Interaction of the nuclear GTP-binding protein Ran with its regulatory proteins RCC1 and RanGAP1.
The guanine nucleotide dissociation and GTPase reactions of Ran, a Ras-related nuclear protein, have been investigated using different fluorescence techniques to determine how these reactions are stimulated by the guanine nucleotide exchange factor RCC1 and the other regulatory protein, RanGAP1 (GTPase-activating protein). The intrinsic GTPase of Ran is one-tenth of the rate of p21ras and is even lower in the Ran(Q69L) mutant. Under saturating conditions the rate constant for the RanGAP1 stimulated GTPase reaction is 2.1 s-1 at 25 degrees C, which is a 10(5)-fold stimulation, whereas RanGAP1 has no effect on Ran(Q69L). The intrinsic guanine nucleotide dissociation rates of Ran are also very low and are likewise increased 10(5)-fold by the exchange factor RCC1. Methods to describe the reaction kinetically are presented. The Ran(T24N) mutant, which is analogous to the S17N mutant of p21ras, has decreased relative affinities for both GDP/GTP and favors GDP binding. However, it was found to interact almost normally with RCC1. The combination of these properties leads to stabilization of the Ran(T24N)-RCC1 complex and may result in vivo in depletion of RCC1 available for stimulating guanine nucleotide exchange.
Klebe
C
C
Max-Planck-Institut für molekulare Physiologie, Dortmund, Germany.
Bischoff
F R
FR
Ponstingl
H
H
Wittinghofer
A
A
D000818:Animals; D001483:Base Sequence; D018797:Cell Cycle Proteins; D004247:DNA; D004268:DNA-Binding Proteins / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D011994:Recombinant Proteins; D013050:Spectrometry, Fluorescence; D014981:Xenopus; D029867:Xenopus Proteins; D020931:ran GTP-Binding Protein; D020703:ras GTPase-Activating Proteins
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins; D011506:Proteins; C499304:RCC1 protein, Xenopus; D011994:Recombinant Proteins; D029867:Xenopus Proteins; D020703:ras GTPase-Activating Proteins; D004247:DNA; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020931:ran GTP-Binding Protein