what is post-translational translocation?
when proteins are fully translated in the cytosol
what is co-translational translocation
when proteins begin translation in the cytosol, before completing it in the RER
describe how proteins’ movements were first tracked
pulse-labelling
1) cells were shortly pulsed with radioactive aa, then chased with non-radioactive aa
2) the location of the radiolabeled proteins was detected using autoradiography
3) this showed that in secretory tissues, proteins associate with the rough ER immediately during translation
what does protease do
destroys proteins
what does detergent do
disrupt the lipid bilayer or membrane
describe how it was proved that secretory proteins entered the ER lumen
1) cells from a secretory tissue were homogenised to break membranes and release organelles
2) centrifugation was used to separate organelles by density and size
3) in vitro translation was performed with microsomes, mRNA, and radiolabelled aa’s
4) after translation, one test tube had protease + detergent, the other only protease
5) results showed that proteins were protected from protease unless detergent was added, confirming proteins were inside the ER lumen
what was the readout experiment for
to confirm that a signal sequence to direct the protein to the ER is present in mRNA
describe the readout experiment
1) in vitro translation of mRNA of a secretory protein was performed with added radiolabelled aa
2) one tube had no microsomes, and the other did
3) with microsomes, a shorter protein was produced, indicating cleavage of signal sequence at the ER
4) without microsomes, a longer protein was produced, indicating the signal sequence was still present
is the signal sequence highly conserved
yes
what is the general sequence of a signal peptide
a positive amino acid, then 6-12 hydrophobic amino acids
are secreted proteins hydrophobic or hydrophilic
hydrophilic
describe the sec61 translocon
- highly conserved
- forms a channel in the membrane
- has a hydrophilic pore between it
describe how a type 1 protein reaches the ER
1) once a signal sequence is detected on the nascent polypeptide (N-terminal), SRP binds and pauses translation
2) SRP brings the translation complex to the SRP receptor on the ER membrane
3) SRP + SRP receptor bind GTP
4) GTP hydrolysis releases SRP and transfers the ribosome to the translocon
5) translation resumes with the growing polypeptide entering the ER lumen
6) a signal peptidase eventually cleaves off the signal sequence
describe a type 1 protein’s hydrophobic parts
1) a hydrophobic stop-transfer sequence is synthesised as the protein enters the ER
2) this causes the translocon to open laterally, allowing the sequence to embed in the hydrophobic ER membrane
3) translation into the lumen stops, and the rest of the protein is synthesised in the cytosol, forming the C terminal
describe how a type 2 protein reaches the ER
1) type 2 proteins don’t have a signal sequence, but a signal-anchor sequence (N-terminal)
2) once this sequence is synthesised, the SRP binds, translation pauses, and the polypeptide reaches the SRP receptor in the ER membrane
3) the SRP complex binds then hydrolyses GTP, releasing SRP and moving the translation complex to the translocon
4) the signal-anchor is hydrophobic, so the translocon opens laterally to allow embedding
5) this establishes the cytosolic N-terminal, and translation of the luminal C-terminal continues
what side are the N and C terminals on in type 1 protiens
N terminal: lumen
C terminal: cytosol
what side are the N and C terminals on in type 2 protiens
N terminal: cytosol
C terminal: lumen
