Chapter 12 Flashcards
What are the main features that distinguishes eukaryotes from prokaryotes?
The presence of a nucleus and membrane bound compartments
Each organelle contains a distinct set of what that defines their cellular function?
Enzymes
What is tightly packed with organelles and contains very little free space?
The cytosol
What organelle contains many metabolic pathways and is the main site for protein synthesis?
Cytosol
Intracellular membrane bound organelles occupy how much of the cell?
Half the volume of a cell
What is this organelle?
- It is one of the most distinct organelles within the cell
- It typically occupies a central location within a cell
- It is used to distinguish prokaryotes from eukaryotes
The nucleus
What organelle houses the majority of the genomic material in the cell, is the site of DNA and RNA synthesis and uncouples transcription and RNA processing from translation?
The nucleus
Positioning of many intracellular organelles is dependent on their association with what?
Cytoskeletal elements (microtubules and actin filaments)
What organelle is responsible for sorting of endocytosed material?
Endosomes
What organelle synthesizes most of the lipids and synthesizes proteins for distribution to many organelles and the plasma membrane?
Endoplasmic Reticulum (ER)
What are continuous with the nuclear envelope and exist as an extensive network?
ER tubules
What are the four kinds of endoplasmic reticulum?
- Smooth ER2. Rough ER3. Sarcoplasmic Reticulum4. Transitional ER
Which ER is ribosome free and is responsible for lipid metabolism?
Smooth ER
Which ER has bound ribosomes and is responsible for protein synthesis?
Rough ER
Which ER is responsible for calcium handling and storage?
Sarcoplasmic reticulum
Which ER is smooth ER subtype, ER to golgi traffic?
Transitional ER
What are the main functions of the ER?
- Storage and production of glycogen2. Carbohydrate addition to proteins3. Protein folding, assembly and degradation4. Lipid and protein biosynthesis (for other organelles)
In polarized cells, what is always positioned between the nucleus and the direction of cell movement?
The Golgi
What appears as a series of flattened stacks appear as a nuclear-cap in mammalian cells?
The Golgi apparatus
What organelle is responsible for the modification, sorting and packaging of proteins and lipids for either secretion or delivery to another organelle?
Golgi Apparatus
What organelle is responsible for Oligosaccharide processing and assembly; Phosphorylation and sulfation modification of proteins?
Golgi apparatus
What organelle is responsible for Intracellular degradation, nutrient and waste storage (plant & fungi) and maintenance of cytosolic pH and turgur pressure (plant & fungi)?
Lysosome/vacuole
What organelle is responsible for the production of ATP (oxidative phosphorylation) and the catabolism of branched chain amino acids (Leu, Ile, Val)?
Mitochondria
What organelle is responsible for heme biosynthesis and the assembly of Fe and Fe-S complexes?
Mitochondria
What organelle is responsible for calcium storage, heat production (mitochondrial uncoupling), steroid biosynthesis (along with the ER) and apoptosis
Mitochondria
What organelle is responsible for oxidation of toxic molecules?
Peroxisomes
What organelle generates hydrogen peroxide which is used for the detoxification of phenols, formaldhyde and alcohol by peroxidation?
RH2 + O2 = R + H2O2
H2O2 + R’H2 = R’ + 2H2O
- Peroxisome
- glyoxysome
- glycosome
What organelle is involved with the beta-oxidation of fatty acids and the synthesis of cholesterol and polyunsaturated fatty acids?
Peroxisome
What organelle is involved in the photorespiration in plants (‘dark reaction’ of carbon fixation) and contains catalase (the hallmark enzyme for this organelle)
Peroxisome
What organelle converts fatty acids into sucrose (glyoxylate cycle)
Glyoxysomes (germinating seeds)
What organelle contains many of the enzymes involved in glycolysis?
Glycosomes (Trypanosoma)
Catalase converts hydrogen peroxide to what?
Water and oxygen
Catalase is responsible for the detoxification of toxins using what?
Hydrogen peroxide
What organelle uses ATP synthesis and carbon fixation by photosynthesis?
Chloroplasts (in plant cells)
What organelle is the site of photosynthesis (ATP production) and CO2 reduction by the Calvin cycle for use in amino acid, fatty acid, carbohydrate synthesis?
- Chloroplasts
- Plastid
What organelle is the site of nitrite and sulfate reduction (amino acid incorporation), storage of starch and oil compounds and has a role in environmental sensing (gravity, pathogen infection)?
- Chloroplasts
- Plastid
True or False: Each organelle carries out the same basic set of functions even among diverse cell types
TRUE
However, organelles can have additional what?
enzymatic properties (peroxisomes)
How do organelles vary between different types of organisms?
They can also vary in size, morphology and abundance depending on cellular demand, environmental stage of development or a cells condition
What cells are the bulk of the cells in the liver and are involved in protein, cholesterol, bile salt and phospholipid biosynthesis which have a high energy demand?
Hepatocytes
What cells are involved in the production of enzymes necessary for the digestion of proteins and fats; these cells are filled with secretory granules containing digestive enzymes?
Pancreatic exocrine cells
It believed that all eukaryotic cells developed from what?
An ancient procaryotic cell
An early invagination of the plasma membrane of a prokaryote formed, with time, what developed next?
The nuclear envelope and its associated ER membrane
The attachment of what to the plasma membrane and the location of all transmembrane events to the plasma membrane facilitated the formation of these organelles?
The bacterial genome
The acquisition of mitochondria and plastids occurred through what events?
Independent endosymbiotic
Early anaerobic eukaryotic cells acquired a what at a time when oxygen levels in the atmosphere increased substantially?
A purple photosynthetic bacterium
Nuclear transfer of bacterial genes occurred over millions of years to generate the what?
Mitochondrion
Today of the ~1,000 proteins in mitochondria, only how many of them are still encoded by the mitochondrial genome in humans?
13
The acquistion of plastids are thought to have occurred in a similar but independent endosymbiotic event. The engulfed organism was likely a what?
A cyanobacterium, a blue-green algae
Many of the bacterial genes escaped to the what over time?
The nucleus
A membrane invagination event likely occured for the development of what?
Chloroplasts
In immature plant cells the precursor to chloroplasts are what, an undeveloped double-membraned organelle that contains few internal structures?
Proplastids
As the immature plant cells differentiate, the proplastid develops based on what?
The needs of the cell
For chloroplasts, a portion of proplastid inner membrane invaginates to form what?
The thylakoid membrane and space
The thylakoid membrane and space are structurally and functionally distinct from the other membrane compartments; these membrane houses all of the what?
The photosynthetic machinery.
Proplastid development is what type dependent? It is under what control?
Tissue type dependent and nuclear control
What are found in green tissues and are responsible for photosynthesis?
Chloroplasts
What are colorless and are found in seeds and root tissue and are specialized for the storage of starch?
