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1.
Nat Commun ; 14(1): 2645, 2023 05 08.
Artículo en Inglés | MEDLINE | ID: mdl-37156835

RESUMEN

Many proteins involved in eukaryotic phosphate homeostasis are regulated by SPX domains. In yeast, the vacuolar transporter chaperone (VTC) complex contains two such domains, but mechanistic details of its regulation are not well understood. Here, we show at the atomic level how inositol pyrophosphates interact with SPX domains of subunits Vtc2 and Vtc3 to control the activity of the VTC complex. Vtc2 inhibits the catalytically active VTC subunit Vtc4 by homotypic SPX-SPX interactions via the conserved helix α1 and the previously undescribed helix α7. Binding of inositol pyrophosphates to Vtc2 abrogates this interaction, thus activating the VTC complex. Accordingly, VTC activation is also achieved by site-specific point mutations that disrupt the SPX-SPX interface. Structural data suggest that ligand binding induces reorientation of helix α1 and exposes the modifiable helix α7, which might facilitate its post-translational modification in vivo. The variable composition of these regions within the SPX domain family might contribute to the diversified SPX functions in eukaryotic phosphate homeostasis.


Asunto(s)
Difosfatos , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Difosfatos/metabolismo , Transporte Biológico , Homeostasis , Fosfatos de Inositol/metabolismo
2.
EMBO J ; 42(10): e113320, 2023 05 15.
Artículo en Inglés | MEDLINE | ID: mdl-37066886

RESUMEN

The eukaryotic vacuolar transporter chaperone (VTC) complex acts as a polyphosphate (polyP) polymerase that synthesizes polyP from adenosine triphosphate (ATP) and translocates polyP across the vacuolar membrane to maintain an intracellular phosphate (Pi ) homeostasis. To discover how the VTC complex performs its function, we determined a cryo-electron microscopy structure of an endogenous VTC complex (Vtc4/Vtc3/Vtc1) purified from Saccharomyces cerevisiae at 3.1 Å resolution. The structure reveals a heteropentameric architecture of one Vtc4, one Vtc3, and three Vtc1 subunits. The transmembrane region forms a polyP-selective channel, likely adopting a resting state conformation, in which a latch-like, horizontal helix of Vtc4 limits the entrance. The catalytic Vtc4 central domain is located on top of the pseudo-symmetric polyP channel, creating a strongly electropositive pathway for nascent polyP that can couple synthesis to translocation. The SPX domain of the catalytic Vtc4 subunit positively regulates polyP synthesis by the VTC complex. The noncatalytic Vtc3 regulates VTC through a phosphorylatable loop. Our findings, along with the functional data, allow us to propose a mechanism of polyP channel gating and VTC complex activation.


Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Microscopía por Crioelectrón , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Vacuolas/metabolismo , Polifosfatos/metabolismo
3.
mBio ; 14(3): e0010223, 2023 06 27.
Artículo en Inglés | MEDLINE | ID: mdl-37074217

RESUMEN

Cells stabilize intracellular inorganic phosphate (Pi) to compromise between large biosynthetic needs and detrimental bioenergetic effects of Pi. Pi homeostasis in eukaryotes uses Syg1/Pho81/Xpr1 (SPX) domains, which are receptors for inositol pyrophosphates. We explored how polymerization and storage of Pi in acidocalcisome-like vacuoles supports Saccharomyces cerevisiae metabolism and how these cells recognize Pi scarcity. Whereas Pi starvation affects numerous metabolic pathways, beginning Pi scarcity affects few metabolites. These include inositol pyrophosphates and ATP, a low-affinity substrate for inositol pyrophosphate-synthesizing kinases. Declining ATP and inositol pyrophosphates may thus be indicators of impending Pi limitation. Actual Pi starvation triggers accumulation of the purine synthesis intermediate 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), which activates Pi-dependent transcription factors. Cells lacking inorganic polyphosphate show Pi starvation features already under Pi-replete conditions, suggesting that vacuolar polyphosphate supplies Pi for metabolism even when Pi is abundant. However, polyphosphate deficiency also generates unique metabolic changes that are not observed in starving wild-type cells. Polyphosphate in acidocalcisome-like vacuoles may hence be more than a global phosphate reserve and channel Pi to preferred cellular processes. IMPORTANCE Cells must strike a delicate balance between the high demand of inorganic phosphate (Pi) for synthesizing nucleic acids and phospholipids and its detrimental bioenergetic effects by reducing the free energy of nucleotide hydrolysis. The latter may stall metabolism. Therefore, microorganisms manage the import and export of phosphate, its conversion into osmotically inactive inorganic polyphosphates, and their storage in dedicated organelles (acidocalcisomes). Here, we provide novel insights into metabolic changes that yeast cells may use to signal declining phosphate availability in the cytosol and differentiate it from actual phosphate starvation. We also analyze the role of acidocalcisome-like organelles in phosphate homeostasis. This study uncovers an unexpected role of the polyphosphate pool in these organelles under phosphate-rich conditions, indicating that its metabolic roles go beyond that of a phosphate reserve for surviving starvation.


