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Open Access Highly Accessed Research article

The intrinsic GTPase activity of the Gtr1 protein from Saccharomyces cerevisiae

Palanivelu Sengottaiyan1, Cornelia Spetea2, Jens O Lagerstedt3, Dieter Samyn1, Michael Andersson1, Lorena Ruiz-Pavón1 and Bengt L Persson14*

Author affiliations

1 School of Natural Sciences, Linnaeus University, SE-391 82, Kalmar, Sweden

2 Department of Biological and Environmental Sciences, University of Gothenburg, SE-405 30, Gothenburg, Sweden

3 Department of Experimental Medical Science, Lund University, SE-221 84, Lund, Sweden

4 Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, Katholieke Universiteit Leuven, and Department of Molecular Microbiology, Flanders Institute of Biotechnology (VIB), Kasteelpark Arenberg 31, BE-3001, Leuven-Heverlee, Flanders, Belgium

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Citation and License

BMC Biochemistry 2012, 13:11  doi:10.1186/1471-2091-13-11

Published: 24 June 2012

Abstract

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.

Keywords:
Gtr1; GTPase; Intrinsic tryptophan fluorescence; Rag GTPase; Cysteine mutagenesis; Switch region