Lecture 10 Flashcards

Question

describe calnexin binding

Answer 1

N linked glycan = branched mannose polymer with 3 glucose residues - always the same polysaccharide glucose and mannose are trimmed off step by step CNX specifically binds the glycan with 1 glucose - keeps polypeptide in ER, single glucose is a signal for incomplete folding but is eventually removed

Answer 2

3 glucose = immature protein that just entered er IN er = ENZYMES trim glucose - 1st and 2nd off then when one glucose left = recognized by cnx and crt going to hold protein by binding polysaccharide with only one glucose and gives time to fold cnx and crt do not interact with protein they interact with this ptm but only when has one glucose give time for protein to fold once released = glucose trimmed then only mannoses left = then if mannoses trimmed = protein seeped out into cytosol and degraded by proteasome = bc if less mannoses = indicates it didnt fold properly

Answer 3

UDP-glucose:glycoprotein glucosyltransferase Binds non-native polypeptides and reattaches a glucose to the glycan – CNX can bind again Glucosidase removes Glc from native and non-native polypeptides Mannosidase trims sugars further- does not recognize shortened glycosylation

Answer 4

glucose trimmed, one left + mannose --> calnexin binds ptm and thioredoxin ERp57 helps with disulfide bonding --> glucosidase = trimmed, unfolded = hydrophobic aaas on surface = recognized by UGGT --> if successfully folded = exit from er --> if not = adds another glucose and goes through another cycle given many cycles since v taxing to make protein = given many chances

Answer 5

1. CNX keeps polypeptide in ER 2. Glucosidase removes the last Glc 3. UGGT restores Glc on misfolded polypeptides – CNX binding 4. Folded polypeptides do not have Glc restored and exit to Golgi 5. Mannosidase trims glycans without Glc = slow, irreversible and proteins retained in ER by chaperones likely to get trimmed 6. Mannose-binding lectins (EDEM) select short glycans for degradation

Answer 6

Native folded polypeptides are not recognized by UGGT

Answer 7

ER associated degradation

Answer 8

both lumenal (soluble and transmemebrane polypeptides) many substrates are misfolded proteins = quality control, before proteins are sent to rest of secretory pathway also regulated degradation in response to signals - regulates metabolism

Answer 9

1- substrates are recognized and brought to E3 ub ligase complexes (of misfolded) 2- E3 complex polyubs substrate and retrotranslocates (dislocates) substrate into cytosol (kinda like pore = brings er lumen/membrane proteins from er to cytosol) 3- substrate is deglycosylated and degraded by proteasomes *ubiquitination degradation

Answer 10

recognition - BiP to ERAD

Answer 11

Substrates which cannot fold are prevented from aggregating by BiP BiP binds substrate in complex with specialized DNAJ (ERdj5) and lectin (EDEM)= ERdj5: J domain and thioredoxin domain, catalyzes breakage of disulfide in substrate, reverse of PDI Complex targets substrate to E3 want to make not bulky = fold as much as possible

Answer 12

lectin= recognizes polysaccharides when no 3 glucose and trimming mannoses disulfide isomerases = breaks there E3 ub ligase = polyubs protein = want to be degraded - targeted N glycanase cuts polysaccharides AAA atpase helps pull polypeptide out - through retrotransposon like in between 1st and 2nd polyubs

Answer 13

TM E3 LIGASES (HRD1 and gp78) form complexes with substrate recognition adaptors - many adaptors allow misfolded proteins to be targeted = misfolded lumenal proteins (SEL1L), mannose binding lectins (EDEM), misfolded TM proteins (erlin 1/2, derlins), chaperones (BiP) other interactions = derlins, p97 receptors

Answer 14

still in question if retrotranslocon requires a true pore regulated opening and closing is pore like but large enough to allow n linked glycans bigger so can fit protein - get through retrotranslocon to cytosol

Answer 15

polyubiquitination in cytosol needed

Answer 16

large tm E3 ligase complexes are thought to perform all of these functions HRD1 and gp78 E3 ligases = homologous, multiple TM helices

Answer 17

cytosolic protein p97/VCP - pulling or unfolding activity

Answer 18

n end rule chip SCF E3 ligases

Answer 19

n linked glycans and polyub too large to fit through pore helps pull protein out of er p97 forms complexes with other proteins = ub binding adaptors, peptide: N glycanase (PNGase) removes glycans (before goes into aaa atpase), DUBs remove poly ub - but E3s re ub after extraction to complete targeting to proteasome hexamer = limited diameter so if have poly ub wont fit into shape

Answer 20

large diverse superfam of atpases with many diff functions usually hexameric rings

Answer 21

unfoldase subunits of proteaseome 19s regulatory p97: homo hexamer of 97kda subunits = uses atpase energy to extract proteins from membrane, substrate is also threaded through the central pore (hole) ub then de ub --> aaa atpases (unfolded here) --> polyubs --> goes to proteasome

Answer 22

1. Misfolded polypeptides are recognized by lectins and chaperones 2. Disulfide bonds are broken by a thioredoxin DNAJ 3. Adaptors bring misfolded polypeptides to E3 ligases – lectins, chaperones, lumenal and TM adaptors 4. Transmembrane E3 ligases polyubiquitinate substrates and start retro-translocation 5. p97 ATPase helps extract substrates from membrane 6. Substrates are de-glycosylated (PNGase) 7. Substrates are recognized by proteasome or shuttling receptors