Quality control and the glycocode

Question 1

early evidence for signal mediated

Answer 1

ER export = rate limiting step and export is not correlated with abundance
- induced artificial bacterial proteins are exported at minimum rate

Question 2

N-glycans

Answer 2

• specific to ER proteins
• N-linked glycoproteins blocked export on tunicamycin treatment
• allow proteins to interact with ERGIC53 and VIP36/L lectin receptors (binding is saturable and limited)

Question 3

Measuring the rate of bulk flow:

Answer 3

1) radiolabelled N-octyl-Asn-Tyr9128)-Thr tripeptides
2) diffuses into cell and ER lumen
3) glycosylated in ER
4) measure rate of secretion of Golgi-modified peptide

Question 4

Observations when measuring the rate of bulk flow:

Answer 4

1) no export signal identified
2) rate of bulk flow was at rate of fastest native proteins but in very low quantities (was there a retention signal?)

Question 5

ER-residents

Answer 5

• > 90% are reticuloplasmins that interact extensively and loosely using calcium-binding domains to build calcium cross-bridges
• K/HDEL retrieval system

Question 6

mutant proteins

Answer 6

CFTR DF508 = usually helps w mucous in lungs; cystic fibrosis

Question 7

What did the QC explain?

Answer 7

• why there was a low quantity of bulk flow: incorrectly folded
• misfolding on tunicamycin treatment
• diffuse mutations caused by folding

Question 8

QC glycocode

Answer 8

• massive conservation across phylogeny
• all nascent proteins transiently interact with ER chaperones

Question 9

CNX and CRT

Answer 9

highly specific lectins that bind GlcI glycans

Question 10

How does the QC glycocode work?

Answer 10

1. high mannose tree is bound to Asp in nascent chain via oligosaccharide transferase N-linkage
   2) Glucosidases I and II remove terminal glucoses
   3) CNX binds end-glucose residue
   4) Glu1 removed as protein folds
   5) UDP-glucose:glycoprotein glycosyltransferase I selectively adds glucose to misfolded proteins
   6) continues CNX association; retention

Question 11

How does UGT discriminate?

Answer 11

• hydrophobic sequence exposure
• glycan core accessibility

Question 12

ER mannosidase I

Answer 12

slow-acting; gives time for proper folding, but begins elimination of misfolding proteins

Question 13

ER mannosidase II

Answer 13

acts on unfolded proteins

Question 14

cargo protein distribution

Answer 14

• high Golgi concentration prior to export
• measured using quantitative immune-electron microscopy

Question 15

COPI recycling

Answer 15

• from Golgi and vesicular-tubular clusters
• selectively returns escaped ER residents and membrane proteins

Question 16

COPII cycling

Answer 16

• diffusion of residents is reduced via calcium cross-bridges
• specialised ER exit site

Question 17

CRT deltaKDEL

Answer 17

secreted slowly

Question 18

CRT deltaCaBD + deltaKDEL

Answer 18

secreted rapidly

Question 19

COPII-Sec24p

Answer 19

• Sec12p ER resident activates Sar1p via GTP hydrolysis
• Sar1p embeds in the membrane
• membrane cargo binds via cytoplasmic tails; bending
• Sec13p/Sec31p bind
• Recruits Sec24p/Sec25p via the cytosolic domain
• forms a coat

Question 20

Looking in COPII cargo for an export signal

Answer 20

1) short cytoplasmic tails
2) Sec24p interaction
3) selective coat
- identify over-represented proteomic motifs

Question 21

Soluble cargo receptors

Answer 21

• large ER lumen domains for cargo binding

Question 22

Experimental evidence for soluble cargo receptors

Answer 22

1) loss of function yeast mutants
2) rapid export

Question 23

Lumen cargo receptors

Answer 23

• ERG1C53 and VIP36/L
• N-glycan specific lectins
• broad specificity
• low affinity
• compete with UGT, EDEM and ERAD lectins
• accelerate packaging and export