Translation and targeting

Question 1

ribosomes moving through the NPC

Answer 1

yeast cryo-EM tomography

Question 2

translation

Answer 2

chain moves by EF-G/eET-2 GTP hydrolysis

Question 3

post-translation

Answer 3

30% of proteins are targeted to the ER and secreted

Question 4

give an example of an ER-associated protein

Answer 4

IgG light chains require ER-membrane for N-termini cleavage

Question 5

SP in vitro assay

Answer 5

1) add radiolabelled precursors into a system and they are integrated into cost proteins
2) autoradiography shows
3) time shift assay via detergent and conduct SDS-page
4) initially, processed proteins have low molecular weight
5) later, larger (higher band)
6) sequences produced are not further modified
7) signal sequence is only identified in late-emerging proteins
8) conduct protease protection tests to show vesicles
9) chimeric SP-introduced proteins are targeted to the ER

Question 6

ER proteins

Answer 6

charged regions map onto polar head groups of water molecules

Question 7

SRP

Answer 7

Elements are defined by their molecular weight:
- SRP-54: signal recognition (relatively low affinity, GTP-hydrolysis)
- SRP-9 and -14: translational arrest (plug domain)
- SRP-68 and -72: ER docking (receptor recognition, conformational change)
- long, wraps around ribosome
- falls off at N-terminal end

Question 8

SRP double-selection

Answer 8

• binds to non-translating ribosomes and elongation factor Ribosome-Nascent-Chain Complices
• gives 100fold selectivity

Question 9

SRP docking conformational change

Answer 9

• Alu domain competes with nascent polypeptide associated complex to bind to the ribosome
• Alu domain dissociation results in continued elongation

Question 10

NAC

Answer 10

higher affinity for hydrophilic sequences than the SRP

Question 11

all polypeptides must either be bound with

Answer 11

NAC/SPR

Question 12

experimental evidence for the Sec61 channel:

Answer 12

• ribosome membranes fuse with black lipid bilayer when added to cis, cytosolic side
• puromycin causes chain termination and release
• reveals high conductance aqueous pore big enough for a protein

Question 13

fluorescence data

Answer 13

aqueous environment of SPR and nascent polypeptide translocation

Question 14

conductance is measured with

Answer 14

electrodes

Question 15

Yeast genetics

Answer 15

• his4 mutants
• • ER-associated His4p
• screen 5x10^7 colonies for growth
• 2000His+ recovered: ‘leaky mutants’
• 37degrees sensitive growth and unglycosylated secretory protein accumulation
• 63 mutants, 3 genes

Question 16

Sec61p

Answer 16

most abundant ER protein

Question 17

Mammalian homologue?

Answer 17

• truncated mRNAs without a stop-codon get stuck in the exit channel
• assaying shows lysine cross-linking around SP and ribosome; recover it
• 3 associated proteins

Question 18

artificial liposomes

Answer 18

become translocation competition if you include Sec61-alpha,beta and gamma, as well as SRalpha and beta

Question 19

axial gating: translocation

Answer 19

• negative leucine residues on flexible helices on cytosolic side of the mouth interact with the positive reduces in the SP
• opens pore
• extended conformation translocation

Question 20

lateral gating: insertion

Answer 20

• clam-shell opens laterally for insertion into lipid bilayer
• pore = hydrophilic, env. = hydrophobic
• hydrophobic TMD becomes membrane-spanning domain in lipid phase

Question 21

secretion across PM in prokaryotes

Answer 21

facilitated by SecYEG

Question 22

Post-translational insertion mediated by Sec61in eukaryotic organisms

Answer 22

• BiP holds loop
• ATP ratchet pulls through

Question 23

Post-translational insertion mediated by Sec61in prokaryotic organisms

Answer 23

• SecD/F protein holds loop
• cytoplasmic SecA ATPase pushes through