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BIOL 2020 > 4: Endomembranes > Flashcards

4: Endomembranes Flashcards

1
Q

Endomembrane system

A

-interconnected system of cytoplasmic membranes

  • includes:
    • ER
    • golgi
    • endosomes
    • lysosomes
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2
Q

Movement out of cell

A
  • biosynthetic or secretory pathways

- ER —> Golgi —> various locations

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3
Q

Movement into cell

A
  • endocytic pathways

- plasma membrane —> endosomes —> lysosomes

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4
Q

ER

A
  • continuous with outer nuclear membrane
  • rough ER folds into sheets
  • smooth ER is more tubular branched shape (coral)
  • inside of ER called lumen
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5
Q

Smooth ER

A
  • not involved in protein synthesis
  • functions:
    1. Drug detoxification: enzymes add -OH to hydrophobic compounds to become water soluble and easier to excrete
    2. Carbohydrate metabolism: glycogen -> glucose (maintain blood glucose
    3. Calcium storage: important for muscle cells
    4. Steroid biosynthesis: cholesterol gets made
  • lots of smooth ER in liver
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6
Q

Rough ER

A
  • has ribosomes
  • important in synthesizing proteins in secretory pathways
  • first step of glycosylation/folding

-site for protein processing/modification/quality control

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7
Q

Ribosomes

A
  • large (60S) and small (40S) subunits in eukaryotes
  • made of ribosomal RNA (rRNA) and protein

-function: protein synthesis

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8
Q

Where are ribosomes found in the cell

A
  • mitochondria and chloroplasts
  • attached to membranes
  • free in cystol

-ribosomes found in cystol and attached to membranes are THE SAME - structurally identical

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9
Q

Free ribosomes vs membrane-bound ribosomes

A
  1. Free:
    • proteins destined to remain in cytosol
    • proteins that go into nucleus
    • proteins that go into mitochondria
    • eg. Actin/tubulin
  2. Membrane bound
    • proteins that are to be secreted or reside inside organelles in the endomembrane system (ER/golgi/lysosomes)
      • protein/peptide hormones
    • proteins that are integral membrane proteins of organelles in the endomembrane system

-ALL proteins start off as free ribosomes in cytoplasm, and then go to ER if needed/destined

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10
Q

How does a ribosome know to go to ER or not

A
  • Gunter Blobel 1999
  • Signal hypothesis:
    1. All ribosomes are the same
    2. An amino acid signal on a new protein directs the growing polypeptide and ribosome to the ER
    3. That protein will be fed into the ER lumen as it is translated (co-translationally)
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11
Q

Signal mechanism for cotranslational import

A
  1. A Signal Recognition Particle (SRP) temporarily binds to the signal sequence
    • SRP = 6 proteins and a piece of RNA
    • once SRP binds, translation stops
  2. The entire complex binds to a translocon in the ER membrane
  3. GTP is hydrolyzed, SRP leaves, and the new polypeptide is fed through the pore in the translocon.
    • signal sequence is cleaved by signal peptidase
  4. Completed polypeptide released inside the ER lumen
    • ribosome leaves
    • pore closes
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12
Q

What is a translocon

A
  • SRP receptor = binds SRP
  • ribosome receptor = binds ribosome
  • pore - channel (new protein feeds through)
  • signal peptidase = enzyme that cuts off signal sequence AAs
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13
Q

Experimental evidence that Blobel was right

A
  • experiment 1 = subcellular fractionation/cell-free systems
    • ie. squish a bunch of cells, spin them at different speeds to separate different cell components

-experiment 2= modify a cytosolic protein by adding a signal sequence to its end and watch where it goes

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14
Q

How transmembrane proteins with C-terminus in cytosol get made

A
  1. Signal sequence targets polypeptide to translocon
  2. Stop transfer sequence halts translocation
    • signal sequence cleaved by signal peptidase
  3. Protein released laterally into ER membrane
    • N terminus is in ER lumen
    • C terminus in cytosol
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15
Q

How a transmembrane protein with N-terminus in cytosol gets made

A
  1. The start-transfer sequence in the MIDDLE of polypeptide directs complex to ER
    • start-transfer sequence locks the polypeptide in the translocon in the correct orientation
  2. Protein continues translocation until the c-terminus moves through the translocon
  3. Protein released laterally into ER membrane
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16
Q

How multipass transmembrane proteins get made

A
  1. Strat-transfer sequence starts polypeptide transfer
  2. Protein continues translocation until stop-transfer sequence encountered
  3. Portion of protein released laterally into ER
    • next start-transfer sequence repeats process to initiate second transmembrane region
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17
Q

