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The intrinsic GTPase activity of the Gtr1 protein from Saccharomyces cerevisiae

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Author

  • Palanivelu Sengottaiyan
  • Cornelia Spetea
  • Jens Lagerstedt
  • Dieter Samyn
  • Michael Andersson
  • Lorena Ruiz-Pavon
  • Bengt L. Persson

Summary, in English

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 [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 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.

Publishing year

2012

Language

English

Publication/Series

BMC Biochemistry

Volume

13

Document type

Journal article

Publisher

BioMed Central (BMC)

Topic

  • Biochemistry and Molecular Biology

Keywords

  • Gtr1
  • GTPase
  • Intrinsic tryptophan fluorescence
  • Rag GTPase
  • Cysteine
  • mutagenesis
  • Switch region

Status

Published

Research group

  • Cellular Biomechanics

ISBN/ISSN/Other

  • ISSN: 1471-2091