The intrinsic GTPase activity of the Gtr1 protein from Saccharomyces cerevisiae

Abstract

BackgroundThe Gtr1 protein 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. Particularly, the Switch regions in the GTPase domain of Gtr1 are essential for TORC1 activation and amino acid signaling. Therefore, knowledge about the biochemical activity of Gtr1 is required to understand its mode of action and regulation.ResultsBy employing tryptophan fluorescence analysis and radioactive GTPase assays, we 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.ConclusionsThe extremely low intrinsic GTPase activity of Gtr1 implies requirement for interaction with activating proteins to support its physiological function. 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.

Genetic variants of TORC1 signaling pathway affect nitrogen consumption in Saccharomyces cerevisiae during alcoholic fermentation

“…However, the SIFT prediction indicates that these mutations would be tolerated for protein function, and further analysis should be performed to determine whether these SNPs affect protein function. In consequence, the observed differences in nitrogen consumption could be due to mutations in the coding or regulatory regions, which could affect the GTPase activity of Gtr1p and the activation of the TORC1 complex, since its activation depends on the GTP-bound conformation of Gtr1p [68,69].…”

The Architecture of the Rag GTPase Signaling Network

“…The G domain in Gtr1 GTP is, similarly to other Ras-related GTPases, composed of six α helices, six β strands, two switch regions that interact with the γ-phosphate of GTP via hydrogen bonds, and a Mg 2+ ion in the nucleotide-binding site [ 44 ] ( Figure 2 b). Biochemical experiments have demonstrated that Gtr1, like Rag A, exhibits an extremely low intrinsic GTP hydrolysis rate when compared to that of other small GTPases like G i α 1 or Ras [ 27 , 49 , 50 ], which may be explained by at least two structural differences. Firstly, Gtr1, like members of the Arf GTPase family, is missing an important tyrosine (Tyr) in the switch I region that is otherwise conserved in Ras, Rho, and Ran GTPases [ 44 ].…”