Inorganic Phosphate and Sulfate Transport in S. cerevisiae.

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.

A novel blood-based biomarker for detection of autism spectrum disorders.

Autism spectrum disorders (ASD) are classified as neurological developmental disorders. Several studies have been carried out to find a candidate biomarker linked to the development of these disorders, but up to date no reliable biomarker is available. Mass spectrometry techniques have been used for protein profiling of blood plasma of children with such disorders in order to identify proteins/peptides that may be used as biomarkers for detection of the disorders. Three differentially expressed peptides with mass-charge (m/z) values of 2020 ± 1, 1864 ± 1 and 1978 ± 1 Da in the heparin plasma of children with ASD that were significantly changed as compared with the peptide pattern of the non-ASD control group are reported here. This novel set of biomarkers allows for a reliable blood-based diagnostic tool that may be used in diagnosis and potentially, in prognosis of ASD.

A new Alkalitolerant Yarrowia lipolytica yeast strain is a promising model for dissecting properties and regulation of Na+ -dependent phosphate transport systems.

A newly isolated osmo-, salt-, and alkalitolerant Yarrowia lipolytica yeast strain is distinguished from other yeast species by its capacity to grow vigorously at alkaline pH values (9.7), which makes it a promising model organism for studying Na+-dependent phosphate transport systems in yeasts. Phosphate uptake by Y. lipolytica cells grown at pH 9.7 was mediated by several kinetically discrete Na+-dependent systems specifically activated by Na+. One of these, a low-affinity transporter, operated at high concentrations of extracellular phosphate. The other two, high-affinity systems, maximally active in phosphate-starved cells, were repressed or derepressed depending on the prevailing extracellular phosphate concentration and pH value. The contribution of Na+/P(i)-cotransport systems to the total cellular phosphate uptake progressively increased with increasing pH, reaching its maximum at pH >/= 9.

Dual regulation of proton- and sodium-coupled phosphate transport systems in the Yarrowia lipolytica yeast by extracellular phosphate and pH.

In this study we used a newly isolated osmo-, salt-, and alkali-tolerant Yarrowia lipolytica yeast strain, with a unique capacity to grow over a wide pH range (3.5-10.5). A procedure was elaborated to follow phosphate accumulation by Y. lipolytica cells grown at different pH values. In this paper we demonstrate that Pi-starved Y. lipolytica cells are endowed by derepressible high-affinity, high-capacity H(+)- and Na(+)-driven Pi uptake systems and that activities of these transport systems are under the dual control by the prevailing extracellular Pi concentrations and pH values.

Isolation and characterization of a novel leaf-inhabiting osmo-, salt-, and alkali-tolerant Yarrowia lipolytica yeast strain.

Salt-excreting leaves of Atriplex halimus plants harvested in the central Negev Highlands of Israel were screened for yeasts inhabiting their surfaces. Several aerobic, moderately salt- and alkali-tolerant yeasts were isolated. One of the isolates (tentatively designated S-8) was identified as Yarrowia lipolytica (Wick.) van der Walt and Arx, on the basis of its morphological, biochemical/physiological characteristics, and of quantitative chemotaxonomic and molecular marker analyses. However, the strain is distinguished from the known members of the type Y. lipolytica strain by its pronounced osmo-, salt-, and pH tolerance. Cells displayed a unique capacity to grow over a wide pH range (from 3.5 to 11.5) with a pH optimum at 4.5 to 7.5. It is proposed that the S-8 strain be assigned to a single Y. lipolytica species as its anamorpha, or as a new variety, Y. lipolytica var. alkalitolerance. The ecophysiological properties and biotechnological potentials of the new strain are discussed.

Properties of the cysteine-less Pho84 phosphate transporter of Saccharomyces cerevisiae.

