Import of proteins into the nucleus requires binding to Importin-? in the cytoso
ID: 37973 • Letter: I
Question
Import of proteins into the nucleus requires binding to Importin-? in the cytosol and release inside the nucleus by association with RanGTP (due to the action of RanGEF). All import receptors then leave the nucleus bound to RanGTP. A key element of the model talked about in class is that these factors leave the nucleus and thus would be effectively depleting the nucleus of Ran. The only way Ran would stay high in the nucleus (and in GTP bound form) would be if it is re-imported back into the nucleus. The experiments below were designed to identify how Ran was imported back into the nucleus.
First an in vitro assay to analyze import of protein Ran was set up. In this assay cells in culture are treated with a mild nonionic detergent Digitonin. Digitonin permeabilizes the membrane such that the cytosolic constituents leak out BUT leaves the nuclear envelope and NPCs intact!
Now experimenters can control what they are adding back to assay. And import of proteins can be visualized by using fluorescent versions of proteins being imported
A) The researchers attached a flourescent tag to RanGDP and observed for RanGDP localization in just the presence of an energyregenerating system (needed for import) and without any further addition (minus cytosol image) and in the presence of energy-regenerating system + addition of cytosolic extract. (+ cytosol image)
(1.5pts) Where does RanGDP localize in the absence of cytosol?
(1.5pt) Where does RanGDP localize in the presence of cytosol?
They then compared the import of fluorescent Ran pre loaded with GDP with the import of a mutant version of Ran- RanQ69L pre-loaded with GTP. This Ran is always in its GTP bound state regardless of RanGAP presence.
(3pts) Why do you think the researchers wanted to compare RanGDP with RanQ69LGTP and what can you conclude from their results?
Explanation / Answer
in absence of cytosol, the Ran GDP importin complex will not be separated by Ran BP and thus, RanGDP will not translocate to the nucleus. stay out of the nucleus.
when cytosol is present the RanGDP importin complex can be separated and thus, it will then translocate into the nucleus.
they compared the two to find out if GTP or bound GDP plays a role in transport into the nucleus. also, they compared Ran GDP with Ran Q69LGTP mutant form to determin if the function of GAP affects the import or it is the Ran GDP or RanGTP that affects import.
since mutant Ran Q69LGTP remains in GTP form regardless of presence of GAP it indicates that GTP bound Ran will not translocate into the nucleus.