Studying Protein Targeting

Question 1

What are the two ways of studying protein targeting using the Microscopy/Fluorescence approach?

Answer 1

1. Immunostaining of dead cells fixed on a glass slide

2. Fluorescent imaging of live cells using fusions of test proteins with GFP

Question 2

Name antibody fluorescent dyes you can use

Answer 2

fluorescein (FITC), rhodamine, Cy3, Cy5 or the Alexa dyes

Question 3

What can you use GFP fusions for?

Answer 3

• tracking the progress of specific events in live cells, and see the effect of inhibiting steps on how long they take/whether they happen
• monitor the movelement of proteins themselves using FRAP

Question 4

What is FRAP?

Answer 4

fluorescence recovery after photobleaching
photobleach a patch of membrane with GFP-tagged proteins using intense illumination.
measure in real time how quickly the area is repopulated with GFP-fusion proteins

Question 5

What is VSVG?

Answer 5

A viral coat protein that enables entry into host cells.

Question 6

Why is VSVG important in protein targeting studies

Answer 6

You can do pulse chase experiments with fluorescent VSVG:
Heat HeLa cells to 40C to prevent proteins leaving the ER (cease secretory pathway)
Transfect HeLa cells with fVSVG and let it accumulate.
Lower temperature to 32 and secretory pathway resumes.

Question 7

How would you find out how long it takes for a protein to diffuse across the cytosol?

Answer 7

Using FRAP at the region of interest

The recovery curve is a proxy for how fast the protein is diffusing.

Question 8

What does FRET stand for?

Answer 8

Förster resonance energy transfer

Question 9

What is FRET used for?

Answer 9

monitoring the interactions between two proteins

Question 10

How does FRET work?

Answer 10

the two potentially interacting proteins need to be tagged with fluorophores or the appropriate fluorescent protein; one that absorbs a slightly lower energy than the other
You excite the protein tagged with higher energy FP and see if the other protein fluoresces by absorbing the light emitted from the protein you excited.
Measure lifetime of fluorescence with a photon counting detector

Question 11

What are the three approaches to investigating protein targeting?

Answer 11

• Microscopy/Fluorescence
• Biochemical approach
• Genetic approach

Question 12

How would you use biochemical assays to investigate protein targeting? (to identify targeting sequences or analyse conditions for a translocation mechanism)

Answer 12

1. In vitro transcription
2. In vitro translation
3. Post translational incubations
4. Run different post translational incubations on a gel and compare

Question 13

What happens in In vitro transcription

Answer 13

Mix your plasmid with gene of interest with RNA pol and nucleotides to make mRNA.

Question 14

What happens in In vitro translation?

Answer 14

Mix mRNA with cytosol preparation from rabbit reticulocytes lysates or wheat embyro lysates (containing ribosomes, initiation, elongation and termination factors etc).
Either add membranes or entire organelles to see if protein is targeted to it.
Add radioactive methionine to visualise it on a gel as only small amounts of proteins can be made in vitro.

Question 15

What happens with post translational incubations?

Answer 15

When translated with an organelle present, if a protein can successfully translocate into the membrane, then it will be protected in the membrane.
Add proteases to degrade all proteins outside the membrane.
Then add detergent to remove the membrane and analysis will tell us whether the protein had translocated

Question 16

What control tests need to be done during biochemical assays?

Answer 16

Two control lanes:
one with just the protein, and one with the protein and protease to show that the protein is intrinsically sensitive to the protease.

Question 17

What tests need to be done to show that the organelle protects the protein?

Answer 17

Two tests in the presence of the organelle, one with the protease and one without to show that in both cases, the protein is not degraded.
Another test adding a detergent to remove the membrane should show that the protein is degraded.

Question 18

What tests need to be done to show whether the protein translocates post-translationally or co-translationally?

Answer 18

Repeat the tests but instead, add the organelle after translation. If you get the same results, translocation is post translational. If the protein is not preserved, it was co-translational.

Question 19

What does the genetic approach to studying protein targeting?

Answer 19

Using mutant yeast cells akak /sec/ mutants which cannot secrete proteins at 37C, so proteins accumulate and the density of the cells increase

Question 20

What are the 5 classes of /sec/ mutants?

Answer 20

Class A = secretory proteins accumulate in the cytosol
Class B = accumulate in the ER
Class C = accumulate in ER-Golgi transport vesicles
Class D = accumulate in the Golgi
Class E = accumulate in secretory vesicles near cell surface

Question 21

How have /sec/ mutants enabled the steps in the secretory pathway to be ordered?

Answer 21

By making double mutants.
e.g. a class A and B mutant had proteins accumulate in the ER like a class A mutant, suggesting that proteins go through the ER before the Golgi.

Question 22

How can you find the mutant gene in a /sec/ mutant?

Answer 22

Nowadays we can compare the mutant genome with a reference wildtype genome.
But in the past, it was done with complementation.

Question 23

How does complementation work to identify mutant genes?

Answer 23

A library of 6000 plasmids, each with a single wildtype yeast gene, were each transfected into /sec/ mutants.
Identify the cells that can grow at 37C.
Rescue the plasmid from wild-type-like cells and identify the gene which is COMPLEMENTING the mutant gene.

Question 24

What is a new approach for the study of the secretome?

Answer 24

Genome-wide RNAi screening

Question 25

What does Genome-wide RNAi screening involve?

Answer 25

Using VSVG, which is normally targeted to the plasma membrane.
Add siRNA with reverse transfection (growing cells on immobilised siRNA) to silence a variety genes and see if VSVG goes to the plasma membrane using immunostaining.
This allows you to screen for new components of the secretion machinery.

Question 26

Why could Genome-wide RNAi screening not identify all components of the secretion machinery?

Answer 26

Because there is a lot of genetic redundancy. Entire families could not be identified because others would replace them.

Question 27

Which paper were the genes required for the secretory pathway in sec mutants discovered?

Answer 27

Novick et al (1980)