You are studying nuclear protein import in human cells. To test the requirement

Question

You are studying nuclear protein import in human cells. To test the requirement for the Ran protein, you have set up an in vitro nuclear import assay. In this essay, human tissus culture cells are treated with digitonin, which permealizies the plasma membrane but leaves the nuclear envelope intact. Since permeabilization results in the release of all soluble cytoplasmic contents, you need to supplement the assay with cytosolic extracts that you have prepared from other cells. In this case, you are using extracts from cells that do not express the Ran protein so that you can supply recombinant ran complexed with various nucleotides. To measure nuclear import you are using fluoresently-labeled protein that contains a classic NLS that you can monitor by fluorescent microscopy. Thus, you add cytosolic extract + Ran + fluorenscent nuclear protein to the permeabilized cells and examine the nuclei for import of the nuclear protein. what do you expect to observe under the following conditions/ Briefly explain you answers to choices.

Note: assume that ALL proteins are in their normal location and that the only source of Ran is the recombinant protein that you add to the assay.

A) what do you predict happens when you add Ran-GTP to the assay?

B) what do you predict happens when you add Ran-GDP to the assay?

C) what do you predict happens when you add Ran-GTP and Sinocol, a small 300 Da RanGAP inhibitor?

Explanation / Answer

A. If you add Ran-GTP to the assay, it needs to be hydrolysed to form Ran-GDP so that it can bind to importin-protein complex for importing the protein into the nucleus as only Ran-GDP can bind to importin-protein complex. So the protein will be imported into the nucleus and will get a positive signal.

B. If you add Ran-GDP to the assay, then also the protein along with importin will bind to Ran-GDP and will be imported into nucleus which will give a positive signal.

C. In this case when you add Ran-GTP along with sinocol which inhibits the RanGAP, then the Ran-GTP cannot be converted into Ran-GDP for binding with importin and protein complex. So the protein will not be imported into nucleus due to the absence of Ran-GDP and you will not get any flourescence signal.

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