Endoplasmic Reticulum Flashcards
What is the first step of the secretory pathway
ER
What is the function of the ER?
- protein synthesis & assembly
- resident, golgi, membran, endosomal & lysosomal proteins made
How are proteins recognised for ER translocation?
- N-terminal signal sequence (short hydrophobic)
- cleaved by signal peptidase on insertion
- sequences sometimes retained
What is the role of SRP ?
- signal sequence appears from ribosome
- SRP recognises signal sequence
How does SRP pause translation?
- prevents proteins with signal sequences from folding
- SRP54 subunit - binds signal sequence
- SRP RNA’s Alu domain - bind ribosome elongation factor bind site
- protein NOT re-fold until in ER lumen
How does SRP deliver ribosome to ER?
- SRP binds to SRPR on ER membrane
- SRP displaced, ribosome docks onto SEC61 (translocon forms pore)
What is the SEC61 translocon?
- translation resumes after binding
- aqueous pore
- co-translational translocation
The role of chaperone BiP?
- HSP70
- binds to proteins as inserted in ER
- helps folding (non-glycosylated especially)
- ATP-dependent
- stops slipping back through pore
How are disulphide bonds formed in the ER?
- Er = oxidising
- disulphide bonds between Cys residues promoted by PDIs
- e.g. immunoglobin folds
How does N-linked glycosylation occur in the ER?
- oligosaccharide chain added by OST to N residues (asparagine)
- OST associated with SEC61 - glycosylates as translocated
What does the oligosaccharide chain comprise of?
2 x N-acetylglucosamine
9 x mannose
3 x glucose
How is protein folding monitored by glycosylation?
- oligosaccharide trimmed leaving 1 glucose
- recognise by chaperone calnexin and ERp57 - bind to oligosaccharide and free cysteines (no aggregation)
- protein release, glucose trimmed
- proper folding = exit ER
What happens if protein fails to fold in ER?
- glucose added by glucosyl transferase
- reenters cycle
What is the ERAD pathway?
- prevents accumulation of mis-folded proteins which would aggregate
- add ubiquitin tags to be degraded, chopped to renter metabolic pathways
What is the role of EDEMs in the monitoring of protein folding?
- mannosidases catalyse slow removal of mannose residues
- stops re-addition of glucose
- marks for degradation by ERAD
How are proteins degraded by the ERAD?
- ERAD recognises mannose trimmed N-glycans
- adaptors recognise and protein is ubiquitinated
- translocated into cytosol
- ubiquitinated recognised by cytosolic proteasome & chops into peptides
Information about cystic fibrosis?
- loss of function in CFTR
- over >1000 possible mutations in gene that encodes CFTR
- most common F508
characteristics of fibrosis?
- Thick mucous secretions in lungs
- infections impair lung function
What is the function of CFTR in cystic fibrosis?
- plasma membran channel transports Cl- ions
- ATP binding & hydrolysis gates
- transports Cl- ions into mucous, increases water movement making it less viscous
- osmotic flow
What is is the F508 mutant?
- loss of phenylalanine in residue 508 in NBD1
- retains some capacity to transport Cl- ions
- ALL DEGRADED BY ERAD PATHWAY - prevent trafficking to plasma membrane
How can we get CFTR F508 out of the ER?
- Lumcaftor combined with Ivacaftor - minor improvement of lung function
- ivacaftor opens chloride channel
- makes conformation not recognised by ERAD
How does HCMV hijack the ERAD pathway?
- US2, US11 membrane proteins
- MHC class I molecules enter ERAD - retro-translocated into cytosol & degraded
- escape detection by cytotoxic T cells
