The thermolability of nuclear protein import in tsBN2 cells is suppressed by microinjected Ran-GTP or Ran-GDP, but not by RanQ69L or RanT24N.

Abstract

The nuclear protein regulator of chromosome condensation 1 (RCC1) stimulates guanine nucleotide exchange on a protein, Ran, that is required for nuclear protein import. In the present report, we confirm that RCC1 is also required for nuclear protein import in tsBN2 hamster cells in vivo. The thermolability of nuclear protein import in tsBN2 cells was suppressed by microinjection of purified Ran-GTP into the cytoplasm, but Ran-GDP also relieved the import deficiency, suggesting either that both forms of Ran are active in import in vivo or that tsBN2 cells at restrictive temperature retain a mechanism to convert Ran-GDP to Ran-GTP. To distinguish between these possibilities, nuclear protein import in tsBN2 cells was tested in the presence of Ran mutants, one deficient in GTP hydrolysis (RanQ69L), and one with weak binding to GDP and 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 inhibited import in vivo at the permissive temperature and failed to stimulate nuclear protein import at the nonpermissive temperature. The implications of these results for the roles of RCC1 and Ran in nuclear protein import in vivo are discussed.