CBS - Protein Targeting

Question 1

What is the reason for protein targeting?

Answer 1

Cellular function in compartmentalised. The function is defined by proteins, and these proteins have to be directed to different parts of cell.

These places can include:

• organelles
• membrane proteins
• secreted proteins
• cytosolic proteins

Question 2

Describe protein synthesis at the ER.

Answer 2

1. Protein synthesis starts with a cytosolic ribosome
2. Signal sequence is first part protein synthesised (N- terminus).
3. Signal sequence recognised by Signal Recognition Particule (SRP) protein complex
4. SRP binds the SRP-receptor on ER membrane
5. Ribosome now bound to ER membrane (rER)
6. New protein is guided through translocon (translocation channel) in ER membrane into the ER lumen
7. Signal sequence cleaved via signal peptidase and SRP is recycled

Question 3

What happens to the proteins once they enter the ER?

Answer 3

The newly synthesised protein can then end up either completely within the lumen of the endoplasmic reticulum, or it could be within the membrane.

For this to happen, as before, the newly synthesised protein is threaded through the membrane, but there is another set of proteins that act as stop transfer sequences(20-22 amino acids), which stop the threading, and that then triggers the termination of the process.

The protein then becomes embedded in the membrane, and the stop transfer sequences remain as transmembrane segments of proteins.

Question 4

How are proteins transported from the ER to the Golgi?

Answer 4

The ER is in close proximity to Golgi, which consists of flat vesicles called cisternae. Things move from the ER to the Golgi via the vesicles, which readily merge with the membrane of the Golgi.

Question 5

Describe the ER-Golgi interactions.

Answer 5

Proteins are synthesised, folded and modified in the ER. They then form vesicles that migrate and merge with cis-Golgi.

Further protein maturation occurs in Golgi. Movement between cisterna of Golgi can occur via vesicles.

In the trans-Golgi, mature proteins are packaged in vesicles for specific compartments.

Vesicles transported along specific cytoskeleton pathways, such as:

• integral membrane proteins / membrane phospholipid
• continuous secretory pathway e.g. extracellular matrix proteins
• stimulated secretory pathway, stored vesicles e.g. insulin

Question 6

How does a vesicle know where it needs to go to?

Answer 6

This is done by proteins called SNARE, v for the one on the vesicle and t for the one on the target. Once they bind to each other, they facilitate the fusion of that membrane.

Question 7

What occurs to proteins that don’t go through the ER?

Answer 7

Some proteins are synthesised in the cytoplasm, and become cytoplasmic proteins.

Others are transported to other organelles, such as the peroxisome, nucleus and mitochondria.

Question 8

How do proteins get targeted to the mitochondria?

Answer 8

Targeting to the mitochondria happens after translation of the protein is completed, but before complete protein folding occurs.

The protein is complexed with a chaperone protein (HSP70, head shock protein 70).

There is an additional signal sequence on the chaperone protein that binds to the receptor on the mitochondria.

The nuclear proteins contain Nuclear Localisation Signal (NLS) - Pro-Lys-Lys-Lys-Arg-Lys-Val (PKKKRKV), which is basic.

The NLS binds to importin, which facilitates transport through the nuclear pore. It requires G-protein Ran and energy by hydrolysis of GTP.

This then facilitates the movement of that protein through a translocator into the mitochondrial matrix, which is where the signal sequence is cleaved off. Then, the proteins is folded into its final state.

Exportin performs the reverse function to exporting proteins from nucleus.

Question 9

How are proteins targeted to lysosomes?

Answer 9

Lysosomal proteins are tagged with mannose-6- phosphate in the Golgi.

There is a mannose-6-phosphate receptor in the Golgi, which then directs proteins into transport vesicles.

This vesicle then becomes the lysosome.

Question 10

Describe the protein targeting disorder, Inclusion-cell (I cell) diseases.

Answer 10

There is a mutation in one of the enzymes that is involved in mannose targetting

It is a mutant N-acetylglucosamine-1- phosphotransferase enzyme which normally phosphorylates mannose.

The proteins for lysosomes are not tagged with mannose-6-phosphate in Golgi and do not reach lysosomes. These enzymes are secreted instead.

Thus, lysosomes do not function properly. This affects all tissues, and results in slow growth and developmental defects.

I-cell disease patients normally die before the age of 10 of heart failure or pneumonia.