| 1. |
- Andersson, Michael R., et al.
(författare)
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Mutational analysis of conserved glutamic acids of Pho89, a Saccharomyces cerevisiae high-affinity inorganic phosphate:Na+ symporter
- 2012
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Ingår i: Biologia. - : Springer Science and Business Media LLC. - 0006-3088 .- 1336-9563. ; 67:6, s. 1056-1061
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Tidskriftsartikel (refereegranskat)abstract
- In Saccharomyces cerevisiae, the high-affinity phosphate transport system comprises the Pho84 and Pho89 permeases. The Pho89 permease catalyzes import of inorganic phosphate in a symport manner by utilizing Na+ ions as co-solute. We have addressed the functional importance of two glutamic acid residues at positions 55 and 491. Both residues are highly conserved amongst members of the inorganic phosphate transporter (PiT) family, which might be an indication of functional importance. Moreover, both residues have been shown to be of critical importance in the hPit2 transporter. We have created site-directed mutations of both E55 and E491 to lysine and glutamine. We observed that in all four cases there is a dramatic impact on the transport activity, and thus it seems that they indeed are of functional importance. Following these observations, we addressed the membrane topology of this protein by using several prediction programs. TOPCONS predicts a 7-5 transmembrane segment organization, which is the most concise topology as compared to the hPiT2 transporter. By understanding the functionality of these residues, we are able to correlate the Pho89 topology to that of the hPiT2, and can now further analyze residues which might play a role in the transport activity.
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| 2. |
- Samyn, Dieter R., et al.
(författare)
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Mutational analysis of putative phosphate- and proton-binding sites in the Saccharomyces cerevisiae Pho84 phosphate:H+ transceptor and its effect on signalling to the PKA and PHO pathways
- 2012
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Ingår i: Biochemical Journal. - : Portland Press. - 0264-6021 .- 1470-8728. ; 445, s. 413-422
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Tidskriftsartikel (refereegranskat)abstract
- In Saccharomyces cerevisiae, the Pho84 phosphate transporter acts as the main provider of phosphate to the cell using a proton symport mechanism, but also mediates rapid activation of the PKA (protein kinase A) pathway. These two features led to recognition of Pho84 as a transceptor. Although the physiological role of Pho84 has been studied in depth, the mechanisms underlying the transport and sensor functions are unclear. To obtain more insight into the structure–function relationships of Pho84, we have rationally designed and analysed site-directed mutants. Using a three-dimensional model of Pho84 created on the basis of the GlpT permease, complemented with multiple sequence alignments, we selected Arg168 and Lys492, and Asp178, Asp358 and Glu473 as residues potentially involved in phosphate or proton binding respectively, during transport. We found that Asp358 (helix 7) and Lys492 (helix 11) are critical for the transport function, and might be part of the putative substrate-binding pocket of Pho84. Moreover, we show that alleles mutated in the putative proton-binding site Asp358 are still capable of strongly activating PKA pathway targets, despite their severely reduced transport activity. This indicates that signalling does not require transport and suggests that mutagenesis of amino acid residues involved in binding of the co-transported ion may constitute a promising general approach to separate the transport and signalling functions in transceptors.
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| 3. |
- Sengottaiyan, Palanivelu, et al.
(författare)
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The intrinsic GTPase activity of the Gtr1 protein from Saccharomyces cerevisiae
- 2012
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Ingår i: BMC Biochemistry. - : BioMed Central (BMC). - 1471-2091. ; 13
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Tidskriftsartikel (refereegranskat)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 [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.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.
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| 4. |
- Basheer, Shabana, et al.
(författare)
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A membrane protein based biosensor: Use of a phosphate - H+ symporter membrane protein (Pho84) in the sensing of phosphate ions.
- 2011
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Ingår i: Biosensors & bioelectronics. - : Elsevier. - 0956-5663 .- 1873-4235. ; 27:1, s. 58-63
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Tidskriftsartikel (refereegranskat)abstract
- A label free biosensor for direct detection of inorganic phosphate based on potential-step capacitance measurements has been developed. The high-affinity Pho84 plasma membrane phosphate/proton symporter of Saccharomyces cerevisiae was used as a sensing element. Heterologously expressed and purified Pho84 protein was immobilized on a self-assembled monolayer (SAM) on a capacitance electrode. Changes in capacitance were recorded upon exposure to phosphate compared to the control substance, phosphate analogue methylphosphonate. Hence, even without the explicit use of lipid membranes, the Pho84 membrane protein could retain its capacity of selective substrate binding, with a phosphate detection limit in the range of the apparent in vivo K(m). A linear increase in capacitance was monitored in the phosphate concentration range of 5-25 mu M. The analytical response of the capacitive biosensor is in agreement with that the transporter undergoes significant conformational changes upon exposure to inorganic phosphate, while exposure to the analogue only causes minor responses.
