they are recruited midway point of the divindng chloroplast by arc6

Question 715

How can pdv2 observed?

Answer 715

using gfp tagging to track position at the equator of divindg chloroplasts

Question 716

What is the role of arc5 in chloroplast division?

Answer 716

arc5 binds to pdv1 and pdv2 at the outer mmebrane of the chloroplast during division. Helps in the fomration of the pd-ring structure

Question 717

How does arc5 contirbute to the pd ring?

Answer 717

assembles into spiral-like structures to form the pd ring around the outer membrane of the chloroplast, aiding its division

Question 718

What does the pd-ring do during chloroplast divison?

Answer 718

pd-ring tightens around the outside of the chloroplasts (via gtp hydrolysis), which leads to constriction of the outer envelope and the divsion of the chloroplast

Question 719

what is a major similarity (and diff) between mitochondrial and chloroplast protein targetting?

Answer 719

both mitochindria and chloroplasts use nuclear endocded proteins, however chloroplasts have more subcompartments than mitochondria making porocess more complex

Question 720

What is the first step in chloroplast protein targetting?

Answer 720

targetting and import of chloroplast stromal proteins, where proteins have a stromal import sequence that directs them to the chloroplast

Question 721

What are the characteristics of the stromal import sequence?

Answer 721

stromal import sequence is located at n-terminuds of the protein, have lots of s/t small hydophobixc residues, makes diff from other targetting signals

Question 722

What happens to the stromal import sequence after the protein enters the chloroplast?

Answer 722

stromal import sequence is cleaved after the protein enters the stroma of the chloroplast, similar to the process in mitochondiral portein import

Question 723

how does hsp93 compare to hsp70?

Answer 723

hsp93 (found in mitochondrial matrix) helps pull portein into stroma, while hsp70 (found in stroma) ensures that the cleaved proteins is properly folded

Question 724

What are the two main thylakoid import pathways for chloroplast proteins?

Answer 724

2 pathways are: 1. srp-dependent pathway (similar to er protein import) 2. ph depenedent pathway

Question 725

what happens in srp dependent lumen?

Answer 725

1. stromal import sequnece is removed by stormal proteases, revealing a thylakoid targetting sequence 2. srp (signal recognition particle) binds to thlyakoid targetting sequence and helps in proteins translocation into the thylakoid lumen via the sec61 translocon

Question 726

What is ph dependt pathway?

Answer 726

1. protein fully folded in stroma 2. folded protein is then imported into thylakoid lumen using a recepotr/trnaslocon complex that recoghnizes a di-arginine containing thylakoif targetting sequence

Question 727

What drives the translocation of proteins in the ph-dependent pathway?

Answer 727

proton gradient across the thylakoid membrane, where the proton (H+) gradient between the stroma and thlakoid lumen provides the enrgy for translocation