Overview of membrane trafficking

Question 1

What are the major organelles of the secretory pathway and their functions?

Answer 1

(1) Endoplasmic Reticulum.
-Newly synthesized proteins inserted
-Folding
- N-linked glycosylation
- Quality Control

(2) Golgi apparatus.
-N-linked oligosaccharides modified.
-Sorting to various destinations occurs (cell surface, secretory granules, endosomes)

Question 2

What was George Palade Analyzing?

Answer 2

• used electron microscopy and membrane fractionation to define the secretory pathway in cells.
• outlined how proteins move through a cell after being synthesized:
  ER -> Golgi apparatus -> secretory granules -> cell surface.

Question 3

What is Autoradiography? How does it work?

Answer 3

• method used to track proteins inside cells.

steps:
1. labelling:
- living cells exposed to tritium-tagged amino acids.
- tritium emits low-energy beta particles, which only travel short distances, making it ideal for precise localization.

2. sample preperation:
   - cells were fixed and embedded in plastic resin (like Epon) for EM.
   - thin sections of the sample were prepared.
3. exposure to emulsion:
   - a photo-sensitive silver emulsion was placed over the sample.
   - the sample was stored in the dark a low temperature to let the radioactive decay expose the emulsion.
4. development:
   - after exposure, the emulsion was developed like a photograph.
   - metallic silver grains formed where radioactivity had been emitted, and these could be seen under electron microscope.

Question 4

What is the pulse-chase technique? how does it work?

Answer 4

• helped track how proteins moved through the secretory pathway over time.

steps:
1. Pulse (label new proteins):
- cells briefly exposed to radioactive amino acids (tritium-labeled).
- cells use those radioactive amino acids to make new proteins.
- all the proteins made during ‘pulse’ are now radioactive and trackable.

2. Chase (stop labeling and follow labeled proteins):
   - the radioactive label was removed and replaced with normal amino acids.
   - now, any new proteins made won’t be radioactive.
   - scientist then watched where the radioactive proteins went at different time points (using techniques such as autoradiography)

Question 5

what findings were gathered from pulse-chase experiment?

Answer 5

• proteins first seen in ER, then golgi, then secretory granules near cell surface.
• first direct evidence for the order of protein movement in secretory pathway.

Question 6

What are the steps of vesicular transport? (moving cargo between organelles inside a cell)

Answer 6

1. Sorting of cargo - selecting the right molecules to package.
2. Budding - forming a vesicle from the source membrane.
3. Separation - vesicles detaches from the membrane.
4. Transfer - movement of the vesicle to the target area.
5. Storage (optional) - vesicles may wait before fusing (synaptic vesicles)
6. Recognition and Fusion - vesicles find the correct target membrane and delivers its cargo.

Question 7

What is the function of coats in vesicle formation?

Answer 7

• coats help shape the budding vesicle and select the right cargo.
• coats; clathirn/adaptin, COPI, COPII, Caveolin, Retromer.

Question 8

What is the function of molecular motors in vesicle formation?

Answer 8

• After budding, vesicles are transported through the cell using motor proteins and cytoskeletal tracks.

1. Dynein (use MTs -> towards (-) end (cell centre))
2. Kinesin (use MTs -> towards (+) end (cell edge))
3. Myosin (use Actin Filaments -> short distance movement)

Question 9

What is the function of tethering proteins and SNARE proteins in vesicle formation?

Answer 9

• once the vesicle reaches its destination, two types of proteins ensure correct fusion.

1. tethering proteins
   - act like ‘docking’ factors
   - work with Rab GTPases to recognize the correct target membrane.
2. SNARE proteins
   - mediate membrane fusion
   - there are two types:
3. v-snares (vesicle)
4. t-snares (target membrane)
   - each organelle has own specific SNAREs, helping ensure correct targeting.