Lectures 1 to 5 ER discoverability

Question 1

How was the SRP discovered

Answer 1

(PPA) Microsome creation
Preprolactin was used
Radiolabelled
Salt washed microsomes and a reconsititition experiments

Question 2

What were the actions of the SRP and how does it fit in with the signal hypothesis

Answer 2

1)emerging nascent chain has a unique set of codons nect to the initiating codons which is required for translocation
SRP54 recgonises this
2)The length of the protein needed to be kept short and targetting needs to happen fast enough so it doesnt get too long or folded
Binding of the SRP14/9 to A site blocking TRNA access

Question 3

THe life cycle of targetting of SRP receptors

Answer 3

GTP cycle of SRPR and SRP
Binding when docks locks the ribsomes onto the translocation machinery, hydroyslis releases SRP from SRPR and nascent chain.

Question 4

How was the channel discovered

Answer 4

Puromycin reaction mimicing 3’end of TRNA, enters A site, cannot enter P site, premature release of nascent chain
Flux of chloride ions froma cis chamber to transside was measured. When washed salt flux stops( shows due to ribosome keeping the channel open)

Question 5

How were the properties of the ER translocation channel Discovered

Answer 5

Truncated Preprolactin

collision quenching to find gate, aqueous or polar enviroment

Question 6

How were machinery discovered and what were they

Answer 6

Phtoactivable crosslinking agent in the chain and ER antibodies used
Discovered the SEC61a, TRAM, SRP

Question 7

How and what did the SEC mutant analysis of yeast reaveal and show

Answer 7

Yeast genetic screen with temp permissive cells, transfection with wild type gene library
Revealed that Sec61 and an essential function
Sec61, 62,62 (post translational Translocation)

coimmuprecipitation of mammalian homologue revelaed a SEC61p complex

Question 8

How were the function and importance of mammalian proteins recognised

Answer 8

Immunodepletion experiments of microsomes(Antibodies and sephose beads after detergent use)
immunodepletion of SEC61A showed in PPA that it is essential for transportation

Question 9

What were the minimal requoremtns for translocation discovered after this

Answer 9

SRPR, Sec61P complex and some require tram

Question 10

ER protein channel
Differences in post and co translational tranlocation in yest and humans
and a bacterial component

Answer 10

Yeast- post- required chaperones, low hydrophobic SP recognised and targetted to lateral gate via Sec62/63,
Sec63 causes wide opening of the lateral gate of the Sec61 channel priming it for the passage of low-hydrophobicity signal sequences into the lipid phase.

metalloproteases for unclogging, driving force comes from ATP-Bip interaction with Sec63
bacterial component is the piston ATP cycle of SecA

Question 11

Transmembrane protein translocation what is needed and in what orientation of the types of TM proteins

Answer 11

N or C terminus(TMD acts as SP) in lumen orietatioed via charge distrubution
ER membrane complex(EMC) required for insertion of 1st TMD of GPCRs in corect orientation and SRP and SEC61 used to to further insertions using lateral gate

Question 12

Alternative ER Targetting routes

Answer 12

Tailed ancorded proteins- Guide Entry of Tail-anchored proteins target TMD
Yeast mutant analysis of all genes- SND1-3 for centralled located TMD transport to translocon