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| 5. |
- Flockhart, Mikael, et al.
(författare)
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Glucosinolate-rich broccoli sprouts protect against oxidative stress and improve adaptations to intense exercise training.
- 2023
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Ingår i: Redox Biology. - : Elsevier. - 2213-2317. ; 67
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Tidskriftsartikel (refereegranskat)abstract
- Oxidative stress plays a vital role for the adaptive responses to physical training. However, excessive oxidative stress can precipitate cellular damage, necessitating protective mechanisms to mitigate this effect. Glucosinolates, found predominantly in cruciferous vegetables, can be converted into isothiocyanates, known for their antioxidative properties. These compounds activate crucial antioxidant defence pathways and support mitochondrial function and protein integrity under oxidative stress, in both Nrf2-dependent and independent manners. We here administered glucosinolate-rich broccoli sprouts (GRS), in a randomized double-blinded cross-over fashion to 9 healthy subjects in combination with daily intense exercise training for 7 days. We found that exercise in combination with GRS significantly decreased the levels of carbonylated proteins in skeletal muscle and the release of myeloperoxidase into blood. Moreover, it lowered lactate accumulation during submaximal exercise, and attenuated the severe nocturnal hypoglycaemic episodes seen during the placebo condition. Furthermore, GRS in combination with exercise improved physical performance, which was unchanged in the placebo condition.
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| 6. |
- Karim, Mahmoud Abdul, et al.
(författare)
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Rab-Effector-Kinase Interplay Modulates Intralumenal Fragment Formation during Vacuole Fusion
- 2018
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Ingår i: Developmental Cell. - : Cell Press. - 1534-5807 .- 1878-1551. ; 47:1, s. 80-97.e6
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Tidskriftsartikel (refereegranskat)abstract
- Upon vacuolar lysosome (or vacuole) fusion in S. cerevisiae, a portion of membrane is internalized and catabolized. Formation of this intralumenal fragment (ILF) is important for organelle protein and lipid homeostasis and remodeling. But how ILF formation is optimized for membrane turnover is not understood. Here, we show that fewer ILFs form when the interaction between the Rab-GTPase Ypt7 and its effector Vps41 (a subunit of the tethering complex HOPS) is interrupted by a point mutation (Ypt7-D44N). Subsequent phosphorylation of Vps41 by the casein kinase Yck3 prevents stabilization of trans-SNARE complexes needed for lipid bilayer pore formation. Impairing ILF formation prevents clearance of misfolded proteins from vacuole membranes and promotes organelle permeability and cell death. We propose that HOPS coordinates Rab, kinase, and SNARE cycles to modulate ILF size during vacuole fusion, regulating lipid and protein turnover important for quality control and membrane integrity.
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| 7. |
- Lundh, Fredrik, et al.
(författare)
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Molecular mechanisms controlling phosphate-induced downregulation of the yeast Pho84 phosphate transporter
- 2009
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 48:21, s. 4497-4505
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Tidskriftsartikel (refereegranskat)abstract
- In Saccharomyces cerevisiae, phosphate uptake is mainly dependent on the proton-coupled Pho84 permease under phosphate-limited growth conditions. Phosphate addition causes Pho84-mediated activation of the protein kinase A (PKA) pathway as well as rapid internalization and vacuolar breakdown of Pho84. We show that Pho84 undergoes phosphate-induced phosphorylation and subsequent ubiquitination on amino acids located in the large middle intracellular loop prior to endocytosis. The attachment of ubiquitin is dependent on the ubiquitin conjugating enzymes Ubc2 and Ubc4. In addition, we show that the Pho84 endocytotic process is delayed in strains with reduced PKA activity. Our results suggest that Pho84-mediated activation of the PKA pathway is responsible for its own downregulation by phosphorylation, ubiquination, internalization, and vacuolar breakdown.
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| 8. |
- Pettersson-Pablo, Paul, 1986-, et al.
