CELL BIO ASSIGNMENT

1. Four proteins (a, b, c & d) are being tested. Cell homogenates are prepared and treated with 0.1M KCl, 2M KCl, lipase, or TX-100 and then centrifuged. The presence of the proteins in either the supernatant or pellet is then determined by immunoblot. From the results it can be seen that some of the proteins are recovered in the pellet fraction under certain conditions. What is causing them to pellet? What can you surmise about each protein based on its behavior under the different conditions?



2. Predict the properties of the lipid bilayer that would result if the following were true:
A. Phospholipids had only one hydrocarbon chain instead of two.
B. The hydrocarbon chains were shorter than normal, say about 10 carbon atoms long.
C. All of the hydrocarbon chains were unsaturated.
D. All of the hydrocarbon chains were saturated.
E. The bilayer contained a mixture of two kinds of lipid molecule, one with two saturated hydrocarbon tails and the other with two unsaturated hydrocarbon tails.
F. Each lipid molecule were covalently attached through the end carbon atom of one of its hydrocarbon chains to a lipid molecule in the opposite monolayer.

3. Drugs are often not as specific as we would hope. An example is cytocholasin B. While it is often used as an inhibitor of actin-based motility, it also potently inhibits D-glucose uptake into cells. When red cell ghosts are incubated with 3H-cytocholasin B and then irradiated with UV light the cytocholasin B becomes covalently crosslinked to GLUT1. In contrast, if the experiment is carried out in the presence of excess D-glucose then cytocholasin B is not crosslinked to GLUT1. However, L-glucose addition does not block the crosslinking. Why does D-glucose, but not L-glucose, prevent crosslinking?
4. Now lets say you want to get fancy. You are trying to demonstrate to your Cell Biology class an important aspect of GLUT1 function. You microinject into intact cells a form of cytocholasin B that is impermeant to the PM. This fills the cells. When you irradiate these cells in glucose free buffer cytocholasin B is not crosslinked to GLUT1. However, if you add normal amounts of glucose to the outside of the cells just before irradiation the cytocholasin becomes crosslinked to the GLUT1 as observed for the ghosts. What principle are you demonstrating and how?

1. How is selectivity achieved in the gating mechanism at the nuclear pore? Explain mechanism.



2. To demonstrate your understanding of the “force” responsible for nuclear transport your idea is to achieve import of a cargo bearing only an export signal (NES) in a assay. To permeabilized cells you add the fluorescent NES-protein, its export receptor and RanQ69L-GTP, a version that is hydrolysis defective. You observe Ran-dependent import! But unlike the 100% accumulation observed for a conventional imported protein with GTP, the maximum nuclear concentration achieved is no higher than the cytoplasmic concentration. Why? And what accounts for the 100% accumulation of conventional proteins?


3. What would happen if wild-type Ran were mutated to a form that can bind GTP but cannot hydrolyze GTP?

4. What experimental steps would you take to define the exact sequence and position of a nuclear import signal in a nuclear protein that you discovered?

5. What steps would you take to identify your protein's import receptor?

6. What is the function of a GEF?

7. What is the function of a GAP?