Lecture 12 - Overview of membrane traffic and methods to study it

Question 1

How are proteins targeted to different organelles?

Answer 1

Post-translational modification tags

Question 2

Protein movement pathway

Answer 2

First stop is the ER before entering the endomembrane system

Question 3

Cell-free assays

Answer 3

Investigation technique that doesn’t use the whole cell

• Permeabilised cells
• Purified membrane fractions + cytosol
• Purified proteins and lipids

Allow manipulation of conditions - temperature, energy, cytosolic/membrane proteins, etc

Question 4

Yeast

Answer 4

Cheap, easy to grow, amenable to genetic analysis (they exist haploids)

Question 5

Defects in either budding or fusion

Answer 5

Larger ER, smaller Golgi - defective in budding

Cytoplasm filled with lots of vesicles - defective in fusion

Question 6

GFP

Answer 6

Green fluorescent protein

Obtained from jellyfish

Intrinsically fluorescent

• No need to have an antibody
• Easy
• Can be used in live cells
• Big, may disrupt folding
• May interfere with assembly of multisubunit protein complexes

Question 7

Super resolution microscpoy

Answer 7

SIM STED PALM STORM

Allow viewing of ~20nm allowing vesicle viewing

Question 8

Electron microscope tomography

Answer 8

Cut a thick section, use powerful electron, tilting the section, eventually form a 3D representation