ER Flashcards
What do proteins targeted to the ER have?
N-terminal signal sequences
What are signal sequences?
Short hydrophobic sequences
What are signal sequences recognised by?
Signal recognition paritcle - SRP
What process does SRP pause and how?
Pauses translation preventing proteins with signal sequences from folding in cytosol. SRP54 subunit binds to singal sequence.
What does the SRP deliver to the ER
The ribosome, SRP binds to STP-receptor on ER membrane, SRP is deplaced and ribosome docks onto SEC61
On binding onto what translocon does translation resumes?
SEC61, which forms a pore across ER membrane.
What is BiP?
Chaperone protein, luminal HSP70. Helps proteins fold by binding to unfolded regions
What type of environment is the ER?
Oxidising
What is correct disulphide bond formation between Cys res is promoted by?
Protein disulphide isomerases
What do most proteins in the ER undergo?
N-linked glycosylation
What is added to proteins in the ER (N-linked glycosylation)
An oligosaccharide chain is added by oligosaccharyltransferase to asparagine residues
What are oligosaccharyltransferases associated with?
SEC61 translocon and glycosulates proteins
What does the oligosaccharide chain is comprised of?
2 N-acetylgluosamines, 9 mannoses and
3 glucoses
Monitoring protein folding with glycosylation
- 1st the N-linked oligosaccharide is trimmed leaving a single glucose
residue - The protein is then recognised by the chaperone calnexin and the
associated protein ERp57, which bind to the oligosaccharide and free
cysteines respectively and prevent unfolded proteins aggregating
together - When the protein is released by calnexin/ERP57 the remaining glucose
is trimmed - If the protein is properly folded it can exit the ER
- If the protein fails to fold a glucose is added to the oligosaccharide by a
glucosyl transferase that recognises unfolded proteins and it reenters the
cycle.
How does ER remove misfolded protein?
ERAD
What is ERAD?
ER-associated degradtion
What catalyses the slow removal of mannose residues from N-linked oligosaccharide chains?
Mannosidases (EDEMs) which stop re-addition of glucose and marks protein for degradation
What is recognised by ERAD machinery?
Mannose-trimmed N-glycans, which are then ubiquitinated and translocated and translocated in the cytosol.
What is recognised by cytosolic proteasome?
Ubiquitinated proteins (mannose-trimmed glycans)
CFTR
Plasma membrane channel that transports Cl- ions, expressed in epithelial cells
CFTR delta-F508 mutant
Autosomal recessive mutation, loss of res508.
Is all of CFTR deltaF508 degraded by ERAD
Yes
Does CFTR deltaF508 retain some capacity to transport chlorine ions
Yes
How can we get CFTR deltaF508 out of the ER?
Lumcaftor which is a chemical chaperone, limited benefit.
What can lumcaftor be used in combination with
Ivacaftor, channel opener
How does cytomegalovirus hijack the ERAD pathway
US2 and US11 are ER membrane proteins and can cause MHC I class molecules to enter ERAD. HCMV infected cells can escape detection by cytotoxic T cells
Molecular mechanism of cholera toxin
B subunit binds to GM1 glycolipid on gut lining cells. CTA-1 once in cytosol activated adenylate cyclase increase cAMP, which activates protein kinase A. PKA phsophorylates CFTR promoting Cl- secretion
