Biochemical characterization of Gyp6p, a Ypt/Rab-specific GTPase-activating protein from yeast.
Gyp6p from yeast belongs to the GYP family of Ypt/Rab-specific GTPase-activating proteins, and Ypt6p is its preferred substrate (Strom, M., Vollmer, P., Tan, T. J., and Gallwitz, D. (1993) Nature 361, 736-739). We have investigated the kinetic parameters of Gyp6p/Ypt6p interactions and find that Gyp6p accelerates the intrinsic GTPase activity of Ypt6p (0.0002 min(-1)) by a factor of 5 x 10(6) and that they have a very low affinity for its preferred substrate (K(m) = 592 micrometer). Substitution with alanine of several arginines, which Gyp6p shares with other GYP family members, resulted in significant inhibition of GAP activity. Replacement of arginine-155 with either alanine or lysine abolished its GAP activity, indicating a direct involvement of this strictly conserved arginine in catalysis. Physical interaction of the catalytically inactive Gyp6(R155A) mutant GAP with Ypt6 wild-type and Ypt6 mutant proteins could be demonstrated with the two-hybrid system. Short N-terminal and C-terminal truncations of Gyp6p resulted in a complete loss of GAP activity and Ypt6p binding, showing that in contrast to two other Gyp proteins studied previously, most of the 458 amino acid-long Gyp6p sequence is required to form a three-dimensional structure that allows substrate binding and catalysis.
Will
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Max Planck Institute for Biophysical Chemistry, Department of Molecular Genetics, D-37070 Göttingen, Germany.
Gallwitz
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D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D020798:Two-Hybrid System Techniques; D020691:rab GTP-Binding Proteins / Q000378:metabolism*
D017931:DNA Primers; D020690:GTPase-Activating Proteins; C079894:GYP6 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D011494:Protein Kinases; C060755:YPT1 protein, S cerevisiae; D020691:rab GTP-Binding Proteins