Problem:

You are interested in how proteins are imported into and eported out of the nucleus. For your experiment you use several mutant cell lines or treat normal cells with abnormal conditions. You examine 2 different proteins - protein A is 100 kD and protein is 25 kD. Both proteins have both an NLS and an NES. Neither NLS or NES is ever masked.

Required:

Question 1: In Mutant 1 you can inactivate all Ran-GEF. You wait until the loss of Ran-GEF function results a steady state for Ran-GEF attached molecules. You then induce this mutant to start tranlating protein A. Where will protein A be found in these cells?Explain thoroughly.

Question 2: Instead of using a mutant cell line, this time you incubate normal cells so that they have an excess of GTP?S in both the cytosol and nucleus. GTP?S is a GTP analog that behaves exactly like GTP except that it cannot be hydrolyzed. You then induce these cells to start translating protein A. Where will protein A be found in these cells? Explain thoroughly.

Question 3: In Mutant 2 you can inactivate all NXF1 and NXT1 so that they have none of their normal functions. You induce this mutant to start translating protein B and inactivate NXF1 and NXT1 simultaneously. Where will protein B be found in these cells? Explain thoroughly.

Question 4: In Mutant 3 you can inactivate all importins instantly so that they have none of their normal functions. You induce this mutant to start translating protein B and inactivate importins simultaneously. Whre will protein B be found in thse cells? Explain thoroughly.