Key role for intracellular K+ and protein kinases Sat4/Hal4 and Hal5 in the plasma membrane stabilization of yeast nutrient transporters

K+ transport in living cells must be tightly controlled because it affects basic physiological parameters such as turgor, membrane potential, ionic strength, and pH. In yeast, the major high-affinity K+ transporter, Trk1, is inhibited by high intracellular K+ levels and positively regulated by two redundant "halotolerance" protein kinases, Sat4/Hal4 and Hal5. Here we show that these kinases are not required for Trk1 activity; rather, they stabilize the transporter at the plasma membrane under low K+ conditions, preventing its endocytosis and vacuolar degradation. High concentrations (0.2 M) of K+, but not Na+ or sorbitol, transported by undefined low-affinity systems, maintain Trk1 at the plasma membrane in the hal4 hal5 mutant. Other nutrient transporters, such as Can1 (arginine permease), Fur4 (uracil permease), and Hxt1 (low-affinity glucose permease), are also destabilized in the hal4 hal5 mutant under low K+ conditions and, in the case of Can1, are stabilized by high K+ concentrations. Other plasma membrane proteins such as Pma1 (H+ -pumping ATPase) and Sur7 (an eisosomal protein) are not regulated by halotolerance kinases or by high K+ levels. This novel regulatory mechanism of nutrient transporters may participate in the quiescence/growth transition and could result from effects of intracellular K+ and halotolerance kinases on membrane trafficking and/or on the transporters themselves.     

A novel mechanism of ion homeostasis and salt tolerance in yeast: the Hal4 and Hal5 protein kinases modulate the Trk1-Trk2 potassium transporter

The regulation of intracellular ion concentrations is a fundamental property of living cells. Although many ion transporters have been identified, the systems that modulate their activity remain largely unknown. We have characterized two partially redundant genes from Saccharomyces cerevisiae, HAL4/SAT4 and HAL5, that encode homologous protein kinases implicated in the regulation of cation uptake. Overexpression of these genes increases the tolerance of yeast cells to sodium and lithium, whereas gene disruptions result in greater cation sensitivity. These phenotypic effects of the mutations correlate with changes in cation uptake and are dependent on a functional Trk1-Trk2 potassium transport system. In addition, hal4 hal5 and trk1 trk2 mutants exhibit similar phenotypes: (i) they are deficient in potassium uptake; (ii) their growth is sensitive to a variety of toxic cations, including lithium, sodium, calcium, tetramethylammonium, hygromycin B, and low pH; and (iii) they exhibit increased uptake of methylammonium, an indicator of membrane potential. These results suggest that the Hal4 and Hal5 protein kinases activate the Trk1-Trk2 potassium transporter, increasing the influx of potassium and decreasing the membrane potential. The resulting loss in electrical driving force reduces the uptake of toxic cations and improves salt tolerance. Our data support a role for regulation of membrane potential in adaptation to salt stress that is mediated by the Hal4 and Hal5 kinases.     

Regulation of Trk-dependent potassium transport by the calcineurin pathway involves the Hal5 kinase

The phosphatase calcineurin and the kinases Hal4/Hal5 regulate high-affinity potassium uptake in Saccharomyces cerevisiae through the Trk1 transporter. We demonstrate that calcineurin is necessary for high-affinity potassium uptake even in the absence of Na⁺ stress. HAL5 expression is induced in response to stress in a calcineurin-dependent manner through a newly identified functional CDRE (nt −195/−189). Lack of calcineurin decreases Hal5 protein levels, although with little effect on Trk1 amounts. However, the growth defect of cnb1 cells at K⁺-limiting conditions can be rescued in part by overexpression of HAL5, and this mutation further aggravates the potassium requirements of a hal4 strain. This suggests that the control exerted by calcineurin on Hal5 expression may be biologically relevant for Trk1 regulation.     

Regulation of yeast H(+)-ATPase by protein kinases belonging to a family dedicated to activation of plasma membrane transporters

