You are studying nuclear protein import in human cells. You have set up an in vitro nuclear import assay. In this assay, normal human tissue culture cells are treated with digitonin, which permeabilizes the plasma membrane but leaves the nuclear envelope intact. You then collect the permeabilized cells and since permeabilization results in the release of all soluble cytoplasmic contents, you need to supplement the assay with cytoplasmic extracts that you have prepared from other cells. To measure nuclear import, you are adding a fluorescently-labeled protein that contains a classic NLS to the permeabilized cells that you can monitor by fluorescent microscopy. Thus, you add cytoplasmic extract fluorescent nuclear protein permeabilized cells and then examine the nuclei of the permeabilized cells for import of the nuclear protein. Do you expect to observe nuclear protein import in the following situations? Briefly explain. A) Using a cytoplasmic extract from wildtype cells.
B) Using a cytoplasmic extract depleted of RanGAP.
C) Using a cytoplasmic extract obtained from wildtype cells but to which a very high concentration of a non- hydrolyzable form of GTP has been added.
D) In a separate line of experiments, you are studying a protein that you suspect shuttles between the nucleus and the cytoplasm. However, when you localize a GFP-tagged fusion protein, it appears to be only in the nucleus. To test whether it shuttles, you set up a heterokaryon assay. You take a cell expressing the GFP fusion protein and partially fuse that cell with another that does not express the GFP protein. The result is a heterokaryon that has a single cytoplasm but two separate nuclei. You treat the heterokaryon with the translation inhibitor cycloheximide for several hours and then examine the localization of the GFP protein. What do you predict will be observed if the GFP protein shuttles between the nucleus and the cytoplasm? Briefly explain.