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Molecular and Cellular Biology, November 2003, p. 8124-8136, Vol. 23, No. 22
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.22.8124-8136.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Biochemical Characterization of the Ran-RanBP1-RanGAP System: Are RanBP Proteins and the Acidic Tail of RanGAP Required for the Ran-RanGAP GTPase Reaction?

Michael J. Seewald, Astrid Kraemer, Marian Farkasovsky, Carolin Körner, Alfred Wittinghofer,* and Ingrid R. Vetter

Max-Planck Institut für Molekulare Physiology, 44227 Dortmund, Germany

Received 7 April 2003/ Returned for modification 28 May 2003/ Accepted 4 August 2003

RanBP type proteins have been reported to increase the catalytic efficiency of the RanGAP-mediated GTPase reaction on Ran. Since the structure of the Ran-RanBP1-RanGAP complex showed RanBP1 to be located away from the active site, we reinvestigated the reaction using fluorescence spectroscopy under pre-steady-state conditions. We can show that RanBP1 indeed does not influence the rate-limiting step of the reaction, which is the cleavage of GTP and/or the release of product Pi. It does, however, influence the dynamics of the Ran-RanGAP interaction, its most dramatic effect being the 20-fold stimulation of the already very fast association reaction such that it is under diffusion control (4.5 x 108 M-1 s-1). Having established a valuable kinetic system for the interaction analysis, we also found, in contrast to previous findings, that the highly conserved acidic C-terminal end of RanGAP is not required for the switch-off reaction. Rather, genetic experiments in Saccharomyces cerevisiae demonstrate a profound effect of the acidic tail on microtubule organization during mitosis. We propose that the acidic tail of RanGAP is required for a process during mitosis.

* Corresponding author. Mailing address: Max-Planck Institut für Molekulare Physiology, Otto-Hahn-Str. 11, 44227 Dortmund, Germany. Phone: 49-231-1332100. Fax: 49-231-1332199. E-mail: Alfred.Wittinghofer{at}mpi-dortmund.mpg.de.

Molecular and Cellular Biology, November 2003, p. 8124-8136, Vol. 23, No. 22
0022-538X/03/$08.00+0     DOI: 10.1128/MCB.23.22.8124-8136.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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