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

Question 1

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

what mediates this?

Answer 1

>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

Question 2

what does transport within secretory pathway?

Answer 2

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

Question 3

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

Answer 3

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

Question 4

what is direction of protein movement in endocytosis?

Answer 4

outside of cell -> inside of cell

Question 5

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

examples?

Answer 5

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

albumbin, IGG, lipoproteins

collagen, fibronectin

Question 6

what are examples of proteins that undergo regulated secretion?

Answer 6

1) peptide hormones: e.g. insulin, glucagon
2) digestive enzymes: e.g. trypsin
3) milk proteins: e.g. caesein, lactalbumin

Question 7

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

Answer 7

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

Question 8

what % of internal cell membranes does ER constitute?

Answer 8

50%

Question 9

what are the different types of ER / their functions?

Answer 9

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

Question 10

why is the rough ER rough?

Answer 10

b/c studded w/ ribosomes

Question 11

what is the nature of proteins in the ER?

Answer 11

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

it is the quality control compartment for proteins

Question 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?

Answer 12

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

Question 13

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

Answer 13

1) nucleus
2) mitochondria
3) peroxisomes
4) secreted or exist in membrane

Question 14

how does a cell know where a protein will go?

Answer 14

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

Question 15

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

Answer 15

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

often cleaved after import into the ER

Question 16

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

Answer 16

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

Question 17

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

Answer 17

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

Question 18

what is the translocon?

Answer 18

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

Question 19

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

Answer 19

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

Question 20

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

Answer 20

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

Question 21

what happens to signal peptide/signal sequence after translocation?

Answer 21

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

Question 22

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

Answer 22

hydrophobic, 20 a.a. long, alpha helical

Question 23

how do membrane-bound proteins interact w/ translocon?

Answer 23

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

Question 24

what is a Type I membrane protein?

Answer 24

membrane protein

N terminus: in lumen

C terminus: in cytosol

Question 25

what is a type II membrane protein?

Answer 25

N terminus: in cytosol

C terminus: in lumen

Question 26

what is a complex membrane protein?

Answer 26

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

Question 27

what is function of memrbane spanning domains of membrane proteins?

Answer 27

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

Question 28

what % of drugs bind to GPCRs?

Answer 28

50%

Question 29

what is a GPI-linked anchor?

Answer 29

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

Question 30

what happens to proteins entering the ER lumen?

Answer 30

1) signal peptide is cleaved
2) protein gets glycosylated
3) new protein binds to ER-specific chaperones and folding enzymes

Question 31

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

how does this addition work?

Answer 31

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

Question 32

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

Answer 32

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

Question 33

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

Answer 33

ER-specific form of molecular chaperones Hsp70 and Hsp90

Question 34

what is calnexin? function?

Answer 34

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

Question 35

what does folding of proteins in ER depend on?

Answer 35

1) chaperones
2) folding enzymes

Question 36

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

Answer 36

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

Question 37

what is protein disulphide isomerase? what does it do?

Answer 37

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

Question 38

what is stress to the ER?

Answer 38

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

Question 39

what happens in type II diabetes re: ER stress?

Answer 39

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

Question 40

how does protein deal w/ stress?

Answer 40

1) activation of the UPR, unfolded protein response
2) ER-associated degradation (ERAD)

Question 41

what is the UPR?

Answer 41

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

Question 42

what is ERAD? how does it work?

Answer 42

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