Endomembranes I: Moving Proteins and Membranes in the Secretory Pathway (L16)

Question 1

unifying principle of vesicle trafficking involves what four things?

Answer 1

1. cargo selection
2. vesicle budding from one compartment
3. transport
4. fusion with a second compartment

Question 2

how can protein transport through the secretory pathway be visualized?

Answer 2

fluorescence microscopy of cells producing a GFP-membrane fusion protein

Question 3

detection of compartment-sensitive oligosaccharide modifications?

Answer 3

transport of a membrane glycoprotein (VSVG) can be assayed by sensitivity to endoglycosidase D

Question 4

cell-free assays for intercompartmental protein transport?

Answer 4

isolated golgi stacks from uninfected WT and infected mutant cell lines are combined in the test tube -> then VSVG is glycosylated

• normally, VSVG is modified in the golgi by GlcNAc transferase
• mutant cells do not perform this rxn

Question 5

what three general ideas are necessary for cargo selection and budding?

Answer 5

1. mechanical force: scission or propulsion/motility
2. target recognition: Rab GTPases
3. membrane fusion: SNAREs

Question 6

what does coat assembly drive?

Answer 6

vesicle budding and selection of cargo

Question 7

three major classes of vesicle coats

Answer 7

COPII
COPI
clathrin and adaptor proteins

Question 8

what are the coats associated with secretory vesicles (TGN -> PM)?

Answer 8

unknown

Question 9

two types of small GTPase associated w/ coating?

Answer 9

SAR1 and ARF - members of Ras GTPase superfamily

Question 10

what are SAR1 and ARF associated with, respectively?

Answer 10

SAR1: COPII coats
ARF: COPI and clathrin coats

Question 11

when is SAR1 active?

Answer 11

when GEF exchanges a GDP for a GTP on the SAR1 protein

Question 12

what makes SAR1 inactive?

Answer 12

when GAP exchanges a GTP for a GDP on the SAR1 protein

Question 13

what is the function of Sec12 with SAR1?

Answer 13

activates it and recruits it to ER membrane - Sec12 is a GEF for SAR1

Question 14

function of Sec23 for SAR1?

Answer 14

GAP - stimulates GTP hydrolysis after coat assembly

Question 15

function of Sec24 for SAR1?

Answer 15

recruits cargo to the vesicle budding site

Question 16

CFTR sorting signal

Answer 16

chloride channel that has a di-acidic sorting signal for packaging in COPII vesicles at the ER

Question 17

mutation that causes cystic fibrosis

Answer 17

deltaF508 - blocks export of CFTR from the ER

Question 18

function of Rab proteins

Answer 18

specify compartment identity

Question 19

how do GTP-Rabs associate with membranes?

Answer 19

undergo a conformational change to expose a prenylated anchor that associates w/ membranes

Question 20

name some Rab effector proteins?

Answer 20

• large protein complexes (exocyst) that mark specific sites
• long, filamentous tethering proteins that anchor to acceptor
• cytoskeletal motor proteins that facilitate targeting/ transport

Question 21

function of SNAREs

Answer 21

• bring vesicle and target membranes into close apposition

- may also confer specificity on targeting-cognate pairs of v- and t-SNARE

Question 22

what happens to SNAREs once fused?

Answer 22

they are disassembled and recycled

Question 23

functions of Golgi

Answer 23

• transport and packaging

- modification and synthesis of oligosaccharides

Question 24

describe Golgi

Answer 24

• has polarity: forming (cis) -> maturing (trans) cisternae
• cis often near ER
• gradations in staining

Question 25

what direction does vesicle mediated protein trafficking occur b/w ER and cis-Golgi?

Answer 25

both directions - anterograde and retrograde

Question 26

two models of maturation/trafficking through the golgi apparatus

Answer 26

cisternal maturation and vesicle shuttling

Question 27

cisternal maturation

Answer 27

• cisterna form on one side, mature and move through the stack
• cargo stay w/ its cisterna (no forward vesicle trafficking)
• enzymes move backward by retrograde flow (COPI vesicles)

Question 28

vesicle shuttling

Answer 28

• fairly static cisternae
• cargo moved forward by vesicle trafficking
• enzymes remain in a given cisterna

Question 29

some evidence for cisternal maturation?

Answer 29

some secreted products are too big to be packaged in secretory vesicles

• algal scales
• collagen fibrils

Question 30

TGN

Answer 30

major sorting center - packages cargo for delivery to PM and lysosome

• has both constitutive and regulated exocytosis
• different coats mediate vesicle formation and cargo selection

Question 31

what is the sorting signal for targeting to lysosome?

Answer 31

M6P