The derepressible Pho84 high-affinity phosphate permease of Saccharomyces cerevisiae, encoded by the PHO84 gene belongs to a family of phosphate:proton symporters (PHS). The protein contains 12 native cysteine residues of which five are predicted to be located in putative transmembrane regions III, VI, VIII, IX, and X, and the remaining seven in the hydrophilic domains of the protein. Here we report on the construction of a Pho84 transporter devoid of cysteine residues (C-less) in which all 12 native residues were replaced with serines using PCR mutagenesis and the functional consequences of this. Our results clearly demonstrate that the C-less Pho84 variant is able to support growth of yeast cells to the same extent as the wild-type Pho84 and is stably expressed under derepressible conditions and is fully active in proton-coupled phosphate transport across the yeast plasma membrane.

Proton- and sodium-coupled phosphate transport systems and energy status of Yarrowia lipolytica cells grown in acidic and alkaline conditions.

In this study we have used a newly isolated Yarrowia lipolytica yeast strain with a unique capacity to grow over a wide pH range (3.5-10.5), which makes it an excellent model system for studying H(+)- and Na(+)-coupled phosphate transport systems. Even at extreme growth conditions (low concentrations of extracellular phosphate, alkaline pH values) Y. lipolytica preserved tightly-coupled mitochondria with the fully competent respiratory chain containing three points of energy conservation. This was demonstrated for the first time for cells grown at pH 9.5-10.0. In cells grown at pH 4.5, inorganic phosphate (P(i)) was accumulated by two kinetically discrete H(+)/P(i)-cotransport systems. The low-affinity system is most likely constitutively expressed and operates at high P(i) concentrations. The high-affinity system, subjected to regulation by both extracellular P(i) availability and intracellular polyphosphate stores, is mobilized during P(i)-starvation. In cells grown at pH 9.5-10, P(i) uptake is mediated by several kinetically discrete Na(+)-dependent systems that are specifically activated by Na(+) ions and insensitive to the protonophore CCCP. One of these, a low-affinity transporter operative at high P(i) concentrations is kinetically characterized here for the first time. The other two, high-affinity, high-capacity systems, are derepressible and functional during P(i)-starvation and appear to be controlled by extracellular P(i). They represent the first examples of high-capacity, Na(+)-driven P(i) transport systems in an organism belonging to neither the animal nor bacterial kingdoms. The contribution of the H(+)- and Na(+)-coupled P(i) transport systems in Y. lipolytica cells grown at different pH values was quantified. In cells grown at pH values of 4.5 and 6.0, the H(+)-coupled P(i) transport systems are predominant. The contribution of the Na(+)/P(i) cotransport systems to the total cellular P(i) uptake activity is progressively increased with increasing pH, reaching its maximum at pH 9 and higher.

Regulation of cation-coupled high-affinity phosphate uptake in the yeast Saccharomyces cerevisiae.

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.

Two systems for phosphate uptake in Yarrowia lipolytica cells grown at acidic conditions.

Inorganic phosphate (Pi) is accumulated by Yarrowia lipolytica cells grown at acidic pH conditions by two kinetically discrete H+/Pi-cotransport systems with apparent K(m) values for Pi of 12-18 microM and 2-3 mM Pi at pH 5.5, respectively. One of these is derepressible and operates at low external Pi concentrations; the other is most likely constitutively expressed and comes into play at high Pi concentrations. The derepression of the high-affinity Pi transport system is under the control of available extracellular Pi as well as the amount of intracellular polyphosphates stores. Characteristics of the Pi transport behavior in Yarrowia lipolytica are discussed.

Phosphate-uptake systems in Yarrowia lipolytica cells grown under alkaline conditions.

In this study we used a newly isolated Yarrowia lipolytica strain with a unique capacity to grow over a wide pH range (3.5-10.5), which makes it an excellent model system for studying phosphate transport systems in cells grown under alkaline conditions. Phosphate uptake by Y. lipolytica yeast cells grown at pH 9.5-10 was shown to be mediated by several kinetically discrete Na+-dependent systems. One of these, a low-affinity transporter, operates at high Pi concentrations and is, to our knowledge, here kinetically characterized for the first time. The other two high-affinity systems are derepressible, come into play under conditions of Pi-starvation, and appear to be controlled by the availability of extracellular Pi. They represent the first examples of high-capacity, Na+-driven Pi transport systems in an organism belonging to neither the animal nor the bacterial kingdoms.

