The intrinsic GTPase activity of the Gtr1 protein from Saccharomyces cerevisiae
© Sengottaiyan et al.; licensee BioMed Central Ltd. 2012
Received: 20 January 2012
Accepted: 24 June 2012
Published: 24 June 2012
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.
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.
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.
KeywordsGtr1 GTPase Intrinsic tryptophan fluorescence Rag GTPase Cysteine mutagenesis Switch region
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 . All Ras-GTPases share conserved amino acid sequences, the G1 to G5 motifs, which regulate the GDP/GTP exchange and GTP hydrolysis, and thus trigger multiple intracellular signaling cascades . Switch I and Switch II are flexible regions around G2 and G3, respectively, which change conformation upon GTP/GDP exchange, thus facilitating interactions with its effectors, GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs) [3, 4]. Ras-GTPases generally exhibit slow dissociation of GDP for GTP and low intrinsic GTP hydrolysis rate, which are strictly enhanced by GEFs and GAPs, respectively .
The Gtr1 protein of Saccharomyces cerevisiae (S. cerevisiae) is a multifunctional GTP-binding protein, involved in phosphate acquisition through modulation of Pho84 transport activity , ribosome biogenesis  and epigenetic control of gene expression . It has also 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 , Gtr1 has been implicated in regulation of the TOR signaling cascade in response to amino acids . Most recently, it has been proposed that the Switch regions are essential for activation of the TOR1 complex (TORC1) . Despite an otherwise low sequence similarity, the G motifs in Gtr1 display high conservation with other Ras GTPases, and are located within the N-terminal GTPase domain of the protein . The C-terminal domain of the protein has been implicated in self-interaction  and protein-protein interactions . In contrast to Ras homologues, the Gtr1 protein lacks lipid modification motifs in its C-terminal region, and the G4 motif (H/NKXD) contains a histidine instead of an asparagine residue .
Gtr1 belongs to the distantly related RagA family, which displays a low sequence similarity with the Ras, Rab, Ran, Arf and Rho proteins [7, 13]. The Gtr1 protein displays 52 % and 47 % sequence identify with the mammalian Rag GTPases RagA and RagB, respectively . Notably, recent studies have shown that the Gtr1 and RagA proteins share a similar mechanism of amino acid-mediated TOR activation, indicating that these proteins are functionally conserved in eukaryotes . S. cerevisiae also contains Gtr2, which corresponds to RagC/RagD in humans. Like the mammalian Rag GTPases, GTP-bound Gtr1 and GDP-bound Gtr2 form a stable heterodimeric complex in vitro. The GTP-bound form but not the GDP-bound Gtr1 interacts with itself, whereas Gtr2 can interact with itself only in the presence of GTP-bound Gtr1 . Most recently, the 3D-structure of the Gtr1-Gtr2 complex in the GMP-PNP bound form was resolved at 2.8 Å resolution . The structure has brought insights into the location of G domains and Switch regions. Based on this, it was proposed that upon nucleotide exchange the Switch regions change conformation, allowing for interaction with Raptor and activation of TORC1 . In the same report, it was shown that the C-terminal domains of the two proteins mediate heterodimeric complex formation that is indispensible for activation of TOR signaling pathways.
Previous studies have localized the Gtr1 protein both to the nucleus and to the cytoplasm , where it interacts in a GTP-dependent manner with diverse cytoplasmic and nuclear proteins, such as Ego1, Yrb2, Nop8 and Ego3 [16–19]. Based on competition studies, it was proposed that the Gtr1 protein displays higher affinity for GTP than for GDP . The GTP-bound form of Gtr1 interacted with the Rpc-19 subunit of RNA polymerases I and III in yeast two-hybrid assays, indicating a role in the assembly of RNA polymerase . Recently, the Vam6 protein, known as a GEF for Ypt7, was shown to assist the exchange of GDP/GTP on the Gtr1 protein in vitro. Moreover, Binda et al.  have reported that Vam6 activates the Gtr1 subunit of the EGO complex during TORC1 function.
Gtr1 harbors all the necessary structural elements for functioning as a GTPase. Previous attempts to estimate the GTPase activity of Gtr1 , RagA [7,] and RagC proteins  resulted in either none or too low activity to be measured. Here we quantify the intrinsic GTP hydrolytic activity of purified recombinant Gtr1 protein and several variants using a radioactive GTPase assay, and in addition study their tryptophan fluorescence emission properties.
Gene cloning, heterologous expression and purification
S. cerevisiae full-length Gtr1 proteins (wild-type, cysteine-less (C-less), Arg37Cys and Val67Cys) were expressed in Escherichia coli and purified in a single step by affinity chromatography essentially as described . The complete procedure is provided in Additional file 1. The protein expression levels and purity of the preparations were assayed as described . The oligomeric state of Gtr1 was assayed by native gel electrophoresis as described 
Assay of GTPase activity
The GTPase assay was carried out using purified recombinant protein at a final concentration of 0.15 mg ml-1 (4 μM), 185 nM GTP and 15 nM [α-32P] GTP (3000 Ci/mmol, 1 Ci = 3.7x1010 Bq; PerkinElmer, Boston, USA) in a 50 μl containing assay buffer (25 mM HEPES/NaOH, pH 7.4, 5 mM KCl, 5 mM MgCl2 and 100 mM sucrose) as described in Ref. .
