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1.  Yeast nutrient transceptors provide novel insight in the functionality of membrane transporters 
Current Genetics  2013;59(4):197-206.
In the yeast Saccharomyces cerevisiae several nutrient transporters have been identified that possess an additional function as nutrient receptor. These transporters are induced when yeast cells are starved for their substrate, which triggers entry into stationary phase and acquirement of a low protein kinase A (PKA) phenotype. Re-addition of the lacking nutrient triggers exit from stationary phase and sudden activation of the PKA pathway, the latter being mediated by the nutrient transceptors. At the same time, the transceptors are ubiquitinated, endocytosed and sorted to the vacuole for breakdown. Investigation of the signaling function of the transceptors has provided a new read-out and new tools for gaining insight into the functionality of transporters. Identification of amino acid residues that bind co-transported ions in symporters has been challenging because the inactivation of transport by site-directed mutagenesis is not conclusive with respect to the cause of the inactivation. The discovery of nontransported agonists of the signaling function in transceptors has shown that transport is not required for signaling. Inactivation of transport with maintenance of signaling in transceptors supports that a true proton-binding residue was mutagenised. Determining the relationship between transport and induction of endocytosis has also been challenging, since inactivation of transport by mutagenesis easily causes loss of all affinity for the substrate. The use of analogues with different combinations of transport and signaling capacities has revealed that transport, ubiquitination and endocytosis can be uncoupled in several unexpected ways. The results obtained are consistent with transporters undergoing multiple substrate-induced conformational changes, which allow interaction with different accessory proteins to trigger specific downstream events.
PMCID: PMC3824880  PMID: 24114446
Nutrient signaling; Transceptor; Co-transport; Proton binding; Endocytosis; Ubiquitination
2.  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
3.  High Complement Factor I Activity in the Plasma of Children with Autism Spectrum Disorders 
Autism Research and Treatment  2011;2012:868576.
Autism spectrum disorders (ASDs) are neurodevelopmental and behavioural syndromes affecting social orientation, behaviour, and communication that can be classified as developmental disorders. ASD is also associated with immune system abnormality. Immune system abnormalities may be caused partly by complement system factor I deficiency. Complement factor I is a serine protease present in human plasma that is involved in the degradation of complement protein C3b, which is a major opsonin of the complement system. Deficiency in factor I activity is associated with an increased incidence of infections in humans. In this paper, we show that the mean level of factor I activity in the ASD group is significantly higher than in the control group of typically developed and healthy children, suggesting that high activity of complement factor I might have an impact on the development of ASD.
PMCID: PMC3420436  PMID: 22928102
4.  Regulation of Cation-Coupled High-Affinity Phosphate Uptake in the Yeast Saccharomyces cerevisiae 
Journal of Bacteriology  2000;182(17):5017-5019.
Studies of the high-affinity phosphate transporters in the yeast Saccharomyces cerevisiae using mutant strains lacking either the Pho84 or the Pho89 permease revealed that the transporters are differentially regulated. Although both genes are induced by phosphate starvation, activation of the Pho89 transporter precedes that of the Pho84 transporter early in the growth phase in a way which may possibly reflect a fine tuning of the phosphate uptake process relative to the availability of external phosphate.
PMCID: PMC111388  PMID: 10940052
5.  Physiological Regulation of the Derepressible Phosphate Transporter in Saccharomyces cerevisiae 
Journal of Bacteriology  1998;180(8):2253-2256.
The extracellular phosphate concentration permissive for the expression of different amounts of the active high-affinity Pho84 phosphate transporter in the plasma membrane as well as the PHO84 messenger RNA levels in low-phosphate-grown Saccharomyces cerevisiae cells is very narrow and essential for a tight regulation of the transporter. The Pho84 transporter undergoes a rapid degradation once the supply of phosphate and/or carbon source is exhausted.
PMCID: PMC107158  PMID: 9555914
6.  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
7.  Functionally Important Amino Acids in the Arabidopsis Thylakoid Phosphate Transporter: Homology Modeling and Site-Directed Mutagenesis† 
Biochemistry  2010;49(30):6430-6439.
The anion transporter 1 (ANTR1) from Arabidopsis thaliana, homologous to the mammalian members of the solute carrier 17 (SLC17) family, is located in the chloroplast thylakoid membrane. When expressed heterologously in Escherichia coli, ANTR1 mediates a Na+-dependent active transport of inorganic phosphate (Pi). The aim of this study was to identify amino acid residues involved in Pi binding and translocation by ANTR1 and in the Na+ dependence of its activity. A three-dimensional structural model of ANTR1 was constructed using the crystal structure of glycerol 3-phosphate/phosphate antiporter from E. coli as a template. Based on this model and multiple sequence alignments, five highly conserved residues in plant ANTRs and mammalian SLC17 homologues have been selected for site-directed mutagenesis, namely, Arg-120, Ser-124, and Arg-201 inside the putative translocation pathway and Arg-228 and Asp-382 exposed at the cytoplasmic surface of the protein. The activities of the wild-type and mutant proteins have been analyzed using expression in E. coli and radioactive Pi transport assays and compared with bacterial cells carrying an empty plasmid. The results from Pi- and Na+-dependent kinetics indicate the following: (i) Arg-120 and Arg-201 may be important for binding and translocation of the substrate; (ii) Ser-124 may function as a transient binding site for Na+ ions in close proximity to the periplasmic side; (iii) Arg-228 and Asp-382 may participate in interactions associated with protein conformational changes required for full transport activity. Functional characterization of ANTR1 should provide useful insights into the function of other plant and mammalian SLC17 homologous transporters.
PMCID: PMC2911078  PMID: 20565143

Results 1-7 (7)