experiments for first midterm

Question 1

How was it determined that nucleoplasmin localizes in the nucleus?

Answer 1

Inject nucleoplasmin into cytosol.
After different amount of times, disrupt membrane and centrifuge.
Nucleus fraction is denser and falls to the bottom.
Separate nucleus and cytosol and run on SDS.
See that nucleoplasmin localizes in nucleus.

Question 2

How was it shown that the core pentamer does not enter the nucleus?

Answer 2

Trypsin digest of nucleoplasmin pentamer leads to removal of tail domains leaving only the core pentamer. When injected into cytosol, unable to enter nucleus.

Question 3

What were the three models for nuclear import regarding nucleoplasmin?

Answer 3

Model 1: tail contains a sequence required for nuclear import.
Model 2: Removal of tail exposes sequence that causes the protein to be retained in the cytoplasm.
Model 3: tail contains a sequence required for nuclear retention.

Question 4

How were models 2 and 3 rejected?

Answer 4

Model 3 rejected by injecting core pentamer into nucleus, saw that it didn’t leave.
Model 2 rejected by limited trypsin digest, resulting in different amount of tails on pentamer, only core pentamer couldn’t be imported into nucleus.

Question 5

Describe the assay to determine proteins required for nuclear import.

Answer 5

HSA cannot normally enter the nucleus but with nls (not mutated) can be imported. (sufficient).
HSA can be added to cells by treating membranes with digitonin. (permeabilized)
Need to add cytosol and ATP to rescue import.
Fractionated cytosol to determine soluble proteins required for import.

Question 6

What did fraction A contain and what did it do alone?

Answer 6

Importin alpha and beta, led to ATP-independent (but nls-dependent) perinuclear accumulation.

Question 7

What did fraction B contain?

Answer 7

Ran-GTPase, leads to import that is ATP-dependent.

Question 8

Describe the Gunter Blobel experiment to identify a signal sequence on proteins that are secreted from cells.

Answer 8

Disrupt membrane with detergent, homogenize cells and reconstitute ER. Centrifuge using sugar density centifugation. Take free ribosomes and rough micosomes.
Take B cells and add radioactively labeled i125.
Take free ribosomes, add translation factors, reticulocyte lysate and radioactively labeled amino acids, same thing with rough microsomes.
Get I125-labeled IgG.
Run on SDS gel with protease treatment, and without and compare to B-cell secreted IgG.
Free ribosome portion is larger and gets degraded by proteases, rough microsome one is mature size and protected from protease degradation

Question 9

How are sec mutants identified?

Answer 9

accumulation of proteins that are normally secreted, like sec, that are detected by an enzyme assay.

Question 10

Why do sec mutants fail to grow?

Answer 10

do not add new membrane to cell

Question 11

Why do cells become dense?

Answer 11

continued biosynthesis of proteins coupled to no increase in size or failure to secrete.

Question 12

Sec mutants can be rescued by?

Answer 12

shifting back to permissive temperature

Question 13

What are the steps for the genetic screen of sec mutants based on the temperature sensitive increase in relative density?

Answer 13

1 - grow billions of haploid wt yeast at permissive temperature
2- add chemical mutagen
3 - shift cells to restrictive for 3 hrs
4 - density gradient centrigugation
5- select densest cells
6 - plate individuals clones at permissive temperature
7 - test for ability to secrete invertase at 37C

Question 14

Complementation test

Answer 14

refer to notes

Question 15

double mutant epistatic test

Answer 15

refer to notes

Question 16

sec 4

Answer 16

rab GTPase, mutant for vesicle accumulation has it on vesicle but not membrane.

Question 17

in-vitro assay for Golgi transport

Answer 17

1 - purify Golgi from cells infected with VSV and that are mutant for glcnac transferase.
2- add recipient golgi that is wt for glcnac transferase and not infected
3- add ATP, cytosol and readiaoctively labeled glcnac
4- golgi transport can be monitored by the accumulation of radioactively labeled VSV-G
5- grind up entire reaction contents and IP using anti-VSV-G bound to beads.
6- measure radioactivity.
add GTPyS and see coated vesicles accumulate,
Don’t add ATP and see uncoated vesicles accumulate.

Question 18

How was the COP1 coat protein purified?

Answer 18

Compare purified vesicles with GTPyS treatment and without, proteins that difer in SDS-PAGE gel are part of coat.