RESUMO
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.
Assuntos
Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Conformação Proteica , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Sistemas de Transporte de Aminoácidos/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Endocitose/fisiologia , Proteínas de Membrana Transportadoras/química , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosfatos/química , Fosfatos/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sódio/metabolismo , UbiquitinaçãoRESUMO
In Saccharomyces cerevisiae, Pho89 mediates a cation-dependent transport of Pi across the plasma membrane. This integral membrane protein belongs to the Inorganic Phosphate Transporter (PiT) family, a group that includes the mammalian Na(+)/Pi cotransporters Pit1 and Pit2. Here we report that the Pichia pastoris expressed recombinant Pho89 was purified in the presence of Foscholine-12 and functionally reconstituted into proteoliposomes with a similar substrate specificity as observed in an intact cell system. The alpha-helical content of the Pho89 protein was estimated to 44%. EPR analysis showed that purified Pho89 protein undergoes conformational change upon addition of substrate.
Assuntos
Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/química , Transporte Biológico , Membrana Celular/química , Dicroísmo Circular , Espectroscopia de Ressonância de Spin Eletrônica , Fosforilcolina/análogos & derivados , Fosforilcolina/química , Pichia/química , Ligação Proteica , Estrutura Secundária de Proteína , Proteolipídeos/química , Proteínas Recombinantes/química , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
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.
Assuntos
Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/metabolismo , Transporte Biológico/efeitos dos fármacos , Western Blotting , Membrana Celular/metabolismo , Cromatografia de Afinidade , Cromatografia em Gel , Técnicas Eletroquímicas , Eletroforese em Gel de Poliacrilamida , Peso Molecular , Monensin/farmacologia , Octoxinol/química , Fosfatos/metabolismo , Fosforilcolina/análogos & derivados , Fosforilcolina/química , Pichia/genética , Multimerização Proteica , Proteolipídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Ionóforos de Sódio/farmacologia , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/química , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/genética , SolubilidadeRESUMO
BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.
Assuntos
Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/metabolismo , Dados de Sequência Molecular , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/genética , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Espectrofotometria UltravioletaRESUMO
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 Arg(168) and Lys(492), and Asp(178), Asp(358) and Glu(473) as residues potentially involved in phosphate or proton binding respectively, during transport. We found that Asp(358) (helix 7) and Lys(492) (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 Asp(358) 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.
Assuntos
Simportadores de Próton-Fosfato/genética , Simportadores de Próton-Fosfato/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fosfatase Ácida/genética , Fosfatase Ácida/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , DNA Fúngico/genética , Genes Fúngicos , Cinética , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosfatos/metabolismo , Simportadores de Próton-Fosfato/química , Prótons , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
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.
Assuntos
Regulação para Baixo/efeitos dos fármacos , Fosfatos/farmacologia , Simportadores de Próton-Fosfato/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Retroalimentação Fisiológica , Espaço Intracelular/metabolismo , Fosfatos/metabolismo , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Simportadores de Próton-Fosfato/química , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/química , Transdução de Sinais/efeitos dos fármacos , Ubiquitina/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
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.
