Conserved Histidine Residues of RCC 1 Are Essential for Nucleotide Exchange on Ran

Abstract

Charged amino acid residues of human RCC 1 were converted to alanine and mutants which were unable to complement tsBN 2 cells (a temperature-sensitive reel- mutant of the hamster BHK 21 cell line) were selected. These RCC 1 mutants were analyzed for the ability to inhibit premature chromatin condensation by microinjection into tsBN 2 cells, and their steady-state kinetic parameters for guanine nucleotide exchange reaction were measured. Examined RCC 1 mutants were unstable in tsBN 2 cells at the restrictive temperature, yet they significantly inhibited premature chromatin condensation. Mutants located on the N-terminus of the RCC 1 repeat showed an increased K_m, while their k_cat values were comparable to that of wild-type RCC 1. In contrast, mutants containing the conserved histidine residues in the C-terminus of the RCC 1 repeat showed a value of K_m similar to that of wild-type RCC 1, while the k_cat values of these mutants were reduced, depending upon the RCC 1 repeats on which the mutation was located. These steady-state kinetic parameters of mutants indicate that the N-terminus and the C-terminus of RCC 1 repeats play different roles in guanine nucleotide exchange on Ran. The comparison of kcal among the histidine mutants suggests that those histidine residues which are conserved in the RCC1 repeats and also through evolution comprise the catalytic site for the guanine nucleotide exchange reaction.