Import into mitochondrial matrix

A
  • example of post translational import
  • proteins first made in cytosol by free ribosomes, then imported to mitochondria if they have TRANSIT sequence
  • TOM=translocase in OUTER membrane
  • TIM=translocase in INNER membrane
  1. Hsp70 binds to new protein so it stays mostly unfolded
  2. New protein goes to mitochondrion and transit sequence binds to the receptor part of TOM
  3. Hsp70 goes away and new protein moves through TOM and TIM
  4. Transit peptidase cuts off transit sequence
  5. Mitochondrial Hsp70 will help pull the protein through the matrix by binding and not allowing protein to go backwards

-various types of TIMs: some allow protein to go straight through and other will allow protein to go sideways and stay in inner membrane

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18
Q

Golgi apparatus

A
  • camillo golgi 1906
  • proteins and membranes can travel from ER to golgi in vesicles

-compartments of golgi called CISTERNAE

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19
Q

Golgi functions

A
  • the “processing plant” of the cell
  • further protein modifications (glycosylation) and trafficking
  • 3 parts:
    1. CGN
    2. medial cisternae
    3. TGN
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20
Q

Why separate compartments?

A

-different enzymes reside in different compartments to ensure processing happens in an organized, sequential manner

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21
Q

How does cargo (i.e.) proteins move through the golgi

A
  • conflicting views!!!
    1. Stationary cisternae model

VS

  1. Cisternal maturation model
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22
Q

Stationary cisternae model

A
  • cisternae are stable compartments

- cargo gets shipped from compartment to compartment and eventually leaves through the trans face

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23
Q

Cisternal maturation model

A
  • more widely accepted
  • cis cisternae formed by fusion of vesicles coming from ER
  • cisternae mature as they move from the cis face to trans face
  • cargo stays in the same compartment and the golgi-resident enzymes are shipped backwards by retrograde transport to return to the “home” compartment
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24
Q

Evidence of cisternal maturation model part 1

A
  • cargo protein is labelled
  • it seems to only be found in cisternae, never in vesicles
  • therefore can only get there because of maturation, NOT being transported
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25
Q

Evidence of cisternal maturation model part 2

A
  • golgi-resident protein labelled

- see it in cisternae AND vesicles, which are moving in the retrograde directions

26
Q

Evidence of cisternal maturation model part 3

A
  • if you use a mutant cells that doesnt allow vesicles to leave the ER, then eventually the golgi will disappear completely
  • if the cell resumes sending out vesicles the golgi will appear again
27
Q

Post-translational modifications

A
  1. Proteolytic cleavage
  2. Glycosylation
    • N linked
    • O linked
28
Q

Membrane carbohydrates (glycoproteins)

A
  1. N-linked: sugar attaches via ASPARAGINE
  2. O-linked: sugar attached via SERINE or THREONINE
  • ALL carbohydrate side chains added in the ER start off with a common core oligosaccharide
  • core oligosaccharide grows on a lipid called DOLICHOL PHOSPHATE
29
Q

How N-lined oligosaccharides are made in ER

A
  • a core oligosaccharide is made in the ER before it gets moved onto a protein
  • carbohydrate chain will grow on DOLICHOL PHOSPHATE in ER membrane
  • GLYCOSYLTRANSFERASES add monosaccharides to dolichol on CYTOPLASMIC side
  • a flippase will flip dolichol and attached sugar chain so that it faces the lumen of ER
  • more sugars get added
  • Oligosaccharide protein transferase will take the core oligosaccharide off dolichol and add it to asparagine
30
Q

Quality control

A

Process of N-linked glycosylation is important in one type of quality control - making sure the proteins are folded properly before leaving the ER

31
Q

Process of quality control

A
  1. after the core oligosaccharide is added to the protein, 2 of 3 glucoses are removed
  2. CLANEXIN will bind to protein with one glucose on core oligosaccharide to give it time to fold
  3. Once folded, glucosidase II will remove 3rd glucose
  4. If NOT folded correctly, a glycosyltransferase called UGGT will ass a glucose back one
  5. Calnexin will recognize it, bind, and give it another chance to fold
  6. If irreparable, the protein will be recognized by a transporter and fed back out to the cytoplasm via reverse translocation to be destroyed by a proteosome
32
Q

Proteasomes

A
  • barrel-shaped protein degrading machines
  • series of ring like subunits with a cap on either end
  • only digests proteins that have been tagged for destruction via UBIQUITINS
33
Q

Ubiquination

A
  • ubiquitin is a small peptide (8.5kDa)
  • 3 enzymes required to add ubiquitin
    • E1 and E2 are ubiquitin carriers
    • E3 is ubiquitin ligase (recognize misfolded proteins and transfer ubiquitins from E1 and E2 to misfolded protein
  • misfolded protein is now POLYUBIQUITINATED and will bind to cap of proteasome
  • proteases in proteasome break down proteins into amino acids
34
Q

