Translation and targeting Flashcards
ribosomes moving through the NPC
yeast cryo-EM tomography
translation
chain moves by EF-G/eET-2 GTP hydrolysis
post-translation
30% of proteins are targeted to the ER and secreted
give an example of an ER-associated protein
IgG light chains require ER-membrane for N-termini cleavage
SP in vitro assay
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
ER proteins
charged regions map onto polar head groups of water molecules
SRP
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
SRP double-selection
- binds to non-translating ribosomes and elongation factor Ribosome-Nascent-Chain Complices
- gives 100fold selectivity
SRP docking conformational change
- Alu domain competes with nascent polypeptide associated complex to bind to the ribosome
- Alu domain dissociation results in continued elongation
NAC
higher affinity for hydrophilic sequences than the SRP
all polypeptides must either be bound with
NAC/SPR
experimental evidence for the Sec61 channel:
- 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
fluorescence data
aqueous environment of SPR and nascent polypeptide translocation
conductance is measured with
electrodes
Yeast genetics
- 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
Sec61p
most abundant ER protein
Mammalian homologue?
- 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
artificial liposomes
become translocation competition if you include Sec61-alpha,beta and gamma, as well as SRalpha and beta
axial gating: translocation
- 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
lateral gating: insertion
- clam-shell opens laterally for insertion into lipid bilayer
- pore = hydrophilic, env. = hydrophobic
- hydrophobic TMD becomes membrane-spanning domain in lipid phase
secretion across PM in prokaryotes
facilitated by SecYEG
Post-translational insertion mediated by Sec61in eukaryotic organisms
- BiP holds loop
- ATP ratchet pulls through
Post-translational insertion mediated by Sec61in prokaryotic organisms
- SecD/F protein holds loop
- cytoplasmic SecA ATPase pushes through
