Conserved histidine residues of RCC1 are essential for nucleotide exchange on Ran.
Charged amino acid residues of human RCC1 were converted to alanine and mutants which were unable to complement tsBN2 cells (a temperature-sensitive rcc1- mutant of the hamster BHK21 cell line) were selected. These RCC1 mutants were analyzed for the ability to inhibit premature chromatin condensation by microinjection into tsBN2 cells, and their steady-state kinetic parameters for guanine nucleotide exchange reaction were measured. Examined RCC1 mutants were unstable in tsBN2 cells at the restrictive temperature, yet they significantly inhibited premature chromatin condensation. Mutants located on the N-terminus of the RCC1 repeat showed an increased K(m), while their kcat values were comparable to that of wild-type RCC1. In contrast, mutants containing the conserved histidine residues in the C-terminus of the RCC1 repeat showed a value of K(m) similar to that of wild-type RCC1, while the kcat values of these mutants were reduced, depending upon the RCC1 repeats on which the mutation was located. These steady-state kinetic parameters of mutants indicate that the N-terminus and the C-terminus of RCC1 repeats play different roles in guanine nucleotide exchange on Ran. The comparison of kcat 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.
Azuma
Y
Y
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka.
Seino
H
H
Seki
T
T
Uzawa
S
S
Klebe
C
C
Ohba
T
T
Wittinghofer
A
A
Hayashi
N
N
Nishimoto
T
T
D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D018797:Cell Cycle Proteins; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism*; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005816:Genetic Complementation Test; D020662:Guanine Nucleotide Exchange Factors; D006150:Guanine Nucleotides / Q000378:metabolism*; D006639:Histidine / Q000502:physiology*; D006801:Humans; D007668:Kidney / Q000166:cytology; D007700:Kinetics; D008647:Mesocricetus; D008845:Microinjections; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism*; D013696:Temperature; D020931:ran GTP-Binding Protein
D018797:Cell Cycle Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; D020662:Guanine Nucleotide Exchange Factors; D006150:Guanine Nucleotides; D009687:Nuclear Proteins; C062521:RCC1 protein, human; D006639:Histidine; D020931:ran GTP-Binding Protein; D000409:Alanine