Amyloplasts
What are involved in pigment synthesis and are found in all colored organs such as fruits and flowers?
Chromoplasts
All nuclear encoded proteins begin translation on what?
Cytosolic ribosomes
The next step depends on a what?
Targeting signal
What direct the protein to utilize one of the three protein trafficking pathways present in eukaryotic cells?
Targeting signals
What are the three protein trafficking pathways present in eukaryotic cells?
- Gated transport2. Transmembrane transport3. Vesicular transport
What transport is this? 1. The trafficking of proteins between the cytosol and the nucleus. 2. This is an active transport process. 3. Proteins contain a nuclear import (or export) signal. 4. They are recognized by specific receptor proteins for transport through nuclear pores
Gated transport
What transport is this? 1. It requires the presence of specific membrane translocators. 2. Pathway used for the transport of proteins from the cytosol into the mitochondria, the ER, plastids, or peroxisomes 3. Polypeptides can only pass through these membrane translocators in an unfolded form (mitochondria, chloroplasts, ER) 4. Peroxisomal proteins are unusual in that folded proteins are capable of transport into this organelle
Transmembrane transport
What transport involves cycles of membrane budding and fusion between the ER and the Golgi, the Golgi and the lysosome and the Golgi and the plasma membrane?
Vesicular
The uptake of extracellular material by endocytosis is also by what?
Vesicular trafficking
Vesicular transport mechanism is responsible for moving material from what?
One membrane bound compartment
Vesicular trafficking Maintains all enclosed proteins in the same what?
Same topology (a soluble protein in the ER is a soluble protein in the Golgi)
With vesicular trafficking, the orientation of a multispanning membrane protein in the ER is maintained as the protein passes through what?
Membrane bound compartments
Protein targeting to an organelle requires what? (3 answers)
- a signal on the protein that specifies its destination, 2. a receptor that recognizes this signal, 3. a transmembrane protein complex responsible for the transport of the protein across the membrane
It is not the specific amino acid sequence of these targeting signals that specify targeting, rather their what?
Physical properties
What are their physical properties are used that specify targeting?
Hydrophobicity or structural motif
What is surrounded by a nuclear envelope which contains pores that allow the passage of materials into and out of the nucleus?
The nucleus
The nucleus is surrounded by a nuclear envelope which contains pores that allow for what kind of traffic?
Bidirectional traffic
What houses the majority of the genomic material and is the site of DNA replication and RNA synthesis?
The nucleus
What is A?
Nuclear envelope
The outer nuclear membrane is continuous with the surrounding membrane of the what?
Endoplasmic reticulum (ER)
The space between the inner and outer nuclear membrane is continuous with what?
The lumen of the ER.
In a typical mammalian cell, the nuclear envelope can contain as many as 3,000 - 4,000 what?
Nuclear pores
Each nuclear pore contains ~30 different proteins (called what?) that assemble into a large complex of about 125 million daltons in mass.
Nucleoporins
The shape and stability of the nucleus is provided by a subset of proteins called the what?
Nuclear lamins.
Nuclear lamins polymerize into a 2D network that sits just beneath the what?
Inner nuclear membrane
Nuclear lamins attach to proteins found in the nuclear pore and the inner membrane and interact with chromatin, thereby providing a physical link what two things?
Between DNA and the nuclear envelope.
What is A?
Nuclear lamins
Nuclear Pore Complexes (NPCs) show what kind of symmetry?
An 8 fold rotational symmetry
Nuclear pore complexes sit between what two membranes without disrupting their continuity
The inner and outer nuclear membranes
What is this structure?
Nuclear pore complex
Identify the parts of the nuclear pore complex
A: Cytosolic fibril
B: Lumenal subunit
C: Ring subunit
D: Nuclear fibril
E: Nuclear basket
F: Nuclear lamina
Identify the 4 ‘substructures’ of the nuclear pore complex and what part of the structure they build:
- Column subunits (form the side walls of the pore), 2. Annular subunits (line the inner portion of the pore), 3. Lumenal subunits (anchor the complex to the membrane), 4. Ring subunits (flank both the cytosolic and nuclear faces of the complex)
Fibril like structures are found where?
On both faces of the pore
How many cytosolic fibrils are present?
8
There are 8 nuclear fibrils that form a what?
A basket-like structure
What are the two types of nuclear transport?
- Passive transport or diffusion 2. Facilitated, Active or Gated Transport:
What kind of transport freely moves molecules less than 5kD and slowly and inefficiently transports of proteins in the 20-40kD size range. So, as the size increases, transport through the pores, slows down ?
Passive Transport
What kind of transport has no difference in metabolite distribution between the cytosol and the nucleus?
Passive Transport
What kind of transport does not require receptors and energy?
Passive Transport
Which kind of transport is the nuclear envelope is not a barrier?
Passive Transport
What type of transport is an energy requiring process?
Facilitated transport or Gated Transport
What kind of energy is required for protein import and export using gated transport?
GTP
What kind of energy is required for mRNA export using gated transport?
ATP
What kind of transport moves molecules against their concentration gradient?
Facilitated or Gated Transport
What kind of transport is used by proteins larger than 60kD?
Facilitated or Gated Transport
What are the four properties of facilitated nuclear transport?
- Requires energy (GTP) 2. Temperature dependent 3. Signal sequence dependent 4. Saturable
The rate of facilitated transport is what dependent?
Temperature
Facilitated transport is selective; only proteins with a what can be transported?
Signal sequence
Facilitate transport is a what event? Only a fixed amount of substrate can be transported in a given period of time
A saturable event
In general ‘facilitated’ nuclear transport can be broken down into what 3 key steps?
- Formation of a cargo:carrier complex in the donor compartment 2. Transport of this complex through the nuclear pore 3. Release of the cargo in the target compartment and recycling of the carrier
What classes of DNA proteins are imported into the nucleus?
- Histones/DNA packaging proteins 2. DNA polymerase (for DNA replication) 3. DNA repair enzymes4. DNA modifying enzymes (e.g. methlyase, topoisomerase I and II, helicase, ligase)
What classes of RNA proteins are imported into the nucleus?
- Transcription factors (for RNA synthesis) 2. RNA processing/transport proteins (e.g. spliceosome, exosome, export factors)3. Ribosomal subunits (for assembly with RNA)
Ribosomal subunits (used in the cytosol for protein translation) require what?
An RNA component
Give the steps of the transport process for the assembly of ribosomal subunits?
- Ribosomal proteins synthesized in the cytosol are imported into the nucleus, 2. They are assembed with ribosomal RNA,3. They are exported back into the cytosol for final assembly
What are the general classes of proteins exported from the nucleus? (5 answers)
- Assembled ribosomal subunits 2. Transcription factors (cell cycle mediated export) E2F4 3. RNA binding/processing proteins (environmental conditions) Pdcd4 4. tRNAs5. Viral proteins and RNAs
What are the general properties of proteins undergoing nucleocytoplasmic shuttling?
