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 your answers.
A) using a cytoplasmic extract from wild-type cells
B) using a cytoplasmic extract depleted of RanGAP
C) using a cytoplasmic extract obtained from wild-type 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 your answer.