LEC33, 34: The Secretory Pathway & Endocytosis (Part A: Overview & Part B: The ER) Flashcards Preview

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Flashcards in LEC33, 34: The Secretory Pathway & Endocytosis (Part A: Overview & Part B: The ER) Deck (43):
1

what % of proteins end up in membranes or secreted from cell? 

what mediates this?

>30% of proteins 

secretory pathway; via the ER for protein folding and QC, Golgi for sorting proteins to diff parts of cell, and Lysosome for recycling cellular materials that can be engulfed by membrane invagination

2

what does transport within secretory pathway?

lipid vesicles that bud from 1 type of membrane & fuse w/ another

3

what are the different types of secretion that new proteins that leave golgi may be destine for? examples of each?

1) constituitive secretion: go straight to plasma membrane 

2) regulated secretion: stay packages into vesicles for a bit then undergo secretion (i.e. hormone signal, neurotransmitter) 

3) internal destinations: to endosome and then lysosome 

these are all movements from inside cell -> outside

4

what is direction of protein movement in endocytosis?

outside of cell -> inside of cell

5

what's the function of proteins that undergo constituitive secretion? 

examples?

proteins w/ house keeping functions that're constantly made by specialized cells, secreted all the time 

albumbin, IGG, lipoproteins 

collagen, fibronectin 

6

what are examples of proteins that undergo regulated secretion?

1) peptide hormones: e.g. insulin, glucagon

2) digestive enzymes: e.g. trypsin

3) milk proteins: e.g. caesein, lactalbumin

7

how do we know that secretory pathway begins in the ER? describe experiment

used radioactive leucine that represented pulse in culture 

at end of 3 min, chased radioactive leucine w/ cold leucine, saw as it became incorporated in different parts of cell 

did x-ray imaging of cells 

saw after 3 min, radioactive labeling was all in ER; after 7, in golgi; after 2 hrs, proteins in sercretory vesicles to be realsed out of cell 

THUS ER = first place newly made proteins go

8

what % of internal cell membranes does ER constitute?

50% 

9

what are the different types of ER / their functions?

1) smooth ER: specailized ER, contains membrane-bound enzymes important for lipid synthesis and metabolism; detoxifying enzymes esp for liver i.e. cytochrome p450s

2) rough ER: contains ribocomes, is a protein-folding compartment

10

why is the rough ER rough?

b/c studded w/ ribosomes

11

what is the nature of proteins in the ER?

proteins that'll be secreted from the cell or exist in membranes fold in the ER 

it is the quality control compartment for proteins 

12

what is the difference between the outside and inside of cell's environments? how does the ER help with this change to-come for proteins?

outside of cell: oxidizing environment 

inside of cell (cytosol): reducing environment

proteins going outside cell need to be prepared for the oxidizing envinroment 

thus the ER = an oxidizing environment; proteins fold in the ER within this oxidizing environment, which prepares them for the outside cell environment later

13

when a ribsome translates, where can protein it creates be targeted to go?

1) nucleus

2) mitochondria

3) peroxisomes 

4) secreted or exist in membrane

14

how does a cell know where a protein will go?

protein sorting: each individual protein has targeting sequences that can be anywhere in the protein; these stretches say where protein is supposed to go 

15

what's the protein targeting sequence for new proteins targeted ot the ER membrane?

N-terminal signal peptide of 8-20 residues enriched in hydrophobic amino acids 

often cleaved after import into the ER 

16

what is the signal peptide? what usually happens to it? 

signal peptide: on the new protein; 8-20 residues, hydrophobic a.a., often cleaved after import into the ER. 

if protein has internal targeting sequence, not cleaved after import

signal peptide binds to the signal recognition particle (SRP)ribonucleoprotein complex that attaches to newly-synthesized proteins while they're being translated

17

what happens when SRP (signal recognition particle) binds to a signal peptide? describe sequence of events

co-translational translocation

1) recognition: binding btwn signal recognition protein and new a.a. coming out of ribosome 

2) arrest of translation: SRP binding to signal peptide arrests translation - physically - b/c it binds to translation center of ribosome and stops it in its tracks 

3) complex of translating ribosome and SRP bind to ER membrane via SRP receptor complex 

SRP receptor sits adjacent to translocation channel, the translocon, an aqueous channel where translation can resume directly into ER lumen 

4) SRP dissociates b/c it binds to SRP-receptor in cell membrane; SRP dissociates; translation resumes in lumen of the ER, while signal peptide remains stuck in translocon and rest of protein loops in to  ER lumen

18

what is the translocon?

an aqueous translocation channel 

adjacent to the SRP receptor, so when SRP w/peptide binds to SRP receptor, translation resumes through the translocon channel, into the lumen of the ER 

 

19

what is the nature of the translocon channel, for membrane proteins?

channel opens sideways, into plane of the membrane, and membrane-spanning domains of proteins become inserted into membrane itself

20

what does co-translational translocation enable for the new protein?

if want to have particle that sequesters all nascent chains of polypeptides, helpful b/c takes ribosome to membrane, and allows translation to resume in context of translocation 

peptides are exposed to oxidizing ER environment this way which is crucial for proteins destined for outside of cell

