The intrinsic GTPase activity of the Gtr1 protein from Saccharomyces cerevisiae

Background The 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...

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Veröffentlicht in:	BMC biochemistry Jg. 13; H. 1; S. 11
Hauptverfasser:	Sengottaiyan, Palanivelu, Spetea, Cornelia, Lagerstedt, Jens O, Samyn, Dieter, Andersson, Michael, Ruiz-Pavón, Lorena, Persson, Bengt L
Format:	Journal Article
Sprache:	Englisch
Veröffentlicht:	London BioMed Central 24.06.2012 BioMed Central Ltd
Schlagworte:	Amino Acid Sequence Amino acids Analysis Binding proteins Biochemistry Biokemi Biologi Biological Sciences Biomedical and Life Sciences Brewer's yeast Crystal structure Cysteine mutagenesis Data processing Enzymes Fluorescence Genetic aspects GTP Phosphohydrolases - chemistry GTP Phosphohydrolases - metabolism GTPase Gtr1 Guanosine triphosphatase Guanosine Triphosphate - metabolism Intrinsic tryptophan fluorescence Life Sciences Molecular Sequence Data Monomeric GTP-Binding Proteins - chemistry Monomeric GTP-Binding Proteins - genetics Monomeric GTP-Binding Proteins - metabolism Mutagenesis, Site-Directed Natural Sciences Naturvetenskap Physiological aspects Protein Structure, Tertiary Proteins Rag GTPase Ragas Recombinant Proteins - chemistry Recombinant Proteins - genetics Recombinant Proteins - metabolism Research Article RNA polymerase Saccharomyces cerevisiae Saccharomyces cerevisiae - metabolism Saccharomyces cerevisiae Proteins - chemistry Saccharomyces cerevisiae Proteins - genetics Saccharomyces cerevisiae Proteins - metabolism Sequence Homology, Amino Acid Spectrophotometry, Ultraviolet Studies Switch region Thiols Yeast Belgium Sweden Intrinsic tryptophan fluorescence Rag GTPase Switch region Cysteine mutagenesis GTPase Gtr1
ISSN:	1471-2091, 1471-2237, 1471-2091
Online-Zugang:	Volltext
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Zusammenfassung:	Background The 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. Results By 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. Conclusions The 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.
Bibliographie:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 14 content type line 23 ObjectType-Article-2 ObjectType-Feature-1
ISSN:	1471-2091 1471-2237 1471-2091
DOI:	10.1186/1471-2091-13-11