Asunto(s)
Difosfatos , Saccharomyces cerevisiae , Difosfatos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Polifosfatos/metabolismo , Inositol/metabolismo , Adenosina Trifosfato/metabolismo
4.
Top Curr Chem (Cham) ; 375(3): 51, 2017 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-28444630

RESUMEN

The complexity of phosphorylation pathways and their downstream effects is vast. Synthetic chemistry has been working side by side with biology to develop phosphate labels for biological processes involving phosphorylated compounds. This chapter discusses recently employed methods for the preparation of several phosphate labels. Synthesis of biomolecules and their analogs and other useful or potentially useful phosphate derivatives is discussed.


Asunto(s)
Fosfatos/química , Estructura Molecular , Fosforilación
5.
J Biol Chem ; 291(42): 22262-22275, 2016 Oct 14.
Artículo en Inglés | MEDLINE | ID: mdl-27587415

RESUMEN

SPX domains control phosphate homeostasis in eukaryotes. Ten genes in yeast encode SPX-containing proteins, among which YDR089W is the only one of unknown function. Here, we show that YDR089W encodes a novel subunit of the vacuole transporter chaperone (VTC) complex that produces inorganic polyphosphate (polyP). The polyP synthesis transfers inorganic phosphate (Pi) from the cytosol into the acidocalcisome- and lysosome-related vacuoles of yeast, where it can be released again. It was therefore proposed for buffer changes in cytosolic Pi concentration (Thomas, M. R., and O'Shea, E. K. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 9565-9570). Vtc5 physically interacts with the VTC complex and accelerates the accumulation of polyP synthesized by it. Deletion of VTC5 reduces polyP accumulation in vivo and in vitro Its overexpression hyperactivates polyP production and triggers the phosphate starvation response via the PHO pathway. Because this Vtc5-induced starvation response can be reverted by shutting down polyP synthesis genetically or pharmacologically, we propose that polyP synthesis rather than Vtc5 itself is a regulator of the PHO pathway. Our observations suggest that polyP synthesis not only serves to establish a buffer for transient drops in cytosolic Pi levels but that it can actively decrease or increase the steady state of cytosolic Pi.


Asunto(s)
Proteínas Portadoras/metabolismo , Chaperonas Moleculares/metabolismo , Polifosfatos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Portadoras/genética , Chaperonas Moleculares/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
6.
Science ; 352(6288): 986-90, 2016 May 20.
Artículo en Inglés | MEDLINE | ID: mdl-27080106

RESUMEN

Phosphorus is a macronutrient taken up by cells as inorganic phosphate (P(i)). How cells sense cellular P(i) levels is poorly characterized. Here, we report that SPX domains--which are found in eukaryotic phosphate transporters, signaling proteins, and inorganic polyphosphate polymerases--provide a basic binding surface for inositol polyphosphate signaling molecules (InsPs), the concentrations of which change in response to P(i) availability. Substitutions of critical binding surface residues impair InsP binding in vitro, inorganic polyphosphate synthesis in yeast, and P(i) transport in Arabidopsis In plants, InsPs trigger the association of SPX proteins with transcription factors to regulate P(i) starvation responses. We propose that InsPs communicate cytosolic P(i) levels to SPX domains and enable them to interact with a multitude of proteins to regulate P(i) uptake, transport, and storage in fungi, plants, and animals.


Asunto(s)
Homeostasis , Inositol/metabolismo , Proteínas de Transporte de Fosfato/química , Fósforo/metabolismo , Polifosfatos/metabolismo , Transferasas (Grupos de Otros Fosfatos Sustitutos)/química , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Cristalografía por Rayos X , Citosol/metabolismo , Humanos , Proteínas de Transporte de Fosfato/genética , Estructura Secundaria de Proteína/genética , Estructura Terciaria de Proteína/genética , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/química , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética
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