Unfolded protein response

A
  • normally BiP (molecular chaperone) binds to unfolded proteins in lumen, some BiP says in ER membrane bound to sensors
  • if there are too many unfolded proteins, BiP will be recruited from membrane
  • protein sensors do 2 things once abandoned by BiP:
    1. Reduce overall protein synthesis in cell
    2. Increase synthesis of helpful proteins
35
Q

Reduce overall synthesis response

A

-sensors add a PO4 to elF2a, which will bind to small subunit of ribosome and reduce synthesis

36
Q

Increase synthesis of chaperones response

A

-a sensor protein is cleaved and acts as a transcription factor to make more proteins that alleviate ER stress

37
Q

Retention and retrieval sequences

A
  • Arg-any AA-Arg tags are retained in ER
    • not shipped out
  • also known as RxR
  • sometimes still accidentally shipped out
  • retrieval sequence: KDEL (lys-asp-Glu-leu)
    • sent back to ER
  • KDEL receptor proteins in golgi
  • receptor changes into vesicle to take protein back to ER
    • retrograde transport
38
Q

Golgi resident proteins

A
  • sorted by their own retention/retrieval signals and length of membrane-spanning domains
    • not all membrane thickness is the same
  • golgi resident proteins will fit into compartment where membrane width matches width of membrane-spanning domain
39
Q

Lysosomes

A
  • lysosomes = membrane bound organelles important for intracellular digestion
    • break down macromolecules via hydrolysis reactions into their smaller monomers so that cell can use monomers
  • many types of enzymes in lysosome
    • all have optimal activity at an acidic pH (acid hydrolases)
      • pump protons to keep pH low
    • all must be directed through secretory pathway
40
Q

Targeting acid hydrolases to lysosome

A
  • uses sugar tags
    • M6P (mannose-6-phosphate)
  • same process as quality control… but many manoses
  • lysosomal enzymes are glycosylated in ER
  • arrives at golgi, one mannose is phosphorylated making M6P tag
  • M6P receptor binds to any tag it encounters
  • buds off to form vesicle (early endosome)
  • low pH in the late endosome/lysosome dissociated the protein from receptor
  • receptor returned to golgi
41
Q

Movement in and out of cell membrane

A
  • exocytosis and endocytosis
  • ER and Golgi involved in exocytosis
    • electron microscopy tecnique (pulse chase experiment)
42
Q

Pulse chase experiment

A
  • incubate tissue in radioactive amino acids (ie. Pulse)
  • amino acids will get incorporated into newly made proteins
  • rinse extra radioactive AAs off
  • wait… (ie. chase)
  • use radiography film chemicals to cover tissue
  • radioactive AA will react and turn black
  • analyze location of black spots to figure out where protein located
  • do it again to see where proteins go
    • use longer chase periods each time
43
Q

Green fluorescent protein

A

-more modern to follow protein movement

44
Q

Sec mutants

A
  • if you dont know how something works, break a part of it and watch to see what happens
  • randy shekman (2013)
  1. Exposed yeast to low levels of mutagens to cause mutations
  2. Screened yeast to find mutation in secretory pathways (endomembrane system)
  3. Only picked mutants that were heat sensitive
    • normal temps: normal yeast
    • high temps: mutated proteins denature
      - found many proteins involved in sending glycoproteins through secretory pathways
45
Q

Exocytosis

A
  1. Vesicle moves toward plasma membrane
  2. Membranes fuse and PM breaks
  3. Vesicle membrane integrates with PM
  4. Vesicle contents dumped to outside of cell
  • 3 types of exocytosis
    1. Constitutive: constant flow of material out of cell
    2. Regulated: vesicle only fuses when receives signal (hormones)
    3. Polarized: only happens on one side of cell (NTs in neuron)

-when vesicle fuses, proteins in exoplasmic leaflet (facing lumen) remain in that exoplasmic leaflet so then face outside of cell

46
Q

Endocytosis

A
  1. PM invagination, forming pocket of materials
  2. Pocket begins to pinch off, enclosing materials
  3. Membrane closes to form vesicle
  4. Vesicle separates

-vesicle derived from PM, therefore lipids and membrane proteins of PM are brought into cell

47
Q

Phagocytosis

A
  • example of endocytosis
  • engulfment of large particles
  • how unicellular cell eats
  • in complex organisms involved in immune system

-during phagocytosis vacuole fuses with lysosome and hydrolysis enzymes break down contents

