Lecture 9: Assembling Proteins into Membranes

Question 1

Explain what the helical hairpin hypothesis is.

Answer 1

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.

Question 2

What is the first helical hairpin hypothesis known as?

Answer 2

The two stage model but is very unlikely.

Question 3

What is pH dependent insertion?

Answer 3

These proteins will only insert into the membrane when the outside environment is acidic

Question 4

Give an example of acidic environment and how could this be exploited?

Answer 4

In tumours as they produce energy through glycolysis and could attach a cargo full of drug to be delivered specifically to tumour cells.

Question 5

What amino acid could spontaneously insert into a membrane in an acidic environment and why?

Answer 5

Aspartic acid will have a negative charge in the acidic environment and will be protonated becoming more hydrophobic.

Question 6

Explain the process by which proteins come to be inserted into the ER

Answer 6

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.

Question 7

What specifically does the SRP recognise?

Answer 7

hydrophobic stretches of amino acids 8-15 long.

Question 8

What does a typical cleavage signal have?

Answer 8

positively charged N terminus, hydrophobic stretch of AAs 1-15 long and a cleavage site for signal peptidase

Question 9

Amino acids in positions -1 and -3 are important for what?

Answer 9

cleavage on the luminal side of the ER

Question 10

What happens to the signal sequence?

Answer 10

It is cleaved off my signal peptidase creating a new N terminus so the signal sequence isn’t in the mature protein.

Question 11

What type of complex is SRP and give another exampe

Answer 11

Ribonuclear complex. Splisosome

Question 12

What does SRP54 have and what does this allow?

Answer 12

A groove with lots of flexible methionine residues in it which allows hydrophobic stretches of AAs to bind

Question 13

What enables the groove to bind to a variety of signal sequences?

Answer 13

The fact that hydrophobic interactions are relatively unspecific. The binding requires hydrophobicity rather than a specific sequence.

Question 14

What size is SRP54?

Answer 14

54kDa

Question 15

Through which domain does SRP bind the signal sequence through?

Answer 15

M domain

Question 16

Which domain halts translation?

Answer 16

Alu domain

Question 17

Explain the approach of Tom Rapoport’s group to identify Sec61 and TRAM

Answer 17

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.

Question 18

What is Sec61 structure?

Answer 18

Heterotrimeric with alpha, beta and gamma subunits

Question 19

What did initial EM studies reveal about Sec61?

Answer 19

Central pore formed by a tetramer of Sec61 which proteins were thought to translocate through.

Question 20

Give an example of a modification that happens cotranslationally.

Answer 20

disaccharyl transferase will add sugars t aspargine X threonine cotranslationally.

Question 21

Name the topology signals

Answer 21

C terminal signal, type1 (stop transfer sequence), 2 (signal anchor) and 3 (reverse signal anchor)

Question 22

What is the c terminal signal?

Answer 22

Hydrophobic domain is at the C terminal so the whole protein is translated before insertion into the membrane called post translational insertion eg synaptobrevin

Question 23

What does a stop transfer sequence do?

Answer 23

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

Question 24

What does the signal anchor do?

Answer 24

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.

Question 25

What is the reverse signal anchor?

Answer 25

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.

Question 26

What is lateral gating?

Answer 26

Allows the hydrophobic sequence to move out the channel into the bilayer

Question 27

What kind of amino acids are usually at the cytoplasmic side? What do they do?

Answer 27

Positively charged ones. They interact with negatively charged AAs or lipids on the inside of the translocon.

Question 28

What does the Sec61 translocon facilitate and what function does it have to maintain?

Answer 28

Movement of proteins through the membrane and integrates proteins into the lipid bilayer through lateral movement. has to maintain barrier function for ions.

Question 29

In what conformation are proteins transported in?

Answer 29

Unfolded

Question 30

What do translocons reduce?

Answer 30

The energy barrier

Question 31

What can a transmembrane protein be linked to when it enters the translocon and how does this change as integration proceeds?

Answer 31

TRAM and Sec61alpha but changes so is only crosslinked to TRAM

Question 32

What can transmembrane domains also be linked to?

Answer 32

Lipids

Question 33

What is TRAM thought to act as?

Answer 33

A chaperone

Question 34

What is autonomous insertion?

Answer 34

When TM segments incorporate into the membrane independently of each other

Question 35

What is heteronomous insertion?

Answer 35

incorporation of TM segments is dependent upon another segment.

Question 36

What does the translocon recognise?

Answer 36

A start/stop hydrophobic sequence.

Question 37

What is it energetically favourable for hydrophobic segments to do?

Answer 37

Move out into the lipid environment

Question 38

How are positive charges topogenic signals?

Answer 38

They interact with the negative charges at the cytoplasmic side of the translocon.