Secretory & endocytic pathways

Question 1

What are the protein coats that form vesicles and incorporate cargo?

Answer 1

clathrin, COPI, COPII

Question 2

What do Rab proteins do?

Answer 2

bring membranes together - tethering proteins

Question 3

What do SNARE proteins do?

Answer 3

bind v-SNARES on t-SNARES = fuse and deliver cargo

Question 4

What does the fusion of SNARE proteins make?

Answer 4

trans-SNARE complex

Question 5

How are proteins synthesises in the ER exported?

Answer 5

COPII vesicles
uncoat
fuse by SNARE interactions
vesicular tubular clusters fuse with cis Golgi

Question 6

How do COPII vesicles form?

Answer 6

• Sec12 - GDP-GTP on Sar1 - binds ER membranes
• Sar 1 - Sec23-24 to membrane
• cargo - Sec24
• Sec13-31 - form coat, drive budding

Question 7

How is cargo recruited into COPII veisicles?

Answer 7

export signals recognised by Sec24

ERGIC-53 receptor binds to soluble glycoproteins

cytosolic di-phenylalance (FF) motif = export signal

Question 8

How are non-selective ER proteins retrieved from the golgi?

Answer 8

ER retrieval motifs

COPI vesicles return

Question 9

What is the retrieval motif of ER resident membrane proteins?

Answer 9

dilysine KKXX

ERGIC-53 is KKFF - FF is what transports it to golgi, KK is what gets it back to ER

Bip sometimes plays role

Question 10

How how ER luminal proteins retrieved?

Answer 10

KDEL motif (BiP)

Question 11

What does the Golgi comprise of?

Answer 11

stacks of membranes (cisterna)

Question 12

How are proteins trafficked through the golgi?

Answer 12

cis to trans direction

stacks mature? vesicles carry proteins?

Question 13

How are proteins modified in the Golgi?

Answer 13

Golgi N-linked oligosaccharides - removal of 3 mannoses

further mods

Question 14

How does sorting take place at the trans-golgi network?

Answer 14

proteins go to final destination
secreted or plasma membrane = no signal
endosome = signal needed

Question 15

What is a signal for trafficking to endosomes?

Answer 15

mannose-6-phosphate
recognised by MPRs
GGAs bind to MPRs

incorporate into clathrin-coated vesicles via AP-1

Question 16

How do early endosomes act as sorting stations in the endocytic pathway?

Answer 16

• recycle back to plasma membrane
• traffic to lysosomes, MVBs
• traffic to the TGN - MPRs

Question 17

What is the endocytosis of receptors?

Answer 17

• LDLs release ligands in early endosomes

- EGF binding - trafficking of receptor to lysosomes to turn off signalling

Question 18

How does clathrin mediated endocytosis occur?

Answer 18

• pinch off plasma membrane
• form vesicles
• recruited via adaptors e.g. AP-2

Question 19

How does endosomal recycling occur?

Answer 19

• endocytosed LDL receptors release ligands in early endosomes
• LDL receptors recycle to plasma membrane
• LDL can capture ligands again

Question 20

How do endosomes help trafficking to lysosomes?

Answer 20

• EGF binding activates signalling cascade
• receptor endocytoses
• trafficked to early endosomes then MVBs
• MVB converted to late endosome- fuse with lysosomes
• degraded

Question 21

What is CLSD (disease)?

Answer 21

• autosomal recessive
• skeletal defects
• facial abnormalities
• mutations in Sec23a
• F382L

Question 22

What does the F382L mutation in SEC23A of CLSD result in?

Answer 22

• dilation of ER
• reduce capacity to generate cargo vesicles
• reduction in collagen export

Question 23

What is I-cell disease caused by?

Answer 23

mutations in N-acetylglucosamine-1-phosphotransferase

Question 24

What is disrupted in I-cell disease?

Answer 24

trafficking of newly synthesised lysosomal hydrolases

don’t form tag - protein is secreted instead of being targeted to lysosome - decrease in degradative capacity

Question 25

What causes familial hypercholesterolaemia?

Answer 25

disruption of LDL receptor
reduces LDL removal from blood
cholesterol deposits in the walls of arteries
JD mutation - in NPVY sequence motif needed to recruit LDL receptor into cc vesicles

Question 26

How does HCV enter cells via the endocytic pathway?

Answer 26

CD81 receptor complex - enter hepatocytes

clathrin & AP-2 adaptor

traffics to early endosomes

Question 27

How does adenovirus hijack the ER retrieval pathway to inhibit MHC class I trafficking?

Answer 27

integral membrane protein - E3/19K - binds to MHC class I by luminal domain

tail has ER retrieval motif - COPI vesicles

prevents MHC class I reaching plasma membrane

Question 28

What does the cholera toxin do?

Answer 28

promotes electrolyte and water movement into the intestinal lumen = diarrhoea

Question 29

How does the cholera toxin act in the body?

Answer 29

2 subunit

B subunit binds to GM1 glycolipid on gut cells

A subunit activates adenylate cyclase - increase cAMP

activates PKA

PKA phosphorylates CFTR promoting Cl- secretion

Question 30

How is cholera toxin trafficked?

Answer 30

retrograde transport through secretory pathways - COPI

dissociation of subunits

A1 - ERAD

not degraded due to lots of lysine in sequence

Question 31

What is Ricin?

Answer 31

castor beans
Nausea, Diarrhoea
2 subunit toxin

Question 32

How does ricin enter the cell?

Answer 32

A-chain - enzymatically active

B-chain - binds to glycolipids & glycoproteins

Ricin trafficked by TGN and ER (but lacks KDEL)

A-chain dissociates in ER - retrotranslocate across membrane

A-chain depurinates 28S ribosomal RNA - cleave bond - release adenine

translation inhibited