48
Q

Receptor mediated endocytosis

A
  • extracellular materials bind to receptors on PM in regions called coated pits
  • requires clathrin (coat protein), adaptor proteins, and dynamin
  • for particular solutes that need to be brought into cell
  1. Ligand binds to coated pits to form receptor-ligand complexes
  2. Lateral diffusion across PM
  3. Clathrin coated pit invaginates
  4. Dynamin pinches vesicle off to form coated vesicle
49
Q

Vesicle formation

A
  • coat proteins act as mechanical device that assemble to produce a force which will then curve the membrane until it forms a bubble
  • coat proteins very selective about what components will get included in vesicle

-clathrin: coat protein
-COPI: vesicles that leave Golgi
COPII: vesicles that leave ER
Caveolin: cholesterol uptake

-coat proteins always form on cytosolic face

50
Q

Clathrin coated vesicles

A
  • forms triskelion structures
    • made from 3 light chains and 3 heavy chains
  • overlap with eachother and cause vesicle to form soccer ball shape
  • clathrin also needs adaptor proteins to form connected between membrane receptors and clathrin coat
    • AP2 and GGA
51
Q

AP2

A
  • forms a connector between membrane receptor and clathrin coat
  • vesicles coming in from plasma membrane
52
Q

GGA

A
  • connecter between receptor and clathrin coat
  • vesicles leaving golgi to go to lysosomes
    • M6P receptor (MPR)
53
Q

Dynamin

A
  • GTP binding protein
  • pinches vesicle off from membrane
  • forms ring around stalk of vesicle
  • hydrolyzes GTP which causes rings to tighten and squeeze off vesicle

-when GTP cannot be hydrolyzed, vesicle never pinches off… dynamin just keeps being added to be elongated

54
Q

COPI

A
  • retrograde transport (backwards)
    • golgi back to ER
    • trans golgi to cis golgi
  • coats made of COPI proteins and small GTP binding protein called ARF
  • ARF in cystol
    • when it binds GTP is inserts into membrane and recruits COPI proteins forcing curvature of membrane
    • after vesicle is formed, GTP hydrolyzes and COPI dissociates
55
Q

COPII

A
  • anterograde transport
    • ER to golgi

-coats made of COPII + Sar1 (small GTP binding protein)

  • Sar1 in cytosol, binds GTP, inserts into membrane and recruits COPII proteins (Sec13, Sec31, Sec 23, Sec24)
    • recruits Sec23+24 first, and then Sec13,31 to complete complex

-after vesicle formed, GTP hydrolyzes and COPII dissociates

56
Q

COPII coated vesicles summary

A
  • Collect proteins that are destined to leave ER
  • ER export signals interact specifically with COPII proteins of the vesicle coats
  1. Sar1 binds GTP and becomes activated
  2. Hydrophobic tail swings out of Sar1 and enters lipidbilayer which starts to curve membrane
  3. Sar1-GTP recruits 4 COPII polypeptides to form coat
  4. Vesicle buds off
  5. Sar1 hydrolyze GTP and coat falls off
57
Q

Summary of coating vesicles

A
  1. clatherin coated vesicles:
    • trans golgi —>out
    • PM —>endosomes
  2. COPI coated vesicles
    • retrograde
      • golgi —> ER
      • trans golgi —> cis golgi
  3. COPII coated vesicles
    • anterograde
      • ER—> golgi
58
Q

Targeting vesicles to specific compartments

A

-SNARE hypothesis

  • Rabs=small, GTP binding proteins, specify vesicle destination
    • 60 types of rabs depending where it has to go
    • rabs associate with membrane via a lipid anchor
    • rabs recruit tethering proteins to loosely attach the vesicle
    • bring SNARES in close proximity
  • targeting and tethering
  • SNARES= membrane proteins that mediate vesicle fusion
    • t-snares = on target membrane (SNAP25 + Syntaxin)
    • v-snares on vesicle membrane (synaptobrevin)
  • involved in docking
  • have alpha helices domains that coil together tightly to bring membranes into close proximity
    • due to closeness membranes fuse together
  • fusion of membranes is spontaneous
  • dissociation of proteins requires energy
59
Q

NSF

A

-required to dissociate SNARES

  • botulin toxin chops up SNARE proteins
    • vesicles cannot fuse
    • paralysis caused due to no NT release in NMJ
60
Q

Autophagy

A
  • destruction of old/sick organelles by isolation in a double membrane vesicle
    • followed by fusion with a lysosome
  • Yoashinori Ohsumi 2016
    • Nobel prize
  • organelle is wrapped in double membrane from ER
    • called autophagosome once encapsulated
    • fuse with lysosome
    • broken down and recycled
    • some indigestible (residual body)

-residual body build up contribute to aging

BIOL 2020 (8 decks)

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