Secretory & endocytic pathways Flashcards
What are the protein coats that form vesicles and incorporate cargo?
clathrin, COPI, COPII
What do Rab proteins do?
bring membranes together - tethering proteins
What do SNARE proteins do?
bind v-SNARES on t-SNARES = fuse and deliver cargo
What does the fusion of SNARE proteins make?
trans-SNARE complex
How are proteins synthesises in the ER exported?
COPII vesicles
uncoat
fuse by SNARE interactions
vesicular tubular clusters fuse with cis Golgi
How do COPII vesicles form?
- Sec12 - GDP-GTP on Sar1 - binds ER membranes
- Sar 1 - Sec23-24 to membrane
- cargo - Sec24
- Sec13-31 - form coat, drive budding
How is cargo recruited into COPII veisicles?
export signals recognised by Sec24
ERGIC-53 receptor binds to soluble glycoproteins
cytosolic di-phenylalance (FF) motif = export signal
How are non-selective ER proteins retrieved from the golgi?
ER retrieval motifs
COPI vesicles return
What is the retrieval motif of ER resident membrane proteins?
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
How how ER luminal proteins retrieved?
KDEL motif (BiP)
What does the Golgi comprise of?
stacks of membranes (cisterna)
How are proteins trafficked through the golgi?
cis to trans direction
stacks mature? vesicles carry proteins?
How are proteins modified in the Golgi?
Golgi N-linked oligosaccharides - removal of 3 mannoses
further mods
How does sorting take place at the trans-golgi network?
proteins go to final destination
secreted or plasma membrane = no signal
endosome = signal needed
What is a signal for trafficking to endosomes?
mannose-6-phosphate
recognised by MPRs
GGAs bind to MPRs
incorporate into clathrin-coated vesicles via AP-1
How do early endosomes act as sorting stations in the endocytic pathway?
- recycle back to plasma membrane
- traffic to lysosomes, MVBs
- traffic to the TGN - MPRs
What is the endocytosis of receptors?
- LDLs release ligands in early endosomes
- EGF binding - trafficking of receptor to lysosomes to turn off signalling
How does clathrin mediated endocytosis occur?
- pinch off plasma membrane
- form vesicles
- recruited via adaptors e.g. AP-2
How does endosomal recycling occur?
- endocytosed LDL receptors release ligands in early endosomes
- LDL receptors recycle to plasma membrane
- LDL can capture ligands again
How do endosomes help trafficking to lysosomes?
- EGF binding activates signalling cascade
- receptor endocytoses
- trafficked to early endosomes then MVBs
- MVB converted to late endosome- fuse with lysosomes
- degraded
What is CLSD (disease)?
- autosomal recessive
- skeletal defects
- facial abnormalities
- mutations in Sec23a
- F382L
What does the F382L mutation in SEC23A of CLSD result in?
- dilation of ER
- reduce capacity to generate cargo vesicles
- reduction in collagen export
What is I-cell disease caused by?
mutations in N-acetylglucosamine-1-phosphotransferase
What is disrupted in I-cell disease?
trafficking of newly synthesised lysosomal hydrolases
don’t form tag - protein is secreted instead of being targeted to lysosome - decrease in degradative capacity
What causes familial hypercholesterolaemia?
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
How does HCV enter cells via the endocytic pathway?
CD81 receptor complex - enter hepatocytes
clathrin & AP-2 adaptor
traffics to early endosomes
How does adenovirus hijack the ER retrieval pathway to inhibit MHC class I trafficking?
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
What does the cholera toxin do?
promotes electrolyte and water movement into the intestinal lumen = diarrhoea
How does the cholera toxin act in the body?
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
How is cholera toxin trafficked?
retrograde transport through secretory pathways - COPI
dissociation of subunits
A1 - ERAD
not degraded due to lots of lysine in sequence
What is Ricin?
castor beans
Nausea, Diarrhoea
2 subunit toxin
How does ricin enter the cell?
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
