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1.  The intrinsic GTPase activity of the Gtr1 protein from Saccharomyces cerevisiae 
BMC Biochemistry  2012;13:11.
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.
PMCID: PMC3477016  PMID: 22726655
Gtr1; GTPase; Intrinsic tryptophan fluorescence; Rag GTPase; Cysteine mutagenesis; Switch region
2.  Functional expression, purification and reconstitution of the recombinant phosphate transporter Pho89 of Saccharomyces cerevisiae 
The Febs Journal  2013;280(3):965-975.
The Saccharomyces cerevisiae high-affinity phosphate transporter Pho89 is a member of the inorganic phosphate (Pi) transporter (PiT) family, and shares significant homology with the type III Na+/Pi symporters, hPit1 and hPit2. Currently, detailed biochemical and biophysical analyses of Pho89 to better understand its transport mechanisms are limited, owing to the lack of purified Pho89 in an active form. In the present study, we expressed functional Pho89 in the cell membrane of Pichia pastoris, solubilized it in Triton X-100 and foscholine-12, and purified it by immobilized nickel affinity chromatography combined with size exclusion chromatography. The protein eluted as an oligomer on the gel filtration column, and SDS/PAGE followed by western blotting analysis revealed that the protein appeared as bands of approximately 63, 140 and 520 kDa, corresponding to the monomeric, dimeric and oligomeric masses of the protein, respectively. Proteoliposomes containing purified and reconstituted Pho89 showed Na+-dependent Pi transport activity driven by an artificially imposed electrochemical Na+ gradient. This implies that Pho89 operates as a symporter. Moreover, its activity is sensitive to the Na+ ionophore monensin. To our knowledge, this study represents the first report on the functional reconstitution of a Pi-coupled PiT family member.
Structured digital abstract
Pho89 and Pho89 bind by molecular sieving (View interaction)Pho89 and Pho89 bind by comigration in gel electrophoresis (View interaction)Pho89 and Pho89 bind by molecular sieving (View interaction)Pho89 and Pho89 bind by comigration in gel electrophoresis (View interaction)
PMCID: PMC3633241  PMID: 23216645
Pho89; phosphate transport reconstitution; Pichia pastoris; Saccharomyces cerevisiae

Results 1-2 (2)