- They are folded (have acquired tertiary structure) 2. They can be associated with other proteins (quaternary structure). Therefore can be large in size; up to 10MDa or 25nm in diameter 3. They contain an amino acid sequence that is recognized by import or export receptors; NLS or NES, respectively, (or they can be associated with a protein that contains anNLS or NES)4. The import/export signal is not removed after transport
What is the primary structure?
Amino acid sequence
What is the secondary structure?
The beta sheet, alpha helical coiled-coil formation
What is the tertiary structure?
The final folded form of a single polypeptide
What is the quaternary structure?
Assembly with other folded subunits
The transport of folded protein occurs for what kinds of transport? (4 answers)
- Peroxisomal transport, 2. Vesicular transport, 3. Plasmadesmata mediated transport,4. Nuclear transport
Nuclear Localization Signal (NLS) is on the surface of what?
A folded protein
Protein import may be through its association with
Another protein that has an NLS
What are the key players in nuclear transport?
- The cargo or protein to be transported 2. The transport receptor or carrier 3. The transport co-receptor or adaptor4. The Ran GTPase system:
What kind of signal will the cargo or protein to be exported contain?
Nuclear Export Signal (NES)
What kind of signal will the cargo or protein to be imported contain?
Nuclear Localization Signal (NLS)
What recognizes the substrate to be transported and are themselves recognized by the transport machinery?
Transport receptor or carrier
What is the transport receptor or carrier for import?
Importin _ (or karyopherin _)
What is the transport receptor or carrier for export?
CRM1 (or exportin 1)
Nuclear import receptors do not always directly bind to nuclear localization signals. What kind of proteins may be used to link the cargo with a receptor?
Adaptor proteins
What is the transport co-receptor or adaptor for import?
Importin _ (binds to the NLS)
What does CRM1 mean?
Chromosome Region Maintenance
The variety of receptors and adaptors enables the recognition of a larger repertoire of what?
Nuclear transport signals
What is Ran?
A monomeric GTPase that acts as a molecuar switch and is regulated by the state of its bound nucleotide
Give the components of the Ran GTPase system:
Ran, a Ran GEF protein, and a Ran GAP protein
Which Ran (Ran GEF or Ran GAP) is this? It promotes the exchange of bound GDP for GTP
Ran GEF
Which Ran (Ran GEF or Ran GAP) is this? It triggers GTP hydrolysis to convert Ran-GTP into Ran-GDP
Ran GAP
Ran-GAP and Ran-GEF are differentially localized in the cell therefore the state of Ran will depend on what?
Its location in the cell
Protein transport into and out of the nucleus requires what?
A transport signal
What are the two transport signals?
- Nuclear Localization Signal (NLS) 2. Nuclear Export Signal (NES)
Which signal (NLS or NES) is comprised of Arg and Lys (basic, postively charged amino acids)?
Nuclear Localization Signal (NLS)
Which signal (NLS or NES) is often bipartite in nature: 2 separate clusters of Arg and Lys residues separated by a ~10 amino acid spacer?
Nuclear Localization Signal (NLS)
Which signal (NLS or NES) is not removed from the protein after nuclear import and is typically part of the protein’s functional domain?
Nuclear Localization Signal (NLS)
Which signal (NLS or NES) is comprised of 5-6 hydrophobic amino acids (Leu and Ile)?
Nuclear Export Signal (NES)
What is the first step in nuclear protein import?
Cytosolic proteins with a NLS are recognized by the co-adaptor, Imp-alpha. The Imp-alpha receptor then binds the substrate-Imp-alpha complex and transports it through the NPC into the
What is the second step in nuclear protein import?
The Impβ receptor then binds the substrate-Impα complex and transports it through the NPC into the
nucleus.
What is the third step of nuclear protein import?
Once in the nucleus, Ran-GTP binds to Impβ causing the trimeric complex to fall apart.
What is the fourth step of nuclear protein import?
Impβ bound to Ran-GTP returns to the cytosol through the NPC (Impβ recycling!).
What is the fifth step of nuclear protein import?
Once in the cytosol, the GTP form of Ran is converted to GDP by a Ran-GAP (guanine activating protein) and Impβ is released to undergo another round of nuclear import.
Nuclear import, cycle using a cargo protein that requires an adaptor for receptor recognition, and what other reason?
Protein movement from the cytosol into the nucleus
What is the first step in nuclear export?
Nuclear proteins with a NES are recognized by CRM1 (an exportin) and Ran-GTP in the nucleus.
Exportins only bind Ran-GTP in the presence of what?
Cargo
What is the second step in nuclear export?
This trimeric protein complex (cargo, CRM1, Ran-GTP) is then transported through the NPC and into the cytosol.
What is the third step in nuclear export?
Once in the cytosol, Ran-GTP releases the cargo protein and the exportin receptor. This release step is mediated by a cytosolic Ran GAP protein that stimulates GTP hydrolysis to generate Ran-GDP.
CRM1 has no affinity for what?
Ran-GDP
What is the fourth step in nuclear export?
Ran-GDP and CRM1 are then reimported into the nucleus for another round of nuclear export.
Localization of Ran GTP sets up the what?
Direction of transport
Ran GTP is high in the nucleus and low in the what?
Cytosol
Ran GTP is high in the nucleus and low in the cytosol is established because Ran-GEF localizes to what and Ran-GAP localizes to the what?
Ran-GEF: nucleus Ran-GAP: cytosol
What two functions is Ran-GTP responsible for?
Binding to incoming receptors with their attached cargo and promoting cargo release in the nucleus
What does Ran-GTP do after it binds incoming receptors with their attached cargo and promotes cargo release in the nucleus?
It escorts the receptor back to the cytosol for an additional round of transport
Nuclear Ran-GTP is also responsible for what other function?
Recognizing NES containing cargo bound to its receptor for nuclear export
Ran in its GDP bound form has no affinity for import or export receptors; unloading occurs only where?
In the cytosol
For the Ran cycle to continue Ran gets reimported back where?
Into the nucleus
The nuclear import of Ran-GDP is mediated what separate transport receptor?
NTF2 (nuclear transport factor 2)
The driving force behind nuclear transport is provided, in part, by what?
The differential subcellular localization of Ran-GTP and Ran-GDP.
What is required in the nucleus for protein export (Including the export of the import receptor, Impβ!).?
Ran-GTP
Ran-GTP is converted to Ran-GDP in the cytosol by
what to promote the release of bound cargo?
Ran-GAP
Ran-GDP is then returned to the nucleus by what, the Ran-GDP specific import receptor?
NTF2
Once in the nucleus, Ran-GDP is converted to Ran-GTP
by what?
The nuclear localized Ran-GEF.