(författare)
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Reference interval for type III procollagen (PIIINP) using the Advia centaur PIIINP assay in adults and elderly
- 2021
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Ingår i: Scandinavian Journal of Clinical and Laboratory Investigation. - : Taylor & Francis. - 0036-5513 .- 1502-7686. ; 81:8, s. 649-652
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: The amino-terminal peptide of type III procollagen (PIIINP) is a byproduct of type III collagen synthesis that exhibits promise as a biomarker of fibrosis, specifically in monitoring hepatic fibrosis in methotrexate treated patients. The Advia Centaur® PIIINP assay is developed for track-based automated laboratory systems and is suitable for large volume analysis. Reference intervals in children and younger adults have been published previously. Here we measured PIIINP to determine reference ranges, specifically including elderly patients, for whom such are currently lacking.METHODS: Samples were collected from subjects ranging from 20 to 98 years of age. Blood donors and clinical samples from primary care patients were used for reference interval calculation. Samples were analysed using the Advia Centaur® PIIINP assay. After exclusion of samples high in alanine transaminase (AST), aspartate transaminase (ALT), and C-reactive protein (CRP) 386 samples were used in the reference interval calculation.RESULTS AND CONCLUSION: We determined the following reference interval for the Advia Centaur® PIIINP assay: the lower limit of the reference interval (2.5% percentile with 95% CI) was 4.42 (4.20-4.65) µg/L and the upper limit of the reference interval (97.5% percentile 95% CI) 16.0 (15.04-17.02) µg/L.No significant differences in mean PIIINP concentrations were found between men and women. While differing mean PIIINP concentrations were seen among subjects in different age groups, the differences were small and partitioning of reference range was determined not to be necessary.
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| 9. |
- Ruiz-Pavon, Lorena, et al.
(författare)
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Functionally important amino acids in the Arabidopsis thylakoid phosphate transporter: Homology modeling and site-directed mutagenesis
- 2010
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Ingår i: Biochemistry. - : American Chemical Society. - 0006-2960 .- 1520-4995. ; 49:30, s. 6430-6439
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 10. |
- Samyn, Dieter R., 1978-, et al.
(författare)
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Inorganic phosphate and sulfate transport in S. cerevisiae
- 2016
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Ingår i: Advances in Experimental Medicine and Biology. - Cham : Springer. - 0065-2598 .- 2214-8019. ; 892, s. 253-269, s. 253-269
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Tidskriftsartikel (refereegranskat)abstract
- Inorganic ions such as phosphate and sulfate are essential macronutrients required for a broad spectrum of cellular functions and their regulation. In a constantly fluctuating environment microorganisms have for their survival developed specific nutrient sensing and transport systems ensuring that the cellular nutrient needs are met. This chapter focuses on the S. cerevisiae plasma membrane localized transporters, of which some are strongly induced under conditions of nutrient scarcity and facilitate the active uptake of inorganic phosphate and sulfate. Recent advances in studying the properties of the high-affinity phosphate and sulfate transporters by means of site-directed mutagenesis have provided further insight into the molecular mechanisms contributing to substrate selectivity and transporter functionality of this important class of membrane transporters. © Springer International Publishing Switzerland 2016.
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| 11. |
- Samyn, Dieter R., 1978-, et al.
(författare)
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Key Residues and Phosphate Release Routes in the Saccharomyces cerevisiae Pho84 Transceptor : THE ROLE OF TYR179 IN FUNCTIONAL REGULATION
- 2016
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Ingår i: Journal of Biological Chemistry. - : Springer. - 0021-9258 .- 1083-351X. ; 291:51, s. 26388-26388
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Tidskriftsartikel (refereegranskat)abstract
- Pho84, a major facilitator superfamily (MFS) protein, is the main high-affinity Pi transceptor in Saccharomyces cerevisiae Although transport mechanisms have been suggested for other MFS members, the key residues and molecular events driving transport by Pi:H+ symporters are unclear. The current Pho84 transport model is based on the inward-facing occluded crystal structure of the Pho84 homologue PiPT in the fungus Piriformospora indica However, this model is limited by the lack of experimental data on the regulatory residues for each stage of the transport cycle. In this study, an open, inward-facing conformation of Pho84 was used to study the release of Pi A comparison of this conformation with the model for Pi release in PiPT revealed that Tyr179 in Pho84 (Tyr150 in PiPT) is not part of the Pi binding site. This difference may be due to a lack of detailed information on the Pi release step in PiPT. Molecular dynamics simulations of Pho84 in which a residue adjacent to Tyr179, Asp178, is protonated revealed a conformational change in Pho84 from an open, inward-facing state to an occluded state. Tyr179 then became part of the binding site as was observed in the PiPT crystal structure. The importance of Tyr179 in regulating Pi release was supported by site-directed mutagenesis and transport assays. Using trehalase activity measurements, we demonstrated that the release of Pi is a critical step for transceptor signaling. Our results add to previous studies on PiPT, creating a more complete picture of the proton-coupled Pi transport cycle of a transceptor.
