Protein transport 2

Question 1

Signal recognition particle

Answer 1

• Signal peptide bound by SRP containing GTP
• Causes pause in translation
• GTP bound forms of SRP + receptor bind
• Complex targets ribosome-nascent chain complex to translocon
• Signal peptide inserts into translocon + initiates co-translation
• GTPase active
• Signal peptide binding inhibits GTPase of SRP
• Signal peptide bound inverse orientation
• SRP M-domain (Meth rich, next to exit tunnel, very hydrophobic)
• SRP ‘Alu domain’ (prevents binding of elongation factors, competes for same bs)

Question 2

Biased diffusion model of PT translocation of eukaryotes

Answer 2

1. Binding of complete polypeptide chain to sex
2. Chaperones released as inserted into channel
3. Bip in T state interacts w/ J domain in Sec63
4. Following ATP hydrolysis, Bip binds polypeptide substrate (ADP), prevents sliding back
5. Bip binds
6. Repeat
7. Nucleotide exchange releases Bip

Question 3

2 models

Answer 3

1. Lever model/ power stroke
   (Bip physically associates w. lumen, binds polypeptide, conf Δ, pulls peptide through, mechanical E)
2. Brownian racket
   (peptide moves either way, overall x effect, Bip restricts diffusion, thermal E)

Question 4

SecA mediated PT translocation in bacteria

Answer 4

• SecB binds preprotein + stabilises
• Binding of sequence signal to secA releases pre-protein
• SecA inserts protein into secY channel
• ATP hydrolysis pushes polypeptide into channel
• After translocation secA released from SecY

Question 5

Experiment

Answer 5

Shows precursor protein transported through gated channel

• Puromycin (tRNA analogue)
• 1st ions x freely pass through translocon when nascent chain is translocated
• 2nd puromycin is incorporated into carboxylate-terminus of nascent chain
• 3rd peptidyl-puromycin is released + translocates across bilayer, unplugs
• 4th ↑ NaCl, channel closes