Lectures

Question 1

Lecture 1, Methods to study

Answer 1

Fluorecent imaging-Immunostaining(fix dead cells to look at stains), GFP fusion(live imaging, may affect signal peptides.), FRAP(kinetics), FRET(interacting partners)
Microsome preparation
BIochemical Assays- Invitro trancription, Translation (lysate+membranes+radiolabelled with [35S] met.)
protein protease assay- post translationally incubated with protease and ran through and sds Page
Genetic experiments- Sec mutant(recusues using library or genome wide RNAi), database search, Antibodies to deduce peptides or product of WT, overexprese and image protein

Question 2

Lecture 3-

Answer 2

Signal peptides- identified via PPA
Co-translational tranlocation- identified via PPA
Signal Hypothesis-The information for targeting a protein to the ER lies at its N-terminus, tranlocation must occur before protein is too long or flooded
SRP discover- 250kda protein added
Action of SRP- P54(SP recognised)-> translational pause doman(A Sire)->SRP68/72 target SRPR.
GTP binds to SRP54 and SRPR to lock(promotes hydrolysis). hydrolysis releases SRP, translation resumes.
Lower eukaryotes and bacteria- targetting is SRP independant.

Protein channel

Question 3

Lecture 4

ER channel and BIP as gate

Answer 3

hydrophobic or aqeous
invitro transcription of preprolactin(truncated to lack stop codon, fluresent probe on only lysine 5’ end), flourecent lifetime 1ns aqueous therefore polar channel (7-8 non polar)
Gating-
Collision quenching of fluorenece(iodide). no drop from cytoplasmic side, drop only on luminal side whne potein was long enough
catalytic or stoichemetric?
Other Machinery?
photoactivated crosslinking and specfic ER protein anibodies- Sec61a TRAM, SPPedtidase.
Sec mutant analaysis- Sec61(yeast-mammalsSec61 complex) essential. Identified SEC62,63
Function- solublise(detergent), immunodepleted, reconstited, translation, PPA- Sec61a depletion not good