Lecture 12 - The Secretory Pathway

Question 1

Role of the rough ER in the secretory pathway?

Answer 1

Vesicle formation/budding to cis-cisternae of the Golgi.

Question 2

Role of the Golgi in the secretory pathway?

Answer 2

Post-translational modifications, budding from the trans-cisternae and vesicle formation.

Question 3

Three key pathways of the secretory system?

Answer 3

1. Constitutive endocytosis.
2. Regulated secretion (external stimulus required).
3. Signal mediated diversion to lysosomes.

Question 4

What happens to proteins in the ER lumen?

Answer 4

Modifications include:

1. Proteolytic cleavages, e.g. removal of the signal sequence by a signal peptidase.
2. Glycosylation (GOLGI).
3. Disulphide bond formation.
4. Folding and assembly of mutlisubunit proteins.

Question 5

Correct folding occurs in the ER. What family of proteins aid this process? How?

Answer 5

Chaperones - they catalyse folding by moving domains. Movement is powered by ATP hydrolysis.

Question 6

Disulphide bond formation?

Answer 6

The lumen of the secretory organelles are oxidising, promoting the formation of S-S bonds. Protein Disulphide Isomerase (PDI) makes sure incorrect S-S bonds are remodelled.

Question 7

Proline isomerisation?

Answer 7

Can either be in the cis or trans conformation. 80% in the trans conformation. Interconversion is accelerated by peptidylpropyl isomerases (PPI).

Question 8

Glycosylation can be N-linked. What does this mean?

Answer 8

Asparagine-linked. Protein in the ER lumen will have the following sequence:

N - any AA (except proline) - Ser/Thr

This sequence is recognised by oligosaccharide transferase. Adds carbohydrate moiety to N. The sugar chain produced will always contain N-acetyl glucosamine, mannose and glucose sugars.

Question 9

Glycosylation can be used to check if the protein has folded correctly. Explain this process.

Answer 9

1. 3 glucose molecules at the end are key.
2. Following folding = glucose trimming = leave 1 glucose molecule.
3. 1 glucose allows calnexin binding (chaperone), which determines whether the protein is folded.
4. Folded = glucosidase cleaves the glucose and the protein exits.
5. Incompletely folded = glucose molecules added back on and the molecule is retained.

Question 10

What response is triggered if unfolded proteins accumulate in the ER? What happens in this response?

Answer 10

Unfolded Protein Response (UPR). Upregulation in chaperone expression (sec61) to increase export into the cytoplasm. Once in the cytoplasm misfolded proteins are ubiquinated –> proteosome –> degraded.

Question 11

Proteins leave the ER via vesicular budding. Folded proteins that want to leave have…

Answer 11

An exit signal, e.g. dileucine motif. Bind to cargo receptors.

Question 12

Vesicles that form from the ER have a COPII coat. What does this consist of?

Answer 12

1. Sar1, which hydrolyses GTP, allowing Sec12 to bind.

2. GTP-Sar1 allows Sec23/24 binding (inner COPII coat) and Sec13/31 binding (outer COPII coat).

Question 13

How do these vesicles travel to the Golgi?

Answer 13

Travel along microtubules to carry proteins.

Question 14

ER resident proteins can get trapped in these COPII coated vesicles. How do they return to the ER?

Answer 14

COPI coat = ER resident proteins have a KDEL sequence that binds to a receptor on the Golgi membrane. Receptors bind to COPI proteins via an ER retrieval signal. Allows packaging into COPI coats.

Question 15

Glycosylation occurs in the Golgi. Functional role of glycans?

Answer 15

1. Folding in the ER (mentioned above).
2. Stability/protease resistance of proteins.
3. Immune response.
4. Signalling
5. Architecture of ECM and cell-to-matrix attachment.

Question 16

If proteins are destined for the lysosome, they are tagged with what as they travel through the Golgi?

Answer 16

Mannose-6-phosphate.

Question 17

Explain how the tagging of proteins with M6P results in lysosome degradation.

Answer 17

1. At the trans Golgi stack there is a M6P receptor.
2. The receptor uses a clathrin coat and directs the vesicle towards an early endosome.
3. In the early endosome, the V ATPase pumps in protons = more acidic pH. Causes hydrolase and M6P to dissociate from the receptor.
4. Early endosome –> late endosome –> lysosome.
5. Retromer coat used to retrieve M6P receptor and take it back to the Golgi.