Microbiology 11: (Prigent) Secretory pathway

Question 1

Types + Examples of secreted proteins? Which cells are they secreted from?

Answer 1

Extracellular Matrix proteins
- Collagen, Fibronectin, Laminin -> Fibroblasts

Serum proteins

• Immunoglobulins (B-cells)
• Apolipoproteins, Albumin (Hepatocytes)

Hormones

• Insulin, Glucagon (Beta, alpha islet cells)
• Beta-endorphin, ACTH (Pituitary)

Digestive Enzymes

• trypsin (Pancreatic)
• Chymotripsin (Acinar)
• Amylase, nucleases

Question 2

Example of proteins sorted by the secretory pathway?

Answer 2

Plasma membrane Proteins - EGF receptor

ER resident proteins - Protein disulphide isomerase

Golgi resident proteins - Golgi mannosidase

Lysosomal enzymes - Cathepsins (proteases)

Question 3

Describe the classic pulse-chase experiments of Jamieson and Palade in 1967

Key findings?

Answer 3

Designed to define kinetics and pathway of transport of newly synthesised proteins

Experiment

1) Slices of guineapig pancreas prepared
2) Pulse to separate contents, medium contains radioactive tritiated leucine -> followed by incubation, microsomes form
3) ‘Chase’ performed, medium switched for non-radioactive leucine
4) Tissue fixed and processed for EM autoradiography

Key Findings:

• Secreted proteins first pass through golgi on way out of the cell
• NEVER mix with cytosolic proteins - remain in vesicles
• Choice of cell model v important (specialised to secrete proteins)

Question 4

Describe experiment studying protein flow from the ER to the plasma membrane using GFP

Answer 4

Use of GFP tagged vesicular stomatitis virus-G protein

• > Insert into cells via transfection
• > Heat to 40 degrees to ensure protein cannot initially leave ER due to misfolded configuration
• > 32 degrees to return to normal function

-> follow movement of protein throughout pathway

Question 5

What happens to ribosomes during translation of:
1) Proteins destined for membrane or secretory pathway / Co-translational insertion

2) Cytosolic proteins / post-translational import into organelles

Answer 5

1)
-> Ribosomes attach to ER membrane

2)
-> Ribosomes remain cytosolic

Question 6

General requirements for protein sorting?

Answer 6

A signal (address) intrinsic to the protein

Receptor which directs the signal and which directs it the the correct membrane

Translocation machinery

Energy to transfer protein to new place

Question 7

Where are signal sequences located? Why is it needed?

Answer 7

N-terminus of secretory / transmembrane proteins
-> needed for transport cotranslational transport into microsomes

(signal sequence removed in process)

Question 8

Structure and function of SRP?

Answer 8

Signal-recognition particle (SRP)

• multidomain riboprotein that mediates a 3-way association with the SRP-receptor in the ER, the ribosome and the signal peptide
  i. e aids insertion of proteins to ER

Hydrophobic pocket of SRP binds to signal sequence

• > interaction of complex and SRP receptor
• > SRPR activation opens translocon -> insertion
• > cleavage of signal sequence by signal peptidase,

Results in full insertion of cleaved protein to ER

Question 9

Mechanism of type I membrane protein synthesis + membrane insertion

Answer 9

Same as secretory protein, but

with Type I membrane protein, a second hydrophobic sequence exists which anchors protein to membrane
-> known as stop transfer anchor sequence

Question 10

What determines orientation of Type II, III, IV proteins in membrane?

Answer 10

Determined by presence of positively charged amino acids adjacent to the hydrophobic membrane spanning region

Positive charge rule = positive residues always on the cytosolic face of the membrane