module 5 Flashcards
question: what’s the difference in func. between RER and SER?
RER
- co-translational transport
- prot. mod.
- formation of vesicles that will transport prot. from ER to golgi
SER
- fatty acid and phospholipid synthesis
- place of carb. metabolism
- place where calcium is sequestered or collected to regulate calcium conc. in the cytosol
question: what are the post-translational mod. that can happen in the ER? (4)
- glycolysation
- forming disulphide bonds
- prot. folding
- proteolytic cleavage
question: what is protein glycolysation?
- adding polysaccharide/sugar
question: what is N-linked glycolysation
- adding polysacch. to NH2 of asparagine
- modified part of the prot. stays on luminal side throughout transport
question: what are disulphide bonds?
- covalent bonds between SH groups of 2 cysteine AA
- help form tertiary or quaternary struc.
- give stability
question: where do disulphide bonds occur?
- in ER lumen
- ER lumen = oxidizing envrt. = good for disulfide bonds (cytoplasm envrt. = opposite)
question: what is the purpose of disulphide bonds?
- common prot. on outside surface of cell mem.
- bc more harsh and denaturing conditions outside
- so bonds can provide more stability
⤷ help prot. stay folded
explain: example of prot. with disulphide bridges
- pancreatic ribonuclease A (RNAse A)
- has 4 disulfide bridges
- acidic conditions of intestine cause prot. to unfold but disulfide bonds help maintain struc.
question: what is protein disulphide isomerase?
- resident ER prot.
- promotes oxidation = good for disulphide birdge formation
- can also correct incorrect disulphide bridge formation
question: how does PDI help form disulphide bonds?
- PDI forms intermediate w/ cysteine AAs to accelerate rxn
- oxidized PDI has a disulphide bridge
- PDI forms intermediate w/ one of the cysteines
⤷ facilitates bond forming - PDI converts back to oxidized form in ER lumen
question: what are lectins?
- recog. mod/ proteins and assit prot. folding
- like chaperones
- ex. calnexin and calreticulin
⤷ calnexin in ER mem.
question: what is BiP?
- ER resident prot.
- can recog. and bind to unfolded prot.
- binds prot. when they appear on luminal side of ER during co-translational transport
question: what is proteolytic cleavage?
- cleavage of peptide backbone of a prot.
- ex. all type I integral mem. prot. have N-term. sig. seq. cleaved by signal peptidase
question: what is the unfolded prot. response for?
- when RER = overwhelmed by unfolded prot.
- bc unfolded prot. can’t leave until properly folded
question: how would the UPR respond?
- 2 resp.:
1. cell tries to restore normal cell func.
⤷ slowing down prot. translation
⤷ removing unfolded prot. for degradation
- increase prod. of chaperones to help fold
question: what prot. are essential in UPR in ER + why?
BiP
- BiP serves as chaperone to assit in folding + prevent aggregation
Ire1
- forms homodimers in ER mem.
⤷ only when active (not bound to BiP)
- serve as activated endonucleases
⤷ makes cuts in nucleic A
question: how do endonucleases work (explain w/ Hac1)?
- make internal cuts in nucleic acids ex. mRNA
- Ire1 specifically targets Hac1
- Hac1 = spliced to remove translation inhibition and allow synthesis of Hac1 prot.
- Hac1 prot = transcription factor to activate transcription for BiP, lectins, PDI, signal peptidases
explain: pulse-chase experiment
- tagging prot. for short period time
- only some prot. labeled
- acinar cells (pancreas) incubated in a medium w/ radioactive methionine that would only be incorporated as they are translated into RER
- cells removed after 3 mins, washed and moved to new medium w/out radioactive
- chase gets visualized to see diff. stages from RER to apical surface of cell
question: what were the results of the pulse-chase experiment?
- found that transport order:
⤷ radioactive prot.
⤷ through golgi complex
post. golgi transport vesicles
⤷ secretory vesicles
question: what were the results of the VSV TEM and GFP?
- vesicular stomatitis virus G-prot = tagged w/ GFP -> VSV-G:GFP prot.
- at permissive T: prot. variant folds
- at restrictive T: prot. variant denatures + stays in ER by UPR
INFECT CELLS
- at permissive T: GFP folds and transports out of ER
- at restrictive T: GFP retained in ER
question: what is the yeast saccharomyces cerevisiae system for prot. transport?
- S.cerevisiae metabolizes sucrose by hydrolysis from prot. = invertase (secreted by yeast)
- hydrolysis prod. glucose and fructose
- gluc. + fruc. moved into cell to feed it
question: what was the experiment on yeast prot. transport?
- random mut. in yeast genomes
- looked for temp. sensitive mut. that failed to secrete invertase at restrictive T
- causes invertase to accumulate
question: what was the result of the experiment on yeast prot. transport?
- invertase accumulated at diff. regions depending on which parts of pathway were defective
INTERTASE IN CYTOSOL ONLY
- means defect in first step of prot. transport
⤷ co-translational transport to ER
- mut. to any part of ER translocon, SRP prot., SRP recep.
question: what are the classes of secretory mutants that cause prot. to accumulate? (5) + where do they cause invertase to accumulate?
- class a mutants
⤷ accumulate in cytosol - class b mutants
⤷ accumulate in ER - class c mutants
⤷ accumulate in ER to golgi transport vesicles - class d mutants
⤷ accumulates in golgi - class e mutants
⤷ accumulates in secretory vesicles
question: what is the pathway for prot. from RER to cell mem.?
- prot. leave RER in vesicles
- through ER -> golgi complex -> cell mem
**from ER, can go towards cis-cisternae or away from trans-cisternae
- trans = constitutive and regulated secretory
question: what is the golgi complex composed of?
