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

Question

You are studying nuclear protein import in human cells. To test the requirements for the Ran protein, you have set up an in vitro nuclear import assay. In this assay, human tissue culture cells are treated with digitonin, which permeabilizes 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 a fluorescently-labeled protein that contains a classic NLS that you can monitor by fluorescent microscopy. Thus, you add cytosolic extract + Ran + fluorescent 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 your 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, the Ran-GTP enters the cell., its lysis releases the NLS containing protein bound to the importin, and thus, the protein will be translocated to the nucleus. Fluorescence can be observed in the nucleus in this case.

B) When Ran-GDP is added, there is no GTP to exchange P or energy. There is no tranlocation of proteins sicne recylcing of GDP-GTP do not occur.

C) When RanGAP inhibitor is added, it inhibits the GAP protein. This protein is required for hydrolysis of GTP so that NLS proteins bind to importin. In the absence of GTP, binding of protein to am importin do not occur.

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