Biochemical characterization of the Ran-RanBP1-RanGAP system: are RanBP proteins and the acidic tail of RanGAP required for the Ran-RanGAP GTPase reaction?
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.
Seewald
Michael J
MJ
Max-Planck Institut für Molekulare Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
Kraemer
Astrid
A
Farkasovsky
Marian
M
Körner
Carolin
C
Wittinghofer
Alfred
A
Vetter
Ingrid R
IR
D000595:Amino Acid Sequence; D001665:Binding Sites; D020690:GTPase-Activating Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D046911:Macromolecular Substances; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D020931:ran GTP-Binding Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D020690:GTPase-Activating Proteins; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D029702:Schizosaccharomyces pombe Proteins; C091775:ran-binding protein 1; D020931:ran GTP-Binding Protein