Uptake of Selenite by Saccharomyces cerevisiae Involves the High and Low Affinity Orthophosphate Transporters

Although the general cytotoxicity of selenite is well established, the mechanism by which this compound crosses cellular membranes is still unknown. Here, we show that in Saccharomyces cerevisiae, the transport system used opportunistically by selenite depends on the phosphate concentration in the growth medium. Both the high and low affinity phosphate transporters are involved in selenite uptake. When cells are grown at low P_i concentrations, the high affinity phosphate transporter Pho84p is the major contributor to selenite uptake. When phosphate is abundant, selenite is internalized through the low affinity P_i transporters (Pho87p, Pho90p, and Pho91p). Accordingly, inactivation of the high affinity phosphate transporter Pho84p results in increased resistance to selenite and reduced uptake in low P_i medium, whereas deletion of SPL2, a negative regulator of low affinity phosphate uptake, results in exacerbated sensitivity to selenite. Measurements of the kinetic parameters for selenite and phosphate uptake demonstrate that there is a competition between phosphate and selenite ions for both P_i transport systems. In addition, our results indicate that Pho84p is very selective for phosphate as compared with selenite, whereas the low affinity transporters discriminate less efficiently between the two ions. The properties of phosphate and selenite transport enable us to propose an explanation to the paradoxical increase of selenite toxicity when phosphate concentration in the growth medium is raised above 1 mm.     

Control of secretory production of human lysozyme from Saccharomyces cerevisiae by incubation temperature and phosphate concentration

A system for the controlled expression of a foreign gene in Saccharomyces cerevisiae by temperature and/or inorganic phosphate (P_i) concentration in the medium was constructed. A DNA fragment bearing the promoter of the PHO84 gene, which encodes a P_i transporter of S. cerevisiae and is derepressed by P_i starvation, was used as promoter. When a cDNA fragment encoding the human lysozyme (h-lysozyme) gene connected with the PHO84 promoter was ligated into a YEp vector, a maximum of 4.5 mg/l of the enzyme was secreted from the host cells in low-P_i medium. When a temperature-sensitive pho81 mutant was used as the host with this vector, 2.6 mg/l of h-lysozyme was secreted in low-P_i medium at 25°C and its production was turned off at 37°C.     

Characterization of the S. cerevisiae inp51 mutant links phosphatidylinositol 4,5-bisphosphate levels with lipid content,membrane fluidity and cold growth

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] and its derivatives diphosphoinositol phosphates (DPIPs) play key signaling and regulatory roles. However, a direct function of these molecules in lipid and membrane homeostasis remains obscure. Here, we have studied the cold tolerance phenotype of yeast cells lacking the Inp51-mediated phosphoinositide-5-phosphatase. Genetic and biochemical approaches showed that increased metabolism of PI(4,5)P₂ reduces the activity of the Pho85 kinase by increasing the levels of the DPIP isomer 1-IP₇. This effect was key in the cold tolerance phenotype. Indeed, pho85 mutant cells grew better than the wild-type at 15 °C, and lack of this kinase abolished the inp51-mediated cold phenotype. Remarkably, reduced Pho85 function by loss of Inp51 affected the activity of the Pho85-regulated target Pah1, the yeast phosphatidate phosphatase. Cells lacking Inp51 showed reduced Pah1 abundance, derepression of an INO1-lacZ reporter, decreased content of triacylglycerides and elevated levels of phosphatidate, hallmarks of the pah1 mutant. However, the inp51 phenotype was not associated to low Pah1 activity since deletion of PAH1 caused cold sensitivity. In addition, the inp51 mutant exhibited features not shared by pah1, including a 40%-reduction in total lipid content and decreased membrane fluidity. These changes may influence the activity of membrane-anchored and/or associated proteins since deletion of INP51 slows down the transit to the vacuole of the fluorescent dye FM4-64. In conclusion, our work supports a model in which changes in the PI(4,5)P₂ pool affect the 1-IP₇ levels modulating the activity of Pho85, Pah1 and likely additional Pho85-controlled targets, and regulate lipid composition and membrane properties.     

Growth kinetics and Pho84 phosphate transporter activity of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> under phosphate-limited conditions

The effect of phosphate (P _i ) concentration on the growth behavior of Saccharomyces cerevisiae strain CEN.PK113-5D in phosphate-limited batch and chemostat cultures was studied. The range of dilution rates used in the present study was 0.08–0.45 h⁻¹. The batch growth of yeast cells followed Monod relationship, but growth of the cells in phosphate-limited chemostat showed change in growth kinetics with increasing dilution rates. The difference in growth kinetics of the yeast cells in phosphate-limited chemostat for dilution rates below and above approximately 0.2 h⁻¹ has been discussed in terms of the batch growth kinetic data and the change in the metabolic activity of the yeast cells. Immunological detection of a C-terminally myc epitope-tagged Pho84 fusion protein indicated derepressive expression of the Pho84 high-affinity P _i transporter in the entire range of dilution rates employed in this study. Phosphate transport activity mediated by Pho84 transporter was highest at very low dilution rates, i.e. 0.08–0.1 h⁻¹, corresponding to conditions in which the amount of synthesized Pho84 was at its maximum.     

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⁺/P_i 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 P_i 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 Δpho89 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 P_i limitation. This is, to our knowledge, the first demonstration that the PHO89 gene codes for the Na⁺/P_i 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 K_m of 0.5 μM 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.     