Assuntos
Dosagem de Genes , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/metabolismo , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Deleção de Genes , Concentração de Íons de Hidrogênio , Cinética , Proteínas de Transporte de Fosfato/genética , Fosfatos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Sódio/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/genética , Desacopladores/farmacologiaRESUMO
In Saccharomyces cerevisiae, nutrient sensing is the major factor controlling cell growth and proliferation. It has been shown that phosphate signalling involves the activation of the protein kinase A (PKA) in response to an elevation of external phosphate when cells have experienced a severe phosphate limitation. Addition of phosphate or its non-metabolized analogue, methylphosphonate (MP), to cells grown under phosphate limitation triggers degradation of the Pho84 phosphate transporter and represses the acidic phosphatase activity. In this study we have shown that of the five inorganic phosphate transporters (Pho84, Pho87, Pho89, Pho90, Pho91) of the plasma membrane, only Pho84 is required for the MP recognition and repression of the acidic phosphatase activity. By use of the PKA inhibitor H89, we demonstrate that down-regulation and degradation of the Pho84 transporter, in response to an elevation of external phosphate, is delayed by the inhibition of PKA. In contrast, down-regulation of the acidic phosphatase is under these conditions not affected by the PKA inhibition. Altogether, these observations suggest that the PKA signalling pathway plays a role in conveying the signal for the down-regulation and degradation of the Pho84 transporter in the vacuolar compartment in response to altered phosphate availability in the external environment.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas de Transporte de Fosfato/metabolismo , Simportadores de Próton-Fosfato/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fosfatase Ácida/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulação para Baixo , Proteínas de Fluorescência Verde/metabolismo , Isoquinolinas/metabolismo , Isoquinolinas/farmacologia , Compostos Organofosforados/metabolismo , Compostos Organofosforados/farmacologia , Fosfatos/metabolismo , Simportadores de Próton-Fosfato/efeitos dos fármacos , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/metabolismo , Sulfonamidas/farmacologia , Fatores de TempoRESUMO
In Saccharomyces cerevisiae, the Pho84 high-affinity transport system is the major phosphate transporter activated when the cells experience a limitation in external phosphate. In this study, we have compared the phosphate-responsive mechanism of cells expressing PHO84 with a Deltapho84 strain by use of a phosphate analogue, methylphosphonate, which was judged to be suitable for assessment of phosphate homeostasis in the cells. Intracellular levels of the analogue, which in several respects mimicks phosphate, were monitored by (31)P NMR spectroscopy. Results show that methylphosphonate is a nonhydrolyzable and nonutilizable analogue that cannot be used to replenish phosphate or polyphosphate in yeast cells grown under conditions of phosphate limitation. However, the presence of methylphosphonate under such conditions represses the Pho5 acidic phosphatase activity of PHO84 cells, a finding that implies a direct role of the analogue in the regulation of phosphate-responsive genes and/or proteins. Likewise, accumulation of the Pho84 protein at the plasma membrane of the same cells is inhibited by methylphosphonate, although the derepressive expression of the PHO84 gene is unperturbed. Thus, a post-transcriptional regulation is suggested. Supportive of this suggestion is the fact that addition of methylphosphonate to cells with abundant and active Pho84 at the plasma membrane causes enhanced internalization of the Pho84 protein. Altogether, these observations suggest that the Pho84 transporter is regulated not only at the transcriptional level but also by a direct molecule-sensing mechanism at the protein level.
Assuntos
Compostos Organofosforados/química , Fosfatos/metabolismo , Simportadores de Próton-Fosfato/biossíntese , Simportadores de Próton-Fosfato/metabolismo , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/metabolismo , Fosfatase Ácida/antagonistas & inibidores , Fosfatase Ácida/metabolismo , Transporte Biológico/genética , Regulação Fúngica da Expressão Gênica , Genes myc , Concentração de Íons de Hidrogênio , Compostos Organofosforados/metabolismo , Simportadores de Próton-Fosfato/antagonistas & inibidores , Simportadores de Próton-Fosfato/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/genética , Supressão GenéticaRESUMO
Energy status of the novel alkalitolerant Yarrowia lipolytica yeast strain grown at alkaline conditions (pH 9.7) was examined. Cells grown under such severe conditions were found to preserve high respiratory activity. The oxidative phosphorylation system dominated in the energy budget of the cell. A procedure was specially design to isolate tightly coupled mitochondria from yeast cells grown at alkaline conditions. The isolated mitochondrial preparations met known criteria of physiological intactness, as inferred from their ability to maintain distinctive state 4-3 respiration transition upon addition of ADP, high respiratory rates, good respiratory control values, and ADP/O ratios close to the theoretically expected maxima for the substrates used.
Assuntos
Metabolismo Energético , Yarrowia/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Respiração Celular , Concentração de Íons de Hidrogênio , Cinética , Potenciais da Membrana , Mitocôndrias/metabolismo , Oxirredução , Fosforilação Oxidativa , Consumo de Oxigênio , Yarrowia/crescimento & desenvolvimentoRESUMO
Membrane transport systems active in cellular inorganic phosphate (P(i)) acquisition play a key role in maintaining cellular P(i) homeostasis, independent of whether the cell is a unicellular microorganism or is contained in the tissue of a higher eukaryotic organism. Since unicellular eukaryotes such as yeast interact directly with the nutritious environment, regulation of P(i) transport is maintained solely by transduction of nutrient signals across the plasma membrane. The individual yeast cell thus recognizes nutrients that can act as both signals and sustenance. The present review provides an overview of P(i) acquisition via the plasma membrane P(i) transporters of Saccharomyces cerevisiae and the regulation of internal P(i) stores under the prevailing P(i) status.