What organelle has a high concentration of Ran-GTP?
Nucleus
What organelle has a low concentration of Ran-GTP?
Cytosol
Facilitated protein export requires what?
Ran-GTP
GTP hydrolysis is required for the release of the cargo after what?
Nuclear export
Protein import also indirectly requires Ran- GTP activity as Ran-GTP is necessary for release of what?
Imported substrate and the export of Imp_.
What is the cellular cost for the transport of one protein?
A single GTP hydrolysis event
In many eukaryotic organisms, the nuclear envelope breaks down during what?
Mitosis
The disassembly of the nuclear envelope during mitosis is triggered by what?
The phosphorylation of both nuclear lamins and inner membrane proteins.
What is the chain of events during the disassembly of the nuclear envelope during mitosis?
- The dispersal of nuclear pore proteins into the cytosol. 2. The movement of membrane proteins into the ER membrane. 3. The removal of the nuclear envelope from the chromatin (by dynein).
Release of nuclear components during mitosis requires that nuclear proteins be what once nuclear envelope formation begins?
Re-imported
The nucleus re-formation is facilitated by the attachment of Ran GEF to what; a high local concentration of Ran-GTP near DNA?
Chromatin
What signals are not removed after transport so that these proteins can be re-imported into the nucleus after every cell division?
Nuclear localization
During protein importation into the nucleus, what amino acids does the NLS have?
Lysine and Arginine
During protein exportation out of the nucleus, what amino acids does the NES have?
Leucine
What carrier/receptor is used for nuclear import?
Importin-_, +/- adaptor
What carrier/receptor is used for nuclear export?
CRM1, +/- adaptor
Where is the donor compartment in nuclear import?
Cytosol
Where is the donor compartment in nuclear export?
Nucleus
Where is the target compartment in nuclear import?
Nucleus
Where is the target compartment in nuclear export?
Cytosol
What are the mechanisms regulating nuclear transport? (5 answers)
- Changes in protein conformation 2. +/- Post-translational modification 3. Blockage of NLS recognition 4. Cytoskeletal tethering to prevent transport 5. Protein cleavage/degradation
Nuclear localization of proteins can be what?
Regulated
Nuclear transport mechanisms give a cell additional levels of regulatory control permitting what?
A quick cellular response
Which is faster?: 1. Transporting an already made protein from the cytosol into the nucleus in response to an external signal 2. A mRNA that has to undergo transcription, followed by translation and then transport
- Transporting an already made protein from the cytosol into the nucleus in response to an external signal
Experimentally, how do you determine whether an amino acid sequence is a nuclear localization signal?
Mutational analysis
How do you perform mutational analysis?
Change a basic amino acid (Arg or Lys) within the putative NLS to an uncharged amino acid such as Thr and monitor the resulting intracellular localization.
Mutational analysis uses experimental identification of the amino acids that act as what?
A nuclear targeting signal
The same strategies used in mutational analysis can be used to study what? (2 answers)
Nuclear export and nuclear import signals
Mutational analysis can be used to identify an amino acid sequence necessary for what?
Nuclear localization
Experimentally, how do you determine whether an amino acid sequence is a nuclear localization signal?
Gene fusion
Give the two steps in gene fusion.
- Transfer the sequence encoding the putative NLS to a cytosolic protein. 2. Follow the resulting intracellular localization of the fusion protein by immunofluorescence microscopy.
Why would you use gene fusion?
To show that an amino acid sequence is sufficient for nuclear localization
How many membranes doe the mitochondria have?
Two
What kind of membranes does the mitochondria have?
Outer and inner membranes
What are the two compartments of the mitochondria?
Intermembrane space (IMS) and Matrix
How many different proteins are distributed between the outer membrane, intermembrane space, inner membrane and matrix space?
Between 900-1,000
Over 98% of the mitochondrial proteins are nuclear encoded and translated on what?
Cytoplasmic ribosomes
Therefore ~880-980 proteins must be targeted to mitochondria and sorted to their correct what?
Subcompartment.
Proteins targeted to mitochondria are roughly broken down into 2 categories based on the nature of their what?
Signal sequence
What are the 2 categories of mitochondrial targeted proteins (translocases) or signal sequences?
- Amino terminal signal sequence containing proteins 2. Internal signal sequence containing proteins
Correct protein targeting requires the activity of separate what?
Mitochondrial membrane translocases
What are two kinds of mitochondrial membrane translocases?
- A translocase of the outer membrane (TOM), and 2. Two separate translocases within the inner membrane (TIM)
What does TOM in the TOM complex mean?
Translocase of the Outer Membrane
What does TIM in the TIM complex mean?
Translocases within the Inner Membrane
The TOM complex is located in what membrane?
Outer membrane
The TIM complex is located in what membrane?
Inner membrane
Explain the system of mitochondrial translocation machinery nomenclature
Tom or “Tim”, followed by the molecular weight of the protein
The number after TIM or TIM refers to what?
Polypeptide molecular weight
A 40kD protein involved in protein translocation across the outer membrane would be named what?
Tom40
Protein targeting to 4 different compartments requires several different what?
Transmembrane translocases
All nuclear-encoded proteins are first recognized by receptor proteins from what?
The outer membrane Tom complex
Ater all nuclear-encoded proteins are first recognized by receptor proteins from the outer membrane Tom complex, proteins are passed off to what?
Tom40, the mitochondrial outer membrane channel
Matrix proteins, and a some inner membrane and intermembrane space proteins, use what?
The Tim23 translocase
All alpha-helix containing multispanning membrane proteins are inserted into the inner membrane by what?
Tim22
Several inner membrane proteins and ALL mitochondrial-encoded proteins are targeted to the inner membrane by what?
Oxa1
Beta-barrel proteins (outer membrane) are inserted and folded by what?
The outer membrane SAM complex
What is A?
Outer membrane
What is B?
Inner membrane
What is C?
Intermembrane space
What is D?
Matrix space
What is A?
TOM complex
What is B?
SAM complex
What is C?
TIM 23 complex
What is D?
TIM 22 complex
What is E?
OXA compex
mRNA encoding a mitochondrial protein is transcribed and processed (5’ and 3’) in the what?
Nucleus
Processed mRNA is exported into the what?
Cytosol
mRNA is translated by
Cytosolic ribosomes
Newly translated proteins associate with to maintain the protein in a unfolded and translocation competent form
Cytosolic chaperones(Hsp70)
Translated pre-proteins are targeted to mitochondria through recognition of their what?
Signal sequence
Protein targeting to mitochondria is when?
Post-translational
Protein import into mitochondria occurs when?
After the entire polypeptide has been synthesized
Most (98%) signal sequences contain what?
Contain an amino terminal signal sequence
Most signal sequences are cleaved by what?
matrix processing peptidase (MPP)
Is the signal sequence transferable or permanent?