The Saccharomyces cerevisiae high affinity phosphate transporter encoded by PHO84 also functions in manganese homeostasis

In the bakers' yeast Saccharomyces cerevisiae, high affinity manganese uptake and intracellular distribution involve two members of the Nramp family of genes, SMF1 and SMF2. In a search for other genes involved in manganese homeostasis, PHO84 was identified. The PHO84 gene encodes a high affinity inorganic phosphate transporter, and we find that its disruption results in a manganese-resistant phenotype. Resistance to zinc, cobalt, and copper ions was also demonstrated for pho84Delta yeast. When challenged with high concentrations of metals, pho84Delta yeast have reduced metal ion accumulation, suggesting that resistance is due to reduced uptake of metal ions. Pho84p accounted for virtually all the manganese accumulated under metal surplus conditions, demonstrating that this transporter is the major source of excess manganese accumulation. The manganese taken in via Pho84p is indeed biologically active and can not only cause toxicity but can also be incorporated into manganese-requiring enzymes. Pho84p is essential for activating manganese enzymes in smf2Delta mutants that rely on low affinity manganese transport systems. A role for Pho84p in manganese accumulation was also identified in a standard laboratory growth medium when high affinity manganese uptake is active. Under these conditions, cells lacking both Pho84p and the high affinity Smf1p transporter accumulated low levels of manganese, although there was no major effect on activity of manganese-requiring enzymes. We conclude that Pho84p plays a role in manganese homeostasis predominantly under manganese surplus conditions and appears to be functioning as a low affinity metal transporter.     

PhoY2 of Mycobacteria Is Required for Metabolic Homeostasis and Stress Response

The ability of pathogenic mycobacteria to adapt to diverse environments is essential for their success as pathogens. Here we describe a transposon-inactivated phoY2 mutant of Mycobacterium marinum. PhoY2 of mycobacteria is a functional homologue of PhoU in Escherichia coli and an important component of the Pho regulon. We found that PhoY2 is required for maintaining intracellular inorganic phosphate (P_i) homeostasis and balanced energy and redox states. Disruption of phoY2 resulted in elevated levels of intracellular poly-P_i and ATP and an elevated NAD⁺/NADH ratio, and the mutant strain exhibited increased sensitivity to environmental stress conditions, including nutrient deprivation as well as SDS and antibiotic treatments. Taken together, our results suggest that PhoY2 is required for maintaining metabolic homeostasis and adaptation to stress conditions, which may provide an explanation for the suggested role of PhoY2 in drug tolerance.     

Maximum glucoamylase production by a temperature-sensitive mutant of Saccharomyces cerevisiae in batch culture

In order to maximize the glucoamylase production by recombinant Saccharomyces cerevisiae in batch culture, first a temperature-controlled expression system for a foreign gene in S. cerevisiae was constructed. A temperature-sensitive pho80 mutant of S. cerevisiae for the PHO regulatory system, YKU131, was used for this purpose. A DNA fragment bearing the promoter of the PHO84 gene, which encodes an inorganic phosphate (P_i) transporter of S. cerevisiae and is derepressed by P_i starvation, was used as promoter. The glucoamylase gene connected with the PHO84 promoter was ligated into a YEp13 vector, designated pKU122. When the temperature-sensitive pho80 ^ts mutant harboring the plasmid pKU122 is cultivated at a lower temperature, the expression of glucoamylase gene is repressed, but at a higher temperature it is expressed. Next the effect of temperature on the specific growth rate, μ, and specific production rate, ρ, was investigated. Maximum values of ρ and ρ at various temperatures were at 30°C and 34°C, respectively. The optimal cultivation temperature strategy for maximum production of glucoamylase by this recombinant strain in batch culture was then determined by the Maximum principle using the relationships of μ and ρ to the cultivation temperature. Finally, the optimal strategy was experimentally realized by changing the cultivation temperature from Tμ (30°C) to Tρ (34°C) at the switching time, t_s. Received 18 September 1997/ Accepted in revised form 07 January 1998     

The absence of cyclin-dependent protein kinase Pho85 affects stability of mitochondrial DNA in yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The cyclin-dependent protein kinase Pho85 is involved in the regulation of phosphate metabolism in yeast Saccharomyces cerevisiae. Mutations in the PHO85 gene lead to constitutive synthesis of Pho5 acid phosphatase, a delay in cell growth on media containing nonfermentable carbon sources, and other pleiotropic effects. In this work, it was shown that the accumulation of respiratory incompetent cells occurs with high frequency in strains carrying pho85 mutations as early as during the first cell divisions, and the number of these cells at the early logarithmic growth phase of the culture promptly reaches virtually 100%. Cytological analysis revealed a high accumulation rate of [rho⁰] cells in the background of gene pho85 that may be related to disturbances in the distribution of mitochondrial nucleoids rather than to changes in morphology of mitochondria and a delay in their transport into the bud. Genetic analysis revealed that secondary mutations pho4, pho81, pho84, and pho87 stabilize nucleoids and prevent the loss of mitochondrial DNA caused by pho85. These results provide an evidence for the influence of intracellular phosphate concentration on the inheritance of mitochondrial nucleoids, but do not exclude the possibility that the occurrence of mutation pho4 in the background of gene pho85 may change the expression level of other genes required for the stabilization of mitochondrial functions.