Endoplasmic Reticulum

Question 1

What is the first step of the secretory pathway

Answer 1

ER

Question 2

What is the function of the ER?

Answer 2

• protein synthesis & assembly

- resident, golgi, membran, endosomal & lysosomal proteins made

Question 3

How are proteins recognised for ER translocation?

Answer 3

• N-terminal signal sequence (short hydrophobic)
• cleaved by signal peptidase on insertion
• sequences sometimes retained

Question 4

What is the role of SRP ?

Answer 4

• signal sequence appears from ribosome

- SRP recognises signal sequence

Question 5

How does SRP pause translation?

Answer 5

• 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

Question 6

How does SRP deliver ribosome to ER?

Answer 6

• SRP binds to SRPR on ER membrane

- SRP displaced, ribosome docks onto SEC61 (translocon forms pore)

Question 7

What is the SEC61 translocon?

Answer 7

• translation resumes after binding
• aqueous pore
• co-translational translocation

Question 8

The role of chaperone BiP?

Answer 8

• HSP70
• binds to proteins as inserted in ER
• helps folding (non-glycosylated especially)
• ATP-dependent
• stops slipping back through pore

Question 9

How are disulphide bonds formed in the ER?

Answer 9

• Er = oxidising
• disulphide bonds between Cys residues promoted by PDIs
• e.g. immunoglobin folds

Question 10

How does N-linked glycosylation occur in the ER?

Answer 10

• oligosaccharide chain added by OST to N residues (asparagine)
• OST associated with SEC61 - glycosylates as translocated

Question 11

What does the oligosaccharide chain comprise of?

Answer 11

2 x N-acetylglucosamine
9 x mannose
3 x glucose

Question 12

How is protein folding monitored by glycosylation?

Answer 12

• 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

Question 13

What happens if protein fails to fold in ER?

Answer 13

• glucose added by glucosyl transferase

- reenters cycle

Question 14

What is the ERAD pathway?

Answer 14

• prevents accumulation of mis-folded proteins which would aggregate
• add ubiquitin tags to be degraded, chopped to renter metabolic pathways

Question 15

What is the role of EDEMs in the monitoring of protein folding?

Answer 15

• mannosidases catalyse slow removal of mannose residues
• stops re-addition of glucose
• marks for degradation by ERAD

Question 16

How are proteins degraded by the ERAD?

Answer 16

• ERAD recognises mannose trimmed N-glycans
• adaptors recognise and protein is ubiquitinated
• translocated into cytosol
• ubiquitinated recognised by cytosolic proteasome & chops into peptides

Question 17

Information about cystic fibrosis?

Answer 17

• loss of function in CFTR
• over >1000 possible mutations in gene that encodes CFTR
• most common F508

Question 18

characteristics of fibrosis?

Answer 18

• Thick mucous secretions in lungs

- infections impair lung function

Question 19

What is the function of CFTR in cystic fibrosis?

Answer 19

• plasma membran channel transports Cl- ions
• ATP binding & hydrolysis gates
• transports Cl- ions into mucous, increases water movement making it less viscous
• osmotic flow

Question 20

What is is the F508 mutant?

Answer 20

• 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

Question 21

How can we get CFTR F508 out of the ER?

Answer 21

• Lumcaftor combined with Ivacaftor - minor improvement of lung function
• ivacaftor opens chloride channel
• makes conformation not recognised by ERAD

Question 22

How does HCMV hijack the ERAD pathway?

Answer 22

• US2, US11 membrane proteins
• MHC class I molecules enter ERAD - retro-translocated into cytosol & degraded
• escape detection by cytotoxic T cells