Intracellular localization of an active green fluorescent protein-tagged Pho84 phosphate permease in Saccharomyces cerevisiae.

Green fluorescent protein (GFP) from Aequorea victoria was used as an in vivo reporter protein when fused to the carboxy-terminus of the Pho84 phosphate permease of Saccharomyces cerevisiae. Both components of the fusion protein displayed their native functions and revealed a cellular localization and degradation of the Pho84-GFP chimera consistent with the behavior of the wild-type Pho84 protein. The GFP-tagged chimera allowed for a detection of conditions under which the Pho84 transporter is localized to its functional environment, i.e. the plasma membrane, and conditions linked to relocation of the protein to the vacuole for degradation. By use of the methodology described, GFP should be useful in studies of localization and degradation also of other membrane proteins in vivo.

Characterization of purified and unidirectionally reconstituted Pho84 phosphate permease of Saccharomyces cerevisiae.

Hydropathy analysis of the amino acid sequence of the Pho84 phosphate permease of Saccharomyces cerevisiae suggests that the protein consists of 12 transmembrane domains connected by hydrophilic loops. The Pho84 protein has been modified by a gene fusion approach, yielding two different N-terminal His-tagged chimeras which can be expressed in Escherichia coli, purified and functionally reconstituted into defined proteoliposomes. The continuous epitopes in the N- and C-terminal sequences of the Pho84 chimeras were shown to be accessible in proteoliposomes containing the purified active Pho84 proteins. Site-specific proteolysis of the immunoreactive N-terminal sequence in the reconstituted protein suggests a unidirectional insertion into liposomes.

Studies of cytochrome c oxidase-driven H(+)-coupled phosphate transport catalyzed by the Saccharomyces cerevisiae Pho84 permease in coreconstituted vesicles.

The proton-coupled Pho84 phosphate permease of Saccharomyces cerevisiae, overexpressed as a histidine-tagged chimera in Escherichia coli, was detergent-solubilized, purified, and reconstituted into proteoliposomes. Proteoliposomes containing the Pho84 protein were fused with proteoliposomes containing purified cytochrome c oxidase from beef heart mitochondria. Both components of the coreconstituted system were functionally incorporated in tightly sealed membrane vesicles in which the cytochrome c oxidase-generated electrochemical proton gradient could drive phosphate transport via the proton-coupled Pho84 permease. The metal dependency of transport indicates that a metal-phosphate complex is the translocated substrate.

Functional analysis and cell-specific expression of a phosphate transporter from tomato.

For a better understanding of the molecular and biochemical processes involved in orthophosphate (Pi) uptake at the root/soil interface, we cloned a Pi-transporter c DNA (LePT1) from a root air-specific cDNA library of tomato (Lycopersicon esculentum Mill.). The corresponding protein belongs to the growing family of ion transporters with twelve putative transmembrane domains. It is highly homologous to recently isolated Pi transporters from higher plants, yeast and fungi. When expressed in a Pi-uptake-deficient yeast mutant, the L. esculentum phosphate transporter 1 (LePT1) protein exhibits an apparent Km of 31 MicroM. The transporter is still active at submicromolar Pi concentrations and mediates highest Pi uptake at pH 5. The activity of LePT1 is dependent on the electrochemical membrane potential mediated by the yeast P-type H + - ATPase. Transcript levels of LePT1 in tomato seedlings are detectable in all vegetative organs under Pi-sufficient conditions, with highest concentrations in root hairs. In situ hybridization studies demonstrate cell-specific expression of LePT1 in the tomato root. The LePT1 mRNA is detectable in peripheral cell layers such as rhizodermal and root cap cells. Under Pi-deprivation condition, mRNA levels are also detectable in young stelar tissue. This work presents molecular and biochemical evidence for distinct root cells playing an important role in Pi acquisition at the root/soil interface.

Phosphate permeases of Saccharomyces cerevisiae.