Transferable
What amino acids are contained in a signal sequence?
Contain positively charged AA (Arg, Lys), hydroxylated (Ser, Thr) and hydrophobic (Val, Leu, Ile) AAs. Few, if any, negatively charged AA (Asp, Glu).
Signal sequences do not have what?
A conserved primary amino acid sequence
What structure does the signal sequence have?
Amphipathic _-helix.
What is the best characterized mitochondrial targeting pathway?
Transport to the mitochondrial matrix
Matrix targeted proteins contain an amino terminal signal sequence which is often removed after transport by the what?
Matrix processing peptidase (MPP)
Matrix signal sequence are sufficient for mitochondrial targeting; they function when transferred onto a what?
Carrier protein
Matrix signal sequences are rich in what?
Basic amino acids, Hydroxylated amino acids, and Hydrophobic amino acids
What kind of amino acids are missing in matrix signal sequences?
Negatively charged amino acids are absent
Is there a conserved amino acid sequence?
No
What is this structure?
Signal sequence alpha helix
In an _-helix, a turn occurs every how many amino acids?
3.6 amino acids
In an _-helix, a turn occurs every 3.6 amino acids. Therefore in an amphipathic _-helix this sets up an arrangement of AA such that every 3rd or 4th residue alternates between what?
A hydrophobic and a hydrophilic one.
In a signal sequence alpha helix, how are the amino acids arranged?
They are arranged so that one side of the helix will contain (+) charged amino acids; the other side is hydrophobic or hydroxylated AAs
Distribution of amino acids is easily visualized if plotted on a ?what
Helical wheel
Distribution of charged amino acids on one side of the helix with what on the other side?
Hydroxylated and hydrophobic amino acids
It is the structural distribution of amino acid properties that is recognized by what?
Mitochondrial receptor proteins
The unfolded precursor protein with its bound cytosolic chaperones is recognized by the what?
TOM receptor protein
The protein is transported through what channel?
Tom40 channel
The N-terminal signal is recognized by the what?
Tim23 inner membrane channel protein
Binding opens the Tim23 channel allowing polypeptide passage into what?
The matrix
Translocation of a polypeptide occurs through what?
Both the outer and inner membrane channels simultaneously
In the matrix, the N-terminal signal sequence is removed by a what?
A matrix localized processing peptidase
The protein is folded with the assistance of
Matrix chaperones
What are the energy dependent steps for matrix targeting?
- Cytosolic localized Hsp70 maintains translocation competent form
- Membrane potential across the inner membrane (ETC driven)
- Matrix localized Hsp70 for ‘pulling’ polypeptide
- Matrix localized Hsp60 for protein folding (not shown on slide)
Directional transport (matrix targeting) requires what?
Energy expenditure
What kind of energy production is essential? Where is this energy produced?
ATP hydrolysis in the cytosol and the mitochondrial matrix
ATP hydrolysis by cytosolic chaperones are required to maintain the precursor in what form?
An unfolded and translocation competent form
A matrix chaperone is also required ; matrix Hsp70 associates with the Tim23 complex and does what?
Helps pull the polypeptide into the matrix
In the matrix, the targeting signal is removed and the protein is folded by Hsp60 in an what driven manner?
ATP dependent manner
Matrix transport also requires a membrane potential across the what?
The inner membrane
The membrane potential is generated by electron transport activity which establishes a what across the inner membrane?
A proton gradient
The proton gradient is used to generate matrix ATP through what?
The inner membrane ATP synthase complex
The charge differential is also thought to electrophoretically drive the positively charged signal sequence through the what?
Tim23 channel
What are the molecular components when proteins are targeted to the mitochondria’s outer membrane?
- Tom40 channel 2. ‘Tiny Tim’ chaperones in the IMS 3. SAM complex in the outer membrane 4. _-signal in carboxy-terminus
β-barrel proteins are only found where?
In the mitochondrial outer membrane
What are pore forming proteins responsible for the passive transport of small molecules?
_-barrel proteins
β-barrel proteins make the cytosol and the mitochondrial intermembrane space equivalent with regards to what?
Small inorganic ions/metabolites
β-barrel proteins prevent the formation of a membrane potential across what?
The outer membrane
β-barrel proteins are transported through the Tom40 channel directly into the what?
The intermembrane space
In the intermembrane space β-barrel proteins associate with small chaperone proteins called what?
Tiny Tims
The chaperone proteins (Tiny Tims) prevent protein aggregation and deliver the protein to what?
The outer membrane SAM complex
All β-barrel proteins contain a conserved targeting signal in their carboxy terminus which is essential for what recognition?
SAM complex recognition
Once bound to SAM components, they insert the protein into the what in the correct conformation?
Outer membrane
ATP hydrolysis by what is required for the transport of _-barrel proteins?
Cytosolic chaperones
Their transport does not require what?
Mitochondrial ATP hydrolysis or membrane potential
What are the molecular components of mechanism 1 for inner membrane targeting?
- Tom40 and Tim23 channel complexes 2. N-terminal, cleavable signal sequence, hydrophobic stop transfer 3. Membrane potential and matrix ATP
The most frequently used pathways is for inner membrane proteins having what kind of targeting signal?
A bipartite targeting signal
Inner membrane proteins have an amino terminal matrix targeting signal followed by what? That act as what?
A hydrophobic stretch of amino acids that act as a ‘stop transfer’ signal
Inner membrane proteins are transported through what? Then make contact with what channel?
The Tom complex; Then, the TIm23 channel
Only the matrix signal sequence enters the matrix; What prevents translocation through the Tim23 channel?
The hydrophobic stop-transfer sequence
The hydrophobic stop-transfer sequence halts transport across the inner membrane but does not block what?
N-terminal signal sequence removal or translocation through the Tom complex
The hydrophobic domain exits laterally from the Tim23 channel to integrate into the inner membrane generating a what?
A membrane tethered protein
The hydrophobic tether can be removed by an inner membrane peptidase to generate what?
A soluble intermembrane space protein
This pathway requires what two things?
The ATP dependent activity of matrix Hsp70 and a membrane potential across the inner membrane
What are the molecular components of mechanism 2 for inner membrane targeting?
- Tom40 and Tim23 channel complexes 2. N-terminal, cleavable signal sequence 3. Hydrophobic retargeting signal sequence 4. Oxa1 complex 5. Membrane potential and matrix ATP
What kind of proteins use the second pathway?
Inner membrane polypeptides
In the second pathway, the inner membrane protein is transported into the matrix by what two complexes?
Tom complex and Tim23 channel
In the matrix, what is removed?
The amino targeting signal sequence
The signal sequence cleavage reveals a hydrophobic targeting signal which re-directs the polypeptide to what?
The Oxa1 complex in the inner membrane
The Oxa1 complex inserts the protein into what?
The inner membrane
What two things are essential for transport and membrane insertion of inner membrane proteins?
