LEC34: The Secretory Pathway and Endocytosis (Part C: The Golgi Apparatus & Pathways for Protein Secretion)

Question 1

how do proteins that fold in ER move to Golgi?

Answer 1

packaging proteins from the ER into vesicles

vesicles bud, are targeted to right organelle, and fuse w/ membrane of Golgi

Question 2

what is the structure of the Golgi apparatus?

Answer 2

stack of flattened membranous disks that have distinct curved appearance

curvature creates polarity w/ a “cis” convex face and “trans” concave face

Question 3

what happens to proteins as they move through the golgi?

which part of golgi does what?

Answer 3

they move and they’re modified via post-translational modification; golgi does transit and modification

cis stack: phosphorylation

medial stack: modifications to carbohydrates, i.e. cutting manose units of carbs that were added as single unit in ER

trans stack: sulphation of tyrosines

Question 4

what is function of PTMs that occur to proteins in the golgi?

Answer 4

1) increase complexity of proteins traveling through the Golgi
2) specify subsequent localization to other membrane systems

Question 5

how do proteins get transported from the ER to the Golgi?

Answer 5

in lipid vesicles that bud from teh ER membrane, are targeted to teh Golgi, and fuse at Cis side of Golgi

Question 6

what is the ER retrieval pathway for?

how does it work?

Answer 6

when proteins go from ER to Golgi, some of the chaperones & folding enzymes that worked on protein in ER go w/ vesicles to the Golgi, and have to be returned to ER via this pathway

operates via a receptor that recognizes peptide seuqnce of **KDEL ** at c-terminus of resident ER proteins that’ve escaped to the Golgi

in cis face of Golgi, KDEL receptor interacts w/ these KDEL sequences, sequester, and return chaperones to the ER in vesicles

Question 7

where in the cell does the Golgi sit?

Answer 7

the centrosome

Question 8

what does glycosylation of proteins in the ER do?

Answer 8

gives proteins added level of complexity, easier to recognize

Question 9

what are the models that explain how proteins that go through stacks of the Golgi maintain their identity, if diff biochemical pathways are occurring in each stack of Golgi?

Answer 9

1) vesicular transport model: vesicles budd and fuse from stacks; proteins bud and fuse in those vesicles as they move from 1 stack to another

but this is discounted model, b/c some of large protein assemblies in ER are too big to go this way

2) cisternal maturation model: stacks themselves are consistently moving as they reach the transgolgi network, enzymes that do these rxns are packaged into vesicles, move backwards to proper destination in cis golgi

Question 10

what is the lysosome?

Answer 10

single-membrane organelle that does recycling

comprises hundreds of different enzymes that chew things up

lysosomal enzymes destined for lysosome start in the ER

pH ~5

Question 11

what is unique about enzymes destined for the lysosome?

Answer 11

synthesized in the ER

sorted in the Golgi

these enzymes = 2 units of glucose, 8 of manose; a phosphotransferase adds a single phosphate to a manose unit while in the cis golgi

marks lysosomal enzymes in the golgi

Question 12

how to lysosomal enzymes get from the cis golgi -> lysosome?

Answer 12

phosphatase adds a phosphate onto a manonse subunit of lysosomal enzymes while they are in the cis golgi

once in the trans golgi, enzyme is connected to a manose-6-phosphate receptor that connnects to the phosphate added onto the lysosomal enzyme

M6P receptor gets bound in a vesicle containing the enzyme; goes to endosome, fuses w/ it

Question 13

how does lysosomal enzyme get delivered from the trans golgi network to the lysosome? describe steps, pH changes

Answer 13

1) M6P receptor binds the phosphate attached to the lysosomal enzyme while it is in trans golgi network
2) M6P gets bound in a vesicle w/ lysosomal enzyme

this goes to endosome, fuses w/ it

3) pH of trans golgi network = 6; pH of endosome = 5.5

this is 5x difference

so when vesicles fuse w/ endosome, pH decrease interferes w/ binding of the lysosomal protein to M6P receptor

4) 2 kinds of vesicles are created:

M6P receptor is removed, bound to vesicle, and transported *usually *back to the trans-golgi network, but several vesicles w/ M6P receptors go to the plasma membrane

protein destined for lysosome are put into vesicles and sent to lysosome, where pH=5 so lysosomal enzymes get dumped once get there

Question 14

what are the pH changes in the lysosomal enzyme delivery system?

Answer 14

pH trans golgi network = 6, M6P binds to lysosomal enzyme’s phosphate

pH endosome = 5.5, allows dissociation of lysosomal enzymes into vesicles and M6P receptors to other vesicles

pH lysosome = 5, lysosomal proteins get delivered there

Question 15

what are lysosomal storage diseases caused by?

Answer 15

missing an enzyme that breaks down glycolipids

so glycolipid is stored, cannot be broken down

i.e. Gaucher’s Disease, Fabry’s Disease

Question 16

what causes Gaucher’s disease?

what is gene therapy for it?

Answer 16

mutation in **glucocerebroside **which breaks down glycolipids

causes glycolipids’ accumulation

gene therapy method: purify glucocerebroside from a mammalian cell in culture, add back to the blood via infusion of purified protein that has M6P and carbs

glucocerebroside gets into cells b/c some errant M6P receptors go to plasma membrane, capture the purified glucocerebroside, take it back into cell via endocytosis

Question 17

what causes fabry’s disease?

what is gene threapy for it?

Answer 17

caused by alpha galactosidase (alpha gal-a) mutation due to mistolding of proteins in the ER

proteins misfolded in the ER will be destroyed by proteasome in cytosol, have no Alpha Gal A

so work w/ DGJ, a competitive inhibotor that binds strongly to alpha Gal-A at pH=7, in the ER; carries alpha gal-A from ER -> Golgi -> lysosome via M6P receptor pathway, and once at lysosome of pH=5, compound falls off enzyme so enzyme now has free active site

this takes advantage of protein folding & competitive inhibitors

Question 18

what happens in the final processing step for proteins that undergo regulated secretion?

describe case of insulin as example of peptide hormone that undergoes regulated secretion

Answer 18

peptide hormones (i.e. insulin) are synthesized as larger precursors, have to be trimmed down before proteins are biologically active

this occurs in vesicles for these peptide hormones

precursor of insulin has a PRO region

when insulin protein precursor is in ER, **signal peptide cleavage **occurs so protein travels through ER and Golgi complex, then to trans golgi network, packaged into secretory vesicle

secretory vesicle contains both the PRO insulin and 2 enzymes that cleave the PRO region out

insulin thus matures in the secretory vesicle; **disulfide bonds **form in the vesicle right before insulin’s secreted

insulin thus exits vesicle in disulfide-bonded form that’s highly concentrated (b/c also have enzymes in vesicle that take out water from insulin)

Question 19

what is the PRO region of a peptide precursor

Answer 19

region that is cleaved out while in vesicle, does not become part of active protein

Question 20

what are examples of acid hydrolases? - lysosomal enzymes?

Answer 20

nucleases, proteases, glycosidases, lipases, phosphatases, sulfatases, phospholipases

Question 21

What are the functions of N-linked glycosylation of proteins entering the ER?

Answer 21

1. It is important for protein folding
2. Important for protein stability - increases the stability of proteins that it’s added to
3. Outside the cell, can act as recognition signals - adds something to the protein that’s complex that’s different from the peptide sequence itself and has a recognition quality
4. Has roles in cell-cell adhesion