The intrinsic GTPase activity of the Gtr1 protein from Saccharomyces cerevisiae

ABSTRACT: BACKGROUND: The Gtr1protein of Saccharomyces cerevisiae is a member of the RagA subfamily of the Ras-like small GTPase superfamily. Gtr1 has been implicated in various cellular processes. The Switch regions in Gtr1 mediate activation of the TORC1 complex [R. Gong, L. Li, Y. Liu, P. Wang, H. Yang, L. Wang, J. Cheng, K.L. Guan, Y. Xu, Genes Dev. 25 (2011) 1668-1673]. Therefore, knowledge about the biochemical activity is required to understand its mode of action and regulation. RESULTS: Here we employ tryptophan fluorescence analysis and radioactive GTPase assays to demonstrate that Gtr1 can adopt two distinct GDP- and GTP-bound conformations, and that it hydrolyses GTP much slower than Ras proteins. Using cysteine mutagenesis of Arginine-37 and Valine-67, residues at the Switch I and II regions, respectively, we show altered GTPase activity and associated conformational changes as compared to the wild type protein and the cysteine-less mutant. CONCLUSIONS: The extremely low intrinsic GTPase activity and distinct conformations upon nucleotide binding imply a strict regulation of Gtr1. These findings as well as the altered properties obtained by mutagenesis in the Switch regions provide insights into the function of Gtr1 and its homologues in yeast and mammals.