- long flat sacs = cisternae
- can be cis or trans cisternae
question: what’s the diff. between constitutive and regulated secretory pathways?
CONSTITUTIVE
- for prot. released immediately after synthesis and transport
- move from trans-golgi to cell mem.
REGULATED
- for prot. that are kept in cell until sig. trigger
- held in secretory granules
define + explain: anterograde transport
- mvt. of prot. from RER to cell mem.
- forward direction (away from nucleus)
- correct model: prot. stay in cisternae and cisternae move through golgi complex
question: what is the cisternal maturation model/cisternal progression model?
- model B
- prot. stay in cisternae
- cisternae themselves move
question: why was model A favoured over B for anterograde transport?
- A = vesicles carry prot. and move from cis to med. and med. to trans
- B = prot. stay in cisternae and cisternae move
B correct bc:
- prot. not in vesicles
- prot. found on med. golgi
⤷ and if cisternae move, med.eventually becomes trans
question: what are the steps to vesicular trafficking? (4)
- budding
⤷ vesicles form
⤷ buds arise from mem. of donor compartment - loading
⤷ cargo prot. loaded into buds - release
⤷ vesicles form + release - docking + fusion
⤷ fuse to mem. of recipient
question: what are the vesicles used in prot. transport?
- clathrin coated
- COPI coated
- COPII coated
question: when are each type of vesicle required in the vesicular transport process?
- clathrin for transport away from trans-golgi to endosomes and cell mem.
- COPI for retrograde (from golgi to ER)
- COPII for transport from RER to cis-golgi
explain: vesicle budding in COPII vesicles
- 1st step
- Sar1-GDP = inactive cytosolic prot.
- Sec12 = transmem. prot. of donor (ER)
- Sec12 = GEF so it facilitates GDP -> GTP on Sar 1
⤷ activates Sar1-GTP - reveals N-term of Sar1 that anchors it to mem. of ER
- COPII accumulates on donor mem. compartment
⤷ amount dep. on ability to bind w/ Sar1
explain: cargo loading in COPII vesicles
- 2nd step
- bud = curved
- cargo natually accumulates
- cargo recep. accumulate in bud
⤷ bc coat prot. interacting w/ cytosolic domains of recep. of cargo
explain: vesicle release in COPII vesicles (w/out uncoating)
- 3rd step
- GTP hydrolysis turns Sar1-GTP -> Sar1-GDP
- releases Sar1 and coat prot. (uncoating)
- uncoated vesicle = loaded w/ cargo
- motor prot. recog. uncoated vesicle
- moved to recipient mem.
question: what would happen if uncoating was prevented? how is uncoating prevented?
- collection of accumulating coated vesicles
- prevents vesicle transport, docking, cargo unloading
⤷ coat is blocking req. prot. - adding non-hydrolyzable GTP or mut. of SarGTP prevents uncoating
question: how is a vesicle uncoated?
- clathrin coat = lattice
- dynamin releases coat
- GTPase hydrolyzes GTP -> GDP -> dynamin changes shape
- release via pinchase or poppase
question: what are the poppase and pinchase models and how do they matter in uncoating?
- poppase = dynamin elongates + pushes vesicle away
- pinchase = dynamin contricts and squeezes mem. to initiate release
- both modles have evi. to be “correct”
explain: basics of fruit fly vesicle model
- vesicle formed during endocytosis at cell mem. of presynaptic neural cell
⤷ assisted by dynamin - shibire gene codes for dynamin
- shibire = temp. sensitive
question: what happens to dynamin at diff. temps.? what happens to the fly?
- permissive T = dynamin expressed, folded, functional
- restrictive T = dynamin denature, nonfunctional (no vesicle formation)
- restrictive T = fly paralyzed
explain: vesicle docking and fusion in secretory vesicles
- Rab GTPase controls vesicle docking
- Rab-GDP = free in cytosol
- Rab-GTP = bound to vesicles
- Rab on vesicle mem. assoc. w/ receptor on target mem.
- Rab-GTP can interact and bind to Rab effector
- mem. need to fuse for cargo to be released into recipient compartment
question: how is vesicle fusion mediated?
- by mem. anchored prot. helices (SNARE prot.)
- vesicle SNAREs (v-SNAREs) = anchored to vesicle mem.
⤷ ex. VAMP - target SNAREs (t-SNAREs) = anchored to target mem.
⤷ ex. syntaxin, SNAP25
explain: vesicle fusion (w/ SNAREs)
- SNARE complex forms + pulls mem. together
- like a ball sitting on a sheet of paper
- hole is created simultaneously through both allow mvt. of contents into target
- OG mems. don’t reseal, instead reseal together as 1 mem.
question: what makes up a SNARE complex?
- 4 helices
- 2 from SNAP25
- 1 from syntaxin
- 1 from VAMP
**helices from target mem. and vesicle mem. weave together and pull mem. close
question: how is the SNARE complex disassembled and why?
- to reuse prot.
- NSF and alpha-SNAP prot. assoc. w/ SNARE
- unwinds helices
question: what is the purpose of retrograde movement?
- if prot. incorrectly brought to golgi from ER
⤷ ex. ER resident prot. - SNARE needs to be recycled to ER
- COPII recep. need to be returned
- unfolded prot. need to be returned
⤷ to be folded, modified, translocated out for degradation
question: how is a resident ER prot. recognized for retrograde?
- look for specific signal to get loaded onto COPI vesicles
- KDEL seq. = lysine, aspartic acid, glutamine, leucine
- lysine, lysine, xx
- asparagine, x, glutamine
question: where is each signal for retrograde found?
- KDEL on soluble ER resident prot.
- KKxx on resident ER mem. prot.
- DxE on cargo acceptor from COPII vesicle