Matrix ATP and a membrane potential
The Oxa1 complex also assembles what proteins into the inner membrane?
Mitochondrially-encoded proteins
In humans, all proteins encoded by the mitochondrial genome are components of the what?
Electron transport chain
Mitochondrial encoded proteins are inserted into the inner membrane in what manner?
Cotranslational manner
Mitochondrial ribosomes have a high affinity for what?
Oxa1
Transport of mitochondrially encoded proteins
- Does not require the Tom complex, 2. Does not require cytosolic or matrix ATP hydrolysis, 3. Requires the Oxa1 complex, and 4. Integration into the inner membrane occurs co-translationally
What are the molecular components of mechanism 3 for inner membrane targeting?
- Tom40 channel complex 2. Tim22 channel complex 3. Membrane potential 4. No cleavable signal sequence
What kind of proteins use the third pathway?
Multispanning transmembrane proteins of the carrier family
Multispanning transmembrane proteins transport what across the inner membrane?
Small molecules
The inner membrane is completely impermeable to what?
Small molecules
Everything that crosses the inner membrane (ATP, ADP, phosphate, etc) must be what?
Actively transported
These multispanning transmembrane proteins contain a what which is not removed after transport?
An internal targeting sequence
The proteins are also transported through the Tom40 channel as a what?
A looped structure
During translocation into the intermembrane space, the polypeptide associates with the “Tiny Tim” chaperone proteins to maintain what state?
An unfolded state
Tiny Tims shuttle the protein to the what?
Tim22 translocase
The Tim22 complex assembles the multispanning membrane domains then inserts the protein into what?
The inner membrane
Transport of these proteins requires what?
Cytosolic ATP hydrolysis and a membrane potential
Orientation of an alpha helix containing transmembrane domains (TMD) follow what?
The (+) end in rule
Positively charged flanking amino acids are likely to be oriented where?
Into the matrix
Negatively charge flanking amino acids will be where?
On the intermembrane space side
What is this structure?
Chloroplast
The chloroplast has 6 distinct compartments; what are they?
3 membranous layers and 3 soluble subcompartments
What is A?
Outer membrane
What is B?
Inner membrane
What is C?
Intermembrane space
What is D?
Stroma (matrix space)
What is E?
Thylakoid membrane
What is F?
Thylakoid space
The Shape and structure of the chloroplast varies depending on what?
The species, tissue and environmental conditions
How do chloroplasts differ from mitochondria?
Chloroplasts have 6 distinct compartments, one more membrane bound compartment than mitochondria
What are the three membrane of the chloroplast?
- outer membrane, 2. inner membrane, 3. the thylakoid membrane
What are the three soluble subcompartments of the chloroplast?
- the intermembrane space, 2. the stroma (similar to the mitochondrial matrix), 3. the thylakoid space
Give four characteristics of the chloroplast transit signal
- N-terminal 2. Cleavable (removed in the stroma) 3. No conserved AA sequence 4. 20-100 AA in length
Like that seen for mitochondria, the targeting signal of the chloroplast contains what?
Positively charged (i.e. basic) and hydroxylated AAs.
Chloroplast signal sequences do not form what in solution?They are modified by what?
An amphipathic _- helix and Ser/Thr phosphorylation
Because plant cells contain both mitochondria and chloroplast the targeting signals for each of these organelles must be what?
Distinct
Nuclear-encoded chloroplast proteins are transported how?
Posttranslationally.
Protein transport into the chloroplast resembles what?
Resembles protein transport into mitochondria
20-100 amino acids transit peptide (targeting signal) found at the what?
The amino terminus
The targeting signal is often removed after what?
Translocation
Transport into the thylakoid membrane requires a second targeting signal which follows what?
The stromal signal (bipartite)
Despite similarities in their transport mechanisms mitochondria and chloroplast evolved how?
Independently
What is not found between any of the proteins that make up the transport machinery of these two organelles?
No sequence conservation
What are the four targeting pathways to the thylakoid?
- Sec pathway 2. SRP-like pathway 3. TAT pathway 4. Spontaneous pathway
Thylakoid space targeting utilizes what two pathways?
The Sec or TAT transport pathways
What pathway is analagous to a pathway found in bacteria for protein transport across the inner membrane?
The Sec pathway
Transport using the Sec pathway requires what?
ATP hydrolysis and a membrane potential across the thylakoid membrane
What pathway is this? “1. Also a pathway found in bacteria. 2. Proteins are folded. 3. Transport requires only a pH difference across the thylakoid membrane.”
TAT pathway
Thylakoid membrane targeting utilizes what two pathways?
The SRP or the spontaneous insertion pathway
What pathway is similar to protein transport into the ER and depends on ATP hydrolysis and a membrane potential?
SRP pathway
What pathway includes proteins whose transport is independent of protein based machinery or energy?
Spontaneous pathway
Which pathways transport soluble proteins? Which pathways transport membrane proteins?
Soluble proteins: Sec and Tat pathways (1 and 3) Membrane proteins: SRP and spontaeous pathways (2 and 4)
What is a single membraned organelle?
Peroxisome
Peroxisomal proteins are localized to what two places?
The peroxisomal matrix or the peroxisomal membrane
All what kind of organisms contain peroxisomes?
Eukaryotic
What is the main function of a peroxisome?
The detoxification of alcohols and phenols
Enzymes in peroxisomes required for detoxification are present at such high concentrations they form what?
Electron dense regions
Approximately how many genes are required for peroxisomal transport?
23 PEX
Peroxin proteins are found where? (3 places)
The cytosol, peroxisomal membrane and the peroxisomal lumen/matrix
What are the three functions of peroxins?
- Recognizing peroxisomal cargo 2. The transport of this cargo 3. The formation of the membrane associated import complex
Where are the transport locations within peroxisomes?
- Matrix/lumen 2. Membrane
All peroxins are nuclear encoded, synthesized on cytoplasmic ribosomes, and transported what?
Post-translationally.
Proteins are fully folded and assembled into a higher order complex before when?
Transport into the organelle
What does PTS mean?
Peroxisome Targeting Signal
What is the most abundant (PTS) signal?
PTS1 (peroxisome targeting signal 1)
Give four characteristics of PTS1
- Carboxy terminal tripeptide sequence: SKL/ARM. 2. Necessary and sufficient for transport. 3. Most abundant signal for matrix targeting. 4. Recognized by the Pex5 cytosolic receptor.
Give three characteristics of PTS2
- Amino terminal or internal sequence: X2RLX5HL. 2. Smaller set of matrix targeted proteins. 3. Recognized by the Pex7 cytsolic receptor.
Where is PTS 1 found?
The carboxy terminus of a protein
PTS 1 has the conserved sequence of what?
Ser, Lys, Leu
Where is PTS found?
