Lecture 9: Assembling Proteins into Membranes Flashcards
Explain what the helical hairpin hypothesis is.
Proteins can either enter the membrane and then fold or can fold then enter membrane. The former takes +46kcal/mol to insert unfolded followed by -106kcal/mol to fold. this because the protein has an exposed polar backbone which would cost energy to insert into the membrane. In the latter, the dehydration energy is compensated for by the new H-bonds formed so no energy cost and insertion into the membrane is -60kcal/mol as the folded protein has hydrophobic regions which prefer to insert into the membrane. No energy barrier with this route.
What is the first helical hairpin hypothesis known as?
The two stage model but is very unlikely.
What is pH dependent insertion?
These proteins will only insert into the membrane when the outside environment is acidic
Give an example of acidic environment and how could this be exploited?
In tumours as they produce energy through glycolysis and could attach a cargo full of drug to be delivered specifically to tumour cells.
What amino acid could spontaneously insert into a membrane in an acidic environment and why?
Aspartic acid will have a negative charge in the acidic environment and will be protonated becoming more hydrophobic.
Explain the process by which proteins come to be inserted into the ER
Protein synthesis in ribosomes occurs in the cytoplasm. If a hydrophobic signal sequence emerges from the ribosome on the N terminus, a signal recognition particle (SRP) recognises it. It binds and halts translation. This complex is then targeted to the ER where is binds SRP receptors in the vicinity of a translocon. SRP hydrolysis GTP to GDP and no longer has affinity for the signal sequence and releases it. This allows translation to continue again and the protein moves through the membrane into the ER lumen through the translocon.
What specifically does the SRP recognise?
hydrophobic stretches of amino acids 8-15 long.
What does a typical cleavage signal have?
positively charged N terminus, hydrophobic stretch of AAs 1-15 long and a cleavage site for signal peptidase
Amino acids in positions -1 and -3 are important for what?
cleavage on the luminal side of the ER
What happens to the signal sequence?
It is cleaved off my signal peptidase creating a new N terminus so the signal sequence isn’t in the mature protein.
What type of complex is SRP and give another exampe
Ribonuclear complex. Splisosome
What does SRP54 have and what does this allow?
A groove with lots of flexible methionine residues in it which allows hydrophobic stretches of AAs to bind
What enables the groove to bind to a variety of signal sequences?
The fact that hydrophobic interactions are relatively unspecific. The binding requires hydrophobicity rather than a specific sequence.
What size is SRP54?
54kDa
Through which domain does SRP bind the signal sequence through?
M domain
Which domain halts translation?
Alu domain
Explain the approach of Tom Rapoport’s group to identify Sec61 and TRAM
They translated artificial mRNA which codes for a protein with a signal sequence and a single lysine (K) codon. The lysine tRNA will incorporate a crosslinkable amino acid to this position on the protein. Ther is no stop codon so when the ribosome reaches the end of the mRAN the whole thing becomes trapped and remains attached to the tRNA. Photoactivate the crosslinker and can identify the proteins that crosslink to the synthetic protein.
What is Sec61 structure?
Heterotrimeric with alpha, beta and gamma subunits
What did initial EM studies reveal about Sec61?
Central pore formed by a tetramer of Sec61 which proteins were thought to translocate through.
Give an example of a modification that happens cotranslationally.
disaccharyl transferase will add sugars t aspargine X threonine cotranslationally.
Name the topology signals
C terminal signal, type1 (stop transfer sequence), 2 (signal anchor) and 3 (reverse signal anchor)
What is the c terminal signal?
Hydrophobic domain is at the C terminal so the whole protein is translated before insertion into the membrane called post translational insertion eg synaptobrevin
What does a stop transfer sequence do?
Cleaving the signal sequence makes a new N terminus and this stops the translocation of the protein into the ER so the N term is in the ER and the C term is in the cytoplasm
What does the signal anchor do?
There is a signal anchor of about 20 hydrophobic amino acids that is not cleaved or recognised by signal peptidase. The C terminus is directed into the lumen and the N remains in the cytoplasm.
What is the reverse signal anchor?
Orientates the protein so the C terminus is in the cytoplasm and the N is in the ER lumen like the stop transfer sequence. same topology but achieved in a different way.
What is lateral gating?
Allows the hydrophobic sequence to move out the channel into the bilayer
What kind of amino acids are usually at the cytoplasmic side? What do they do?
Positively charged ones. They interact with negatively charged AAs or lipids on the inside of the translocon.
What does the Sec61 translocon facilitate and what function does it have to maintain?
Movement of proteins through the membrane and integrates proteins into the lipid bilayer through lateral movement. has to maintain barrier function for ions.
In what conformation are proteins transported in?
Unfolded
What do translocons reduce?
The energy barrier
What can a transmembrane protein be linked to when it enters the translocon and how does this change as integration proceeds?
TRAM and Sec61alpha but changes so is only crosslinked to TRAM
What can transmembrane domains also be linked to?
Lipids
What is TRAM thought to act as?
A chaperone
What is autonomous insertion?
When TM segments incorporate into the membrane independently of each other
What is heteronomous insertion?
incorporation of TM segments is dependent upon another segment.
What does the translocon recognise?
A start/stop hydrophobic sequence.
What is it energetically favourable for hydrophobic segments to do?
Move out into the lipid environment
How are positive charges topogenic signals?
They interact with the negative charges at the cytoplasmic side of the translocon.