21

what happens to signal peptide/signal sequence after translocation?

it remains embedded in the membrane of the ER b/c it was inserted directly into translocation channel 

cleavage by signal peptidase 

polypeptide is released into lumen of the ER, and cleaved signal peptide remains in membrane

22

what is the nature of the membrane-spanning portion of a membrane-bound protein?

hydrophobic, 20 a.a. long, alpha helical 

23

how do membrane-bound proteins interact w/ translocon?

translation resumes on ER membrane until membrane spanning domain hits the translocon

then, b/c that domain is hydrophobic, it triggers a door in translocon to open, and protein swims into plane of the membrane so N and C termini each are either in cytosol or lumen of ER

24

what is a Type I membrane protein?

membrane protein 

N terminus: in lumen

C terminus: in cytosol 

 

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25

what is a type II membrane protein?

N terminus: in cytosol 

C terminus: in lumen 

 

26

what is a complex membrane protein?

protein w/ multiple membrane-spanning domains comprising hydrohobic amino acids that form 20-25 a.a. residues 

27

what is function of memrbane spanning domains of membrane proteins? 

help either inhibit or promote translation of the loops of the protein into the lumen of the ER/the cytosol outside

act as "start transfer" and "stop transfer" sequences 

28

what % of drugs bind to GPCRs?

50%

29

what is a GPI-linked anchor?

glycosylphosphatidyl inositol anchor 

post-translational modification to anchor proteins to apical membrane of epithelial cells 

protein is transferred to the anchor post-translationally rather than having a true membrane-spanning domain

allows much faster diffusion of membrane epithlial cells 

 

A image thumb
30

what happens to proteins entering the ER lumen?

1) signal peptide is cleaved 

2) protein gets glycosylated 

3) new protein binds to ER-specific chaperones and folding enzymes 

 

31

what is signal for N-linked glycosylation of a protein emerging in to the lumen of the ER?

how does this addition work?

sequence that is Asn-x-Threonine/Serine

signals addition of complex carbohydrate moiety of 14 residues 

occurs via linkage to epsilon-amino group of Asn 

follwed w/ 2 residues of N-acetylglucosamine (GlcNac), 9 residues of mannose, 3 residues of glucose 

glucose residues have important roles in protein folding

32

why does N-linked glycosylation of some proteins entering ER lumen occur?

1) important for protein folding 

2) stabilizes proteins it's added to

3) can act as recognition signal for when peptide is outside the cell 

4) has role in cell-cell adhesion

 

33

what aids proteins protein folding for proteins entering the ER lumen?

ER-specific form of molecular chaperones Hsp70 and Hsp90

 

34

what is calnexin? function?

specialized form of a chaperone that binds N-linked glycosylated protein in the ER while it's folded

1) binds to final glucose unit of glycosylation complex carbohydrate moiety, keeps the protein in the ER until it's folded 

2) once folded, signal allows final glucose to be removed 

3) when glucose is removed, folded protein is detached from calnexin, and then conitnues on secretory pathway

35

what does folding of proteins in ER depend on?

1) chaperones 

2) folding enzymes

36

what is peptidyl-prolyl isomerase? what does it do?

enzyme that changes the configuration of proline in loop region of a protein 

changes proline from cis to trans configuration, flipping protein 180 degrees, speeding up rate of folding reaction 

 

A image thumb
37

what is protein disulphide isomerase? what does it do?

aids folding for proteins that form disulphide bonds, commonly proteins that exist outside of cell but not for intracellular proteins

proteins w/ Cysteine residues become disulfide bonds when in the ER b/c it's an oxidizing environment 

S-S- bonds thus form in the ER 

but PDI works to rearrange disulfides once they've arranged in the ER so they are in the cis configuration

 

38

what is stress to the ER?

when level of unfolded protein exceeds the available # of chaperones to fold those proteins = stress; can be toxic accumulation of misfolded proteins that aggregate 

39

what happens in type II diabetes re: ER stress?

type II diabetics' pancrease produces massive quantity of insulin

however, insulin produced is not funcitonal 

so sugar stays in blood, and signal goes back to pancreas to make mroe insulin 

ER thus gets very stressed 

pancreas can undergo programmed cell death b/c of stress

and level of msifolded protein in ER is so high that cells, esp insulin, completely exceeds number of chaperones available to fold the protein

40

how does protein deal w/ stress? 

1) activation of the UPR, unfolded protein response 

2) ER-associated degradation (ERAD) 

41

what is the UPR?

signaling molecules in membrane of ER that're very sensitive to levels of misfolded protein 

when activated, results in expression of genes that encode ER-specific molecular chaperones & components of ubiquitin/proteasome pathway - so proteins can be destroyed 

however proteins must be translocated back to cytosol for destruction; are not destroyed in ER 

gene expression of ERAD components increase during UPR

42

what is ERAD? how does it work?

ER-associated degredation 

proceeds after UPR 

if UPR is massively unregulated, cell goes into suicide program, ERAD 

luminal and membrane proteins are retrotranslocated from ER to cytosol for degradation by the proteasome in ERAD 

 

43

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