The enzymatic reaction was carried out at 37 °C for the indicated time periods (0, 15, 30, 60 and 120 min), and terminated by the addition of an equal volume of 4 M formic acid. A volume of 2.5 μl (62.5 nCi) quenched reaction mixture was spotted onto a poly (ethyleneimine)-cellulose plate (Merck, Germany) and the nucleotides were separated by thin-layer chromatography using 0.75 M KH2PO4 (pH 3.65) as elution buffer. The radioactive nucleotides were detected by phosphorimaging (BAS-1500, Fuji, Japan). The radioactive GDP spots were quantified using MultiGauge software (Fuji, Japan). The data are given as means ± SD and were obtained from 2–3 independent experiments. Initial velocities for GTP hydrolysis were measured using the same assay conditions at varying [α-32P] GTP concentrations (0.1 to 1.2 μM) for 60 min. The data were plotted as a function of GTP concentration, and fitted to the Michaelis-Menten equation. A Lineweaver-Burk plot of the data was used to determine the values for the Michaelis-Menten constant (Km) and the maximum velocity (Vmax) parameters. The catalytic constant kcat was calculated as the Vmax/Et ratio, where Et is the enzyme concentration.
Intrinsic tryptophan fluorescence
Tryptophan (Trp) fluorescence spectra of purified recombinant Gtr1 proteins were recorded using a Fluoromax-3 spectrofluorometer (Horiba Jobin Yvon, Japan) in the presence of guanine nucleotides. The interaction of the protein with guanosine 5’-O-(3-thiotriphosphate) (GTPγS) or GDP was measured using 0.019 mg ml-1 (0.50 μM) protein and 25 μM nucleotides in the GTPase assay buffer. The reaction mixture was incubated for 120 min at 37 °C prior to fluorescence measurements . The intrinsic protein fluorescence was excited at 297 nm, and emission spectra were recorded in the range 297 to 400 nm at 25 °C. The spectra of free nucleotides in assay buffer were subtracted from the collected sample dataset.
Results and discussion
Intrinsic tryptophan fluorescence of the purified Gtr1 protein
The fluorescence emission intensity and λmax of Gtr1 and the mutations were measured as described in Methods
Relative intensity (GTPγS / GDP)
0.91 ± 0.01
329 ± 1
327 ± 1
0.95 ± 0.02
327 ± 1
326 ± 1
0.90 ± 0.01
325 ± 1
324 ± 1
0.96 ± 0.01
321 ± 2
316 ± 2
Intrinsic GTPase activity of the purified Gtr1 protein
Next, we determined the enzymatic kinetic parameters of the protein under similar assay conditions as described in Methods. Values of 1.0 μM and 0.1 nmol GDP mg protein-1 min-1 were obtained for the Km and Vmax parameters by Michaelis-Menten analyses (Figure5B and insert). The obtained values were used to calculate the kcat of the protein, 0.004 min-1. For reference, the kcat values determined for three representative GTPases, namely Gial, Ras and EF-TU are 3, 0.3 and 0.003 min-1, respectively . The finding of an extremely low GTPase activity implies a strict regulation, for example interaction with other proteins, such as Gtr2. In addition, Vam6 has been proposed as a GEF for Gtr1 [14, 21]. It will be important to understand the way this key GTPase is regulated by activating proteins to control various processes.
Role of Arg37 and Val67 in the GTPase activity of the Gtr1 protein
Intrinsic tryptophan fluorescence of the Gtr1 mutants
Role of Arg37 in the crystal structure of Gtr1-Gtr2 complex
Recently, the crystal structure of the Gtr1-Gtr2 complex, with the two proteins bound to GMPPNP, was solved . This structural model served as a template to design and analyze the role of RagA in activating TORC1. Mutational analysis of several residues indicated that the surface charge potential is of great importance for RagA to interact with Raptor, and thus being able to activate TORC1 . Of the two adjacent arginines close to the Switch I region and which are conserved amongst the Gtr1 and RagA region (Arg36, Arg37), we have mutated Arg37 to a cysteine and have observed a change in hydrolytic activity. The removal of one of the two Arg residues might alter the surface charge to a degree where a minimal conformational alteration could influence the nucleotide interaction. Whether this charge alteration effectively results in a conformational change that impairs nucleotide binding is currently not known. Our results indicate that a replacement of Arg37 with a cysteine leads to a decreased hydrolytic activity, supporting the importance of this residue.
Here we have employed a combination of structural, cysteine mutagenesis, radioactive GTPase assays and intrinsic tryptophan fluorescence approaches to study the biochemical activity of Gtr1 from S. cerevisiae. The data obtained reveal a very low intrinsic GTPase activity of the Gtr1 protein as compared to Ras GTPases, implying requirement for activating proteins, as previously reported [14, 21]. This activity was found altered in Arg37Cys and also Val67Cys mutants of the Switch regions and associated with conformational changes, which are distinct from those in the WT and cysteine-less mutant. Despite the fact that both mutated residues are not fully conserved amongst Gtr1 homologues, alterations made in those positions have an influence on the GTPase activity and fluorescence. This indicates that both residues are of importance for the functionality of Gtr1. Our findings provide insights into the function of Gtr1 and its homologues in yeast and mammals.
guanine nucleotide exchange factor
TOR complex 1
We are grateful to Prof. Peter Brodelius for providing us with the pET28a+ expression plasmid. This work was supported by grants from the Swedish Research Council to B.L.P. (621-2003-3558 and 621-2007-6144), C.S. (621-2007-5440 and 621-2010-463) and J.O.L. (522-2008-3724).
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