Found either at the amino terminus or internal to the protein
Proteins destined for the matrix contain what?
One or more peroxisomal targeting signals
Some proteins lack their own signal but are transported through their association with what?
Partner proteins that contain a PTS
Transport requires a soluble cytoplasmic receptor protein: What for PTS1 signals? What for PTS2 signals?
Pex5 for PTS1 signals; Pex7 for PTS2 signals
Pex5 and Pex7 proteins are responsible for trafficking their cargo where?
To the peroxisome for receptor binding
Pex5 may be responsible for forming what?
The transport channel in the peroxisomal membrane
In eukaryotes, the endoplasmic reticulum can make up how much the total membrane in a cell?
Half
Give two functions of the ER
- Lipid and protein biosynthesis 2. The intracellular storage of calcium
Rough ER is studded with what?
Bound ribosomes
Ribosomes are absent on smooth ER which are specialized for the production of what?
Steroid/hormone production
An individual cell contains both smooth and rough ER membranes that are often what?
Interconnected
The percentage of the ER that is smooth versus rough will vary depending on what?
The specialized function of that cell
The bulk of the proteins targeted are what targeted to the ER explaining the association of ribosomes with ER?
Co-translationally
A small subset of proteins are targeted to the ER what?
Post-translationally
In mammalian cells, proteins less than 70 amino acids in size are transported when?
Post-translationally
In mammalian cells, proteins less than 70 amino acids in size are transported post-translationally; the remaining are transported when?
Co-translationally
Identify A and B
A: Co-translational translocation B: Post-translational translocation
Give some examples of co-translational transport pathways
- Endoplasmic Reticulum 2. Mitochondrial-encoded proteins 3. Chloroplast-encoded proteins
Give some examples of post-translational transport pathways
- Endoplasmic Reticulum 2. Nuclear-encoded mitochondrial proteins 3. Nuclear-encoded chloroplast proteins 4. Peroxisomes 5. Nucleus
The idea of a targeting signal originated in the 1970’s by scientists working on protein transport into what?
Isolated ER vesicles
What is the idea that proteins destined for the ER had a stretch of amino acids that were recognized by receptors found only in the ER?
Signal hypothesis
The ER has a stretch of amino acids that were recognized by recepters found only in the ER. This signal peptide was then removed after what was complete?
Translocation
The signal hypothesis has expanded to include transport into what?
Other organelles
Proteins destined for the ER (or later organelles in the secretory pathway) begin translation on what?
Cytoplasmic ribosomes
When their amino terminal signal sequence emerges from the ribosome it is recognized by a what
Signal recognition particle (SRP)
SRP binding halts translation and targets the ribosome with its bound mRNA and polypeptide chain to the what?
ER membrane
The SRP engages with the what?
ER translocation channel
SRP is released and ribosome mediated translation continues with the elongating polypeptide entering the what?
ER lumen
When translation is completed, the ribosome is released from the ER into what? For what reason?
The cytosol for another round of protein synthesis
The ER targeting signal has no what?
Amino acid sequence conservation
ER targeting signals share a common distribution of what? (2 answers)
Charged and nonpolar amino acids
The centrally located hydrophobic stretch of amino acids is recognized by what?
The signal sequence binding pocket on SRP
Following signal sequence binding pocket on SRP is a peptidase cleavage site that is recognized by what?
The ER signal peptidase
What two kinds of proteins are targeted to the ER using similarly organized targeting signals?
Both soluble and transmembrane proteins
Give 5 characteristics of the ER targeting sequence:
- No amino acid sequence conservation 2. 20-30 amino acids in length 3. At the amino terminus of ER targeted proteins 4. Shared pattern of charged and nonpolar amino acids (+ charged N terminus, central hydrophobic domain, polar C-terminus) 5. Peptidase cleavage site follows the signal sequence
What are the two types of proteins targeted to the ER?
- Soluble proteins (lumenal ER, Golgi, secreted proteins) 2. Transmembrane proteins (ER, Golgi, plasma membrane)
What are the two purposes of translational pausing?
- Provides time for targeting to the ER before translation is completed. 2. Prevents the release of potentially dangerous proteins into the cytosol.
Recognition of the amino terminal ER signal sequence soon after its translation by cytosolic SRP is a key step for what?
ER targeting
What is a ribonucleoprotein composed of 6 protein subunits and 1 7S RNA molecule?
The eukaryotic SRP
What does the eukaryotic SRP look like?
An elongated, flexible rod shaped structure
Identify A - E
A: Signal Recognition Particle (SRP) B: Signal sequence bound by SRP C: Small ribosomal subunit D: Large ribosomal subunit E: Signal sequence on nascent polypeptide chain
The SRP allows one end to bind to the emerging
polypeptide chain while the other end does what?
Wraps around the large ribosomal subunit to block the elongation factor binding site.
SRP binding causes what?
Translational arrest
There are how many SRPs for every ribosome?
~1 (cytosolic SRP molecule for every ribosome)
SRP affinity for a ribosome increases 1,000 fold when what is present?
A signal sequence
Predicted that SRP acts by cycling on and off cytosolic ribosomes searching for what?
An ER targeting signal
Translationally paused SRP-ribosome-mRNA-nascent polypeptide complex is targeted to the ER membrane by its association with what?
The integral membrane receptor SR, for SRP receptor.
Ribosome binding to the ER sets up what two populations of ribosomes?
Free cytosolic ribosomes and membrane bound ribosomes
Free cytosolic ribosomes and membrane bound ribosomes are structurally and functionally identical and only differ at any one time in the what?
Protein they are synthesizing
Identify the three cycles:
A: Free ribosome cycle B: Membrane-bound ribosome cycle C: SRP cycle
The SRP bound ribosome is targeted to the cytoplasmic side of the ER membrane through its association with what?
The ER receptor SR
The SRP bound ribosome associated with SR promotes ribosome binding to the ER membrane where it binds to an available what?
Sec61 channel
The transfer of the amino-terminal sequence from SRP to the Sec61 channel triggers hydrolysis of the GTP bound to both the SRP and the SR receptor, inducing what?
A conformational change
After the hydrolysis of the GTP bound to both the SRP and the SR receptor, SRP is released allowing for the what?
Reinitiation of translation
The ER translocon is an unopened position until the what is bound?
Ribosome
The protein conducting channel in the large ribosomal subunit is positioned directly over the Sec61 channel such that the elongating polypeptide chain has direct access to what?
The ER lumen
Co-translational transport into the ER requires what components?
- Cytosolic SRP, 2. A membrane bound SRP receptor, 3. A translationally arrested ribosome, 4. The Sec61 translocon
For co-translational protein transport the driving force for protein translocation is provided by what?
The ribosome
Because of the close contact between the ribosome and the channel, the elongating polypeptide has no where to go but through the ER channel. Therefore, for co-translational transport the energy driving the translocation is what?
