THE THERMOLABILITY OF NUCLEAR-PROTEIN IMPORT IN TSBN2 CELLS IS SUPPRESSED BY MICROINJECTED RAN-GTP OR RAN-GDP, BUT NOT BY RANQ69L OR RANT24N

The nuclear protein regulator of chromosome condensation 1 (RCC1) stim ulates guanine nucleotide exchange on a protein, Ran, that is required for nuclear protein import. In the present report, we confirm that RC C1 is also required for nuclear protein import in tsBN2 hamster cells in vivo. The thermolability of nuclear protein import in tsBN2 cells w as suppressed by microinjection of purified Ran-GTP into the cytoplasm , but Ran-GDP also relieved the import deficiency, suggesting either t hat both forms of Ran are active in impart in vivo or that tsBN2 cells at restrictive temperature retain a mechanism to convert Ran-GDP to R an-GTP. To distinguish between these possibilities, nuclear protein im port in tsBN2 cells was tested in the presence of Ran mutants, one def icient in GTP hydrolysis (RanQ69L), and one with weak binding to GDP a nd little or no binding to GTP (RanT24N). Microinjection of the mutant RanQ69L inhibited import in vivo in either the GTP- or GDP-bound form at both the permissive and nonpermissive temperatures, RanT24N-GDP in hibited import in vivo at the permissive temperature and failed to sti mulate nuclear protein import at the nonpermissive temperature, The im plications of these results for the roles of RCC1 and Ran in nuclear p rotein import in vivo are discussed.