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| 12. |
- Samyn, Dieter R., 1978-
(författare)
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Structure/function relationships of inorganic phosphate transporters
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Of the many nutrients that make the metabolic clock tick, inorganic phosphate fulfills an essential role in all yeast (and other organisms), being necessary for both structural and metabolic purposes. In order to transport phosphate into the cell interior, Saccharomyces cerevisiae makes use of two systems, comprising high- or low-affinity transporters, which are responsible for the cellular accumulation of inorganic phosphate. Depending on the extracellular concentration of inorganic phosphate, one of the two systems will be responsible for scavenging phosphate from the surroundings.The present thesis focuses on the high-affinity transport system in S. cerevisiae, i.e. Pho84, which is induced under low phosphate conditions. The expression and degradation of Pho84 is dependent on the availability of phosphate. When confronted with ample amounts of external phosphate, the Pho84 undergoes phosphorylation, prior to ubiquitylation. These events will eventually lead to the removal of Pho84 from the plasma membrane, followed by vacuolar degradation. The Pho84, together with the ANTR1 high-affinity inorganic phosphate transporter of Arabidopsis thaliana, are integral membrane proteins belonging to the major facilitator superfamily. Both are predicted to have 12 transmembrane helices, which have been confirmed by in silico modeling of both proteins, using the glycerol-3-phosphate transporter as template. The obtained models served as a rational start point for the study of the molecular transport mechanisms by means of site-directed mutagenesis and consequently functional and biochemical characterization.The other high-affinity transporter, Pho89, has been studied less because of its lower activity (as compared to the Pho84) and its preferred alkaline operational conditions. In order to study Pho89 in more detail, a quadruple deletions strain (Pho84Δ Pho87Δ Pho90Δ Pho91Δ) was used.In conclusion, this thesis has contributed to broaden the knowledge of structural and functional aspects of inorganic phosphate transporters.
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| 13. |
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| 14. |
- Schothorst, Joep, et al.
(författare)
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Yeast nutrient transceptors provide novel insight in the functionality of membrane transporters.
- 2013
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Ingår i: Current Genetics. - : Springer. - 0172-8083 .- 1432-0983. ; 59:4, s. 197-206
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 15. |
- Zvyagilskaya, Renata A., et al.
(författare)
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Characterization of the Pho89 phosphate transporter by functional hyperexpression in Saccharomyces cerevisiae
- 2008
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Ingår i: FEMS yeast research (Print). - : Oxford University Press. - 1567-1356 .- 1567-1364. ; 8:5, s. 685-696
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Tidskriftsartikel (refereegranskat)abstract
- The Na(+)-coupled, high-affinity Pho89 plasma membrane phosphate transporter in Saccharomyces cerevisiae has so far been difficult to study because of its low activity and special properties. In this study, we have used a pho84Deltapho87Deltapho90Deltapho91Delta quadruple deletion strain of S. cerevisiae devoid of all transporter genes specific for inorganic phosphate, except for PHO89, to functionally characterize Pho89 under conditions where its expression is hyperstimulated. Under these conditions, the Pho89 protein is strongly upregulated and is the sole high-capacity phosphate transporter sustaining cellular acquisition of inorganic phosphate. Even if Pho89 is synthesized in cells grown at pH 4.5-8.0, the transporter is functionally active under alkaline conditions only, with a K(m) value reflecting high-affinity properties of the transporter and with a transport rate about 100-fold higher than that of the protein in a wild-type strain. Even under these hyperexpressive conditions, Pho89 is unable to sense and signal extracellular phosphate levels. In cells grown at pH 8.0, Pho89-mediated phosphate uptake at alkaline pH is cation-dependent with a strong activation by Na(+) ions and sensitivity to carbonyl cyanide m-chlorophenylhydrazone. The contribution of H(+)- and Na(+)-coupled phosphate transport systems in wild-type cells grown at different pH values was quantified. The contribution of the Na(+)-coupled transport system to the total cellular phosphate uptake activity increases progressively with increasing pH.
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| 16. |
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