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

• DOI: 10.1128/mcb.23.22.8124-8136.2003
• Publication Date: 2003-10-30T04:03:24Z
• Authors: Michael J. Seewald; Astrid Kraemer; Marian Farkasovsky; Carolin Kürner; Alfred Wittinghofer; Ingrid R. Vetter
• Keywords: Models, Molecular; 0301 basic medicine; Binding Sites; Sequence Homology, Amino Acid; Macromolecular Substances; GTPase-Activating Proteins; Molecular Sequence Data; Nuclear Proteins; Saccharomyces cerevisiae; In Vitro Techniques; Models, Biological; Recombinant Proteins; Kinetics; 03 medical and health sciences; Spectrometry, Fluorescence; ran GTP-Binding Protein; Schizosaccharomyces; Mutagenesis, Site-Directed; Humans; Amino Acid Sequence; Schizosaccharomyces pombe Proteins; Sequence Deletion

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 P(i). 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 10(8) 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.