Lecture 13 - Compartmentalisation of the cell and protein sorting

Question 1

What is a nuclear pore?

Answer 1

Formed at the junction of the inner and outer membrane of the nuclear envelope.

It consists of multiple copies of ~ 30 different nucleoporins

Each complex is made of 8 subunits with a central plug

Question 2

What do nuclear pore complexes do ?

Answer 2

They’re involved in moving substances across the nuclear envelope

Facilitate the selective transport of macromolecules, such as proteins, RNA, and ribonucleoproteins (RNPs), across the nuclear envelope. Small molecules and ions can passively diffuse through the nuclear pores, but larger molecules require the assistance of specialized transport factors.

Question 3

How can substances be transported by the pore complex ?

Answer 3

1. Diffusion - 60,00 is the limit
2. Active diffusion

Question 4

What is the signal for proteins for nuclear pores ?

Answer 4

Signals are linked top the peptide signals. Nuclear transport recognition sites are rich in Lys, Arg and Pro

Example is T antigen of SV40 virus, when sequence is disrupted there is no staining in the nucleus

Question 5

What is the experimental evidence that the transport is active ?

Answer 5

In cells mRNA transport out of nucleus, this can be inhibited by cooling the cell to 4 degrees. This shows that ATP hydrolysis is required

In vitro - Import of protein into the nucleus. When ATP is removed in this case the protein will still bind to the pore complex but it will remain outside the nucleus

Question 6

What are the main steps of ER translocation ?

Answer 6

1. Targeting: The nascent polypeptide chain is recognized by a signal recognition particle (SRP), which binds to a hydrophobic signal sequence located near the amino terminus of the protein. The SRP then interacts with the SRP receptor on the ER membrane, which initiates the process of translocation.
2. Translocation channel formation: The SRP receptor binds to the translocon, which is a protein complex that forms a channel in the ER membrane. The binding of the SRP receptor to the translocon triggers the opening of the channel.
3. Translocation: The nascent polypeptide chain is then translocated across the ER membrane in a process that is powered by the hydrolysis of GTP. The polypeptide chain passes through the central channel of the translocon, which serves as a conduit for the nascent protein to enter the lumen of the ER.
4. Co-translational folding and modification: As the polypeptide chain emerges from the translocon, it is co-translationally folded and modified by a variety of chaperones and enzymes. These modifications are important for proper protein folding and function.
5. Release: Once the entire protein has been synthesized and translocated across the ER membrane, the signal sequence is cleaved by a signal peptidase, and the mature protein is released into the ER lumen.

Question 7

What do defects in protein folding give rise to ?

Answer 7

They give rise to disease e.g. Cystic fibrosis (CFTRdelta508)

Misfiled proteins stimulate the Unfolded Protein Response. UPR also occurs in plants but the machinery is different.

Question 8

How do you get proteins ointpo the mitochondrial matrix ?

Answer 8

1. N-terminal signal sequence – recognised by the TOM complex
2. The protein translocates through TOM and TIM23
3. Translocates through TIM23 into matrix
4. Signal is cleaved off

Question 9

What is the nature of the signal ?

Answer 9

It has to get across a membrane so the signals take the form ofamphipharic alpha helices. There is variation but conform to the same structure.

One side os hydrophobic (hydrophobic residues bind in a hydrophobic groove) and ones hydrophilic.

Receptors recognise the structure rather than the amino acids

Question 10

How are proteins translocated into a a bacterial membrane ? What are the 2 methods ?

Answer 10

1. Sec translocation pathway: The Sec pathway is the main pathway for protein translocation across the bacterial cytoplasmic membrane. The pathway is composed of the SecYEG translocon, which forms a protein-conducting channel in the membrane, and a set of accessory proteins that help to target and translocate the protein across the membrane.

The Sec pathway can translocate both soluble and membrane proteins, and can also be used for the insertion of membrane proteins into the membrane.

2. TOM complex translocates in the polypeptide chains, oubneed an additional chaperone to help polypeptide to assemble. SAM complex helps it assemble in the outer mitochondrial membrane