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) When RAN-GTP is added to the assay then RAN-GAP will convert RAN-GTP to RAN-GDP.

B) If RAN-GDP is added to the assay then the gradient of RAN-GTP/RAN-GDP will increase as there will be RAN-GDP apart from the one converted via RAN-GTP.

C) If RAN-GTP and Sinocol are added then RAN-GTP/RAN-GDP gradient will decrease until RAN-GTP amount was equal in the nucleus and the cytosol.

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