The intrinsic GTPase activity of the Gtr1 protein from Saccharomyces cerevisiae

Palanivelu Sengottaiyan,Dieter Samyn,Jens O Lagerstedt,Michael Andersson,Bengt L Persson,Lorena Ruiz-Pavón,Cornelia Spetea

doi:10.1186/1471-2091-13-11

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.

Highlights

The Gtr1 protein of Saccharomyces cerevisiae is a member of the RagA subfamily of the Ras-like small GTPase superfamily
It has been shown that the Gtr1 protein is a subunit of the EGO/GSE complex, which is indispensible for intracellular sorting of the amino acid permease Gap1 [9], Gtr1 has been implicated in regulation of the TOR signaling cascade in response to amino acids [10]
Intrinsic tryptophan fluorescence of the purified Gtr1 protein Trp fluorescence has been widely used as a tool to study structural changes upon nucleotide binding to Ras GTPases [23,24] The Gtr1 protein harbors three Trp residues, namely Trp60, Trp167 and Trp175 (Figures 1 and 2)

Summary

Introduction

The Gtr protein of Saccharomyces cerevisiae is a member of the RagA subfamily of the Ras-like small GTPase superfamily. The Switch regions in the GTPase domain of Gtr are essential for TORC1 activation and amino acid signaling [R. The small GTPases of the Ras superfamily have been implicated in diverse cellular functions, including regulation of ion channel activity, cytoskeleton reorganization, nucleocytoplasmic transport, vesicular trafficking, cell proliferation and differentiation [1]. All Ras-GTPases share conserved amino acid sequences, the G1 to G5 motifs, which regulate the GDP/GTP exchange and GTP hydrolysis, and trigger multiple intracellular signaling cascades [2]. Despite an otherwise low sequence similarity, the G motifs in Gtr display high conservation with other Ras GTPases, and are located within the N-terminal GTPase domain of the protein [12]. In contrast to Ras homologues, the Gtr protein lacks lipid modification motifs in its C-terminal region, and the G4 motif (H/ NKXD) contains a histidine instead of an asparagine residue [6]

Methods

Results

Conclusion