The PHO84 and PHO89 genes of Saccharomyces cerevisiae encode two high-affinity phosphate cotransporters of the plasma membrane. Hydropathy analysis suggests a secondary structure arrangements of the proteins in 12 transmembrane domains. The derepressible Pho84 and Pho89 transporters appear to have characteristic similarities with the phosphate transporters of Neurospora crassa. The Pho84 protein catalyzes a proton-coupled phosphate transport at acidic pH, while the Pho89 protein catalyzes a sodium-dependent phosphate uptake at alkaline pH. The Pho84 transporter can be stably overproduced in the cytoplasmic membrane of Escherichia coli, purified and reconstituted in a functional state into proteoliposomes.

Identification, cloning and characterization of a derepressible Na+-coupled phosphate transporter in Saccharomyces cerevisiae.

Based on the high sequence homology between the yeast ORF YBR296c (accession number P38361 in the SWISS-PROT database) and the PHO4 gene of Neurospora crassa, which codes for a Na+/Pi cotransporter with twelve putative transmembrane domains, the YBR296c ORF was considered to be a promising candidate gene for a plasma membrane-bound phosphate transporter in Saccharomyces cerevisiae. Therefore, this gene, here designated PHO89, was cloned and a set of deletion mutants was constructed. We then studied their Pi uptake activity under different conditions. We show here that a transport activity displayed by PHO89 strains under alkaline conditions and in the presence of Na+ is absent in pho89 null mutants. Moreover, when the pH was lowered to pH 4.5 or when Na+ was omitted, this activity decreased significantly, reaching values close to those exhibited by the deltapho89 mutant. Studies of the acid phosphatase activity of these strains, as well as promoter sequence analysis, suggest that expression of the PHO89 gene is under the control of the PHO regulatory system. Northern analysis shows that this gene is only transcribed under conditions of Pi limitation. This is, to our knowledge, the first demonstration that the PHO89 gene codes for the Na+/Pi cotransporter previously characterized by kinetic studies, and represents the only Na+-coupled secondary anion transport system so far identified in S. cerevisiae. Pho89p has been shown to have an apparent Km of 0.5 microM and a pH optimum of 9.5, and is highly specific for Na+; activation of transport is maximal at a Na+ concentration of 25 mM.

Physiological regulation of the derepressible phosphate transporter in Saccharomyces cerevisiae.

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.

Isolation and characterization of membrane vesicles of Saccharomyces cerevisiae harboring the high-affinity phosphate transporter.

Membrane vesicles with an inside-out orientation were isolated from the plasma membrane of Saccharomyces cerevisiae by an improved aqueous two-phase partitioning technique. The activity of the orthovanadate-sensitive H+-pumping ATPase, the plasma membrane marker, was highly enriched by the partitioning technique. The obtained results suggest that the membrane vesicles produced were predominantly oriented inside-out. The isolated plasma membrane vesicles displayed cross-reactions with antibodies raised against synthetic peptide corresponding to the N-terminal (residues 1-10) and the C-terminal (residues 578-597) regions of the plasma membrane phosphate transporter encoded by the PHO84 gene and the H+-pumping ATPase of S. cerevisiae. The purified membrane vesicles catalyzed a derepressible inhibitor-sensitive phosphate uptake at levels comparable with the situation in intact cells of S. cerevisiae indicating that transport of phosphate across the membrane is both functional and bidirectional. The PHO84 transporter harbored in isolated plasma membranes could moreover be enriched in a high state of purity by immunoaffinity chromatography using immobilized anti-PHO84 antibodies.

Expression and purification of the high-affinity phosphate transporter of Saccharomyces cerevisiae.

The plasma membrane high-affinity phosphate permease of Saccharomyces cerevisiae has been overproduced as a stable membrane-bound chimeric protein in Escherichia coli. Construction of a chimera between the permease and a peptide containing 10 consecutive histidine residues allowed selective binding of the chimera to a chelating column charged with Ni2+, and elution with imidazole in a high state of purity. Approximately 5 mg purified His10-permease was obtained from 3 g (wet mass) cells. The purified phosphate permease chimera catalyzes uncoupler-sensitive phosphate transport after reconstitution into proteoliposomes.