THE ASYMMETRIC DISTRIBUTION OF THE CONSTITUENTS OF THE RAN SYSTEM IS ESSENTIAL FOR TRANSPORT INTO AND OUT OF THE NUCLEUS

The GTPase Ran is essential for nuclear import of proteins with a clas sical nuclear localization signal (NLS), Ran's nucleotide-bound state is determined by the chromatin-bound exchange factor RCC1 generating R anGTP in the nucleus and the cytoplasmic GTPase activating protein Ran GAP1 depleting RanGTP from the cytoplasm, This predicts a steep RanGTP concentration gradient across the nuclear envelope, RanGTP binding to importin-beta has previously been shown to release importin-alpha fro m -beta during NLS import, We show that RanGTP also induces release of the M9 signal from the second identified import receptor, transportin , The role of RanGTP distribution is further studied using three metho ds to collapse the RanGTP gradient, Nuclear injection of either RanGAP 1, the RanGTP binding protein RanBP1 or a Ran mutant that cannot stabl y bind GTP. These treatments block major export and import pathways ac ross the nuclear envelope, Different export pathways exhibit distinct sensitivities to RanGTP depletion, but all are more readily inhibited than is import of either NLS or M9 proteins, indicating that the block of export is direct rather than a secondary consequence of import inh ibition, Surprisingly, nuclear export of several substrates including importin-alpha and -beta, transportin, HIV Rev and tRNA appears to req uire nuclear RanGTP but may not require GTP hydrolysis by Ran, suggest ing that the energy for their nuclear export is supplied by another so urce.