Protein translation
Proteins are translated on cytosolic ribosomes and are bound by cytosolic chaperones to prevent what?
Protein folding and aggregation
They are targeted to the Sec61 translocon by what?
An unknown mechanism
Polypeptide translocation through the Sec61 channel requires what components?
- 4 additional membrane proteins: Sec62, 62, 71 and 72 2. 1 additional ER lumenal protein: Bip/Kar2.
What is an ATP dependent chaperone which grabs the polypeptide as it emerges into the ER lumen?
BiP
Rounds of ATP dependent binding and release by BiP mediates what?
The unidirectional transport of polypeptides into the ER
Post-translational transport occurs in the absence of a what?
Ribosome
Transport of a completely synthesized polypeptide is completely dependent on the ATPase activity of what?
BiP
What organisms use Co-translational translocation?
Bacteria, Archaea, and Eucaryotes
What organisms use Post-translational translocation?
Eucaryotes
Which one (A or B) shows Co-translational translocation?
A
Which one (A or B) shows Post-translational translocation?
B
What triggers Sec61 translocon opening, and ribosome mediated translation pushes the elongating polypeptide through the channel?
Signal sequence binding
For a single pass transmembrane protein a second hydrophobic stretch of amino acids acts as what kind of signal that halts polypeptide elongation through the channel?
Stop transfer signal
The ribosome continues translation to generate a transmembrane protein with domains located where?
On both sides of the ER membrane
The hydrophobic domain exits the Sec61 translocon through a lateral opening in the what?
Sec61 channel
Pathways for the transport of single-pass transmembrane protein containing what?
An internal, noncleavable targeting signal
During translation the hydrophobic targeting signal is recognized by cytosolic SRP leading to a halt in what?
Translation and ER targeting
At the ER membrane, the internal targeting signal is recognized by the Sec61 translocon triggering what?
Its opening
The continuation of ribosome mediated translation promotes polypeptide translocation with the internal hydrophobic signal acting as a what?
A stop transfer sequence
Lateral exit through the Sec61 channel generates a what?
Membrane bound protein
The internal hydrophobic targeting sequence can bind to the Sec61 translocon in one of how many orientations?
One of two orientations
Each protein is inserted into the membrane with a specific orientation that changes or does not change?
Does not change
The orientation of the transmembrane domain is influenced by whath two things?
The length and composition of the hydrophobic anchor and the amino acids that flank the domain
The flanking sequence with the greatest net positive charge will remain on what side of the ER membrane?
The cytosolic side of the ER membrane
What plays a role in protein folding in the ER?
Glycosylation
What proteins are involved in monitoring protein folding in the ER?
- Calnexin and calreticulin (calcium requiring ER chaperone proteins) 2. Glucosyl transferase and glucosidase (soluble ER enzymes)
A newly translocated, and unfolded protein contains a complete 14 sugar what?
Oligosaccharide group
Trimming occurs where one or two terminal glucose moieties are removed by what?
ER glucosidases
Unfolded proteins with a single glucose containing oligosaccharide is recognized by what two chaperones?
Calnexin or calreticulin
Unfolded proteins with a single glucose containing oligosaccharide is recognized by either calnexin or calreticulin chaperones. This binding prevents the protein from doing what?
Exiting the ER
After the third glucose is removed by glucosidase, the protein is released from what
Calnexin/calreticulin
After the third glucose is removed by glucosidase, the protein is released from calnexin/calreticulin. The released protein is then recognized by what?
Glucosyl transferase
What is the monitoring step in the pathway?
Glucosyl transferase
If the protein is unfolded, glucosyl transferase will add another what to the oligosaccharide using UDP-glucose as a substrate?
Glucose
If the protein is properly folded, glucosyl transferase does what?
Releases the glucose free glycoprotein so that it can leave the ER
What kind of proteins can not leave the ER as they could be deleterious to cell function?
Misfolded
Can the cycle of adding and removing a glucose molecule continue forever?
No (There has to be a system in place to get rid of proteins that are unable to fold within a certain period of time)
The state of the oligosaccharide group is used to determine what?
How long a protein has been in the ER
What machinery transports the unfolded/misfolded protein out of the ER and into the cytosol
The retrotranslocation machinery
In the cytosol the oligosaccharide is removed by what?
N-glycanase activity
The polypeptide is then targeted for ubiquitination followed by degradation by the what?
Cytosolic 26S proteosome complex
The export pathway for misfolded protein is referred to as what?
ER associated degradation or ERAD
Retrotranslocation occurs through the Sec61 channel with the help of what?
The Sec63 accessory protein
BiP activity is required for what two functions?
- Maintaining the unfolded protein in an conformation competent for retotranslocation. 2. Driving the polypeptide through the channel and out of the ER.
Some of the cytosolic ubiquitinating machinery is associated with the ER membrane ensuring ubiquitination after emerging from the what?
Sec61 translocon
What contains proteins responsible for the modification, folding and assembly of newly translocated proteins?
The ER lumen
What pathway is responsible for clearing some of these proteins?
The ERAD degradation pathway
The cell has parallel a monitoring system in place that measure the level of unfolded proteins present within the ER
A significant accumulation of misfolded proteins triggers a transcriptional response called the what?
The “Unfolded protein response” (UPR)
What is an intracellular signaling pathway that links defects in ER protein folding to the transcriptional activation of genes required for the alleviation of ER distress?
The “Unfolded protein response” (UPR)
The inability to fold may be due to what?
- Genetic error, 2. Environmental stress, 3. The absence of oligomeric partner proteins, 4. Changes in ER homeostasis
How many pathways are triggered by the accumulation of unfolded ER proteins?
Three
The molecular components of the IRE1 based unfolded protein response pathway include what? (6 answers)
- Unfolded proteins in the ER,
- Ire1p; A
- Ser/Thr kinase ER receptor,
- HAC1 mRNA, a tRNA ligase,
- The Hac1p transcription factor, 6. The UPRE promoter element
Under normal conditions Hac1 mRNA is transcribed where?
In the nucleus and exported into the cytosol
Secondary structure within the Hac1 mRNA prevents complete translation so the mRNA remains where? Associated with what?
In the cytosol associated with polyribosomes
When unfolded proteins accumulate in the ER Ire1p receptor is activated. Activation is mediated by what?
ER lumenal BiP
With low concentrations of unfolded proteins, BiP binds to the lumenal portion of Ire1p preventing its what?
Dimerization
Increased concentrations of unfolded ER proteins titrate BiP off of Ire1p where loss of BiP allows for what?
Ire1p dimerization and phosphorylation
Increased concentrations of unfolded ER proteins titrate BiP off of Ire1p where loss of BiP allows for Ire1p dimerization and phosphorylation. This activates what?
Ire1p endonuclease activity
Activated Ire1p cleaves the intron from what?
Hac1 mRNA