The regulation of electrical membrane potential is a fundamental property of living cells. This biophysical parameter determines nutrient uptake, intracellular potassium and turgor, uptake of toxic cations, and stress responses. In fungi and plants, an important determinant of membrane potential is the electrogenic proton-pumping ATPase, but the systems that modulate its activity remain largely unknown. We have characterized two genes from Saccharomyces cerevisiae, PTK2 and HRK1 (YOR267c), that encode protein kinases implicated in activation of the yeast plasma membrane H(+)-ATPase (Pma1) in response to glucose metabolism. These kinases mediate, directly or indirectly, an increase in affinity of Pma1 for ATP, which probably involves Ser-899 phosphorylation. Ptk2 has the strongest effect on Pma1, and ptk2 mutants exhibit a pleiotropic phenotype of tolerance to toxic cations, including sodium, lithium, manganese, tetramethylammonium, hygromycin B, and norspermidine. A plausible interpretation is that ptk2 mutants have a decreased membrane potential and that diverse cation transporters are voltage dependent. Accordingly, ptk2 mutants exhibited reduced uptake of lithium and methylammonium. Ptk2 and Hrk1 belong to a subgroup of yeast protein kinases dedicated to the regulation of plasma membrane transporters, which include Npr1 (regulator of Gap1 and Tat2 amino acid transporters) and Hal4 and Hal5 (regulators of Trk1 and Trk2 potassium transporters).     

Role of protein phosphatases 2C on tolerance to lithium toxicity in the yeast Saccharomyces cerevisiae

Protein phosphatases 2C are a family of conserved enzymes involved in many aspects of the cell biology. We reported that, in the yeast Saccharomyces cerevisiae, overexpression of the Ptc3p isoform resulted in increased lithium tolerance in the hypersensitive hal3 background. We have found that the tolerance induced by PTC3 overexpression is also observed in wild-type cells and that this is most probably the result of increased expression of the ENA1 Na(+)-ATPase mediated by the Hog1 MAP kinase pathway. This effect does not require a catalytically active protein. Surprisingly, deletion of PTC3 (similarly to that of PTC2, PTC4 or PTC5) does not confer a lithium-sensitive phenotype, but mutation of PTC1 does. Lack of PTC1 in an ena1-4 background did not result in additive lithium sensitivity and the ptc1 mutant showed a decreased expression of the ENA1 gene in cells stressed with LiCl. In agreement, under these conditions, the ptc1 mutant was less effective in extruding Li(+) and accumulated higher concentrations of this cation. Deletion of PTC1 in a hal3 background did not exacerbate the halosensitive phenotype of the hal3 strain. In addition, induction from the ENA1 promoter under LiCl stress decreased similarly (50%) in hal3, ptc1 and ptc1 hal3 mutants. Finally, mutation of PTC1 virtually abolishes the increased tolerance to toxic cations provided by overexpression of Hal3p. These results indicate that Ptc1p modulates the function of Ena1p by regulating the Hal3/Ppz1,2 pathway. In conclusion, overexpression of PTC3 and lack of PTC1 affect lithium tolerance in yeast, although through different mechanisms.     

Pyrroline-5-carboxylate synthase senses cellular stress and modulates metabolism by regulating mitochondrial respiration

Pyrroline-5-carboxylate synthase (P5CS) catalyzes the synthesis of pyrroline-5-carboxylate (P5C), a key precursor for the synthesis of proline and ornithine. P5CS malfunction leads to multiple human diseases; however, the molecular mechanism underlying these diseases is unknown. We found that P5CS localizes in mitochondria in rod- and ring-like patterns but diffuses inside the mitochondria upon cellular starvation or exposure to oxidizing agents. Some of the human disease-related mutant forms of P5CS also exhibit diffused distribution. Multimerization (but not the catalytic activity) of P5CS regulates its localization. P5CS mutant cells have a reduced proliferation rate and are sensitive to cellular stresses. Flies lacking P5CS have reduced eclosion rates. Lipid droplets accumulate in the eyes of the newly eclosed P5CS mutant flies, which degenerate with aging. The loss of P5CS in cells leads to abnormal purine metabolism and lipid-droplet accumulation. The reduced lipid-droplet consumption is likely due to decreased expression of the fatty acid transporter, CPT1, and few β-oxidation-related genes following P5CS knockdown. Surprisingly, we found that P5CS is required for mitochondrial respiratory complex organization and that the respiration defects in P5CS knockout cells likely contribute to the metabolic defects in purine synthesis and lipid consumption. This study links amino acid synthesis with mitochondrial respiration and other key metabolic processes, whose imbalance might contribute to P5CS-related disease conditions.Subject terms: Enzyme mechanisms, Cell biology     

Identification of Isn1 and Sdt1 as Glucose- and Vitamin-regulated Nicotinamide Mononucleotide and Nicotinic Acid Mononucleotide 5��-Nucleotidases Responsible for Production of Nicotinamide Riboside and Nicotinic Acid Riboside

Recently, we discovered that nicotinamide riboside and nicotinic acid riboside are biosynthetic precursors of NAD⁺, which are utilized through two pathways consisting of distinct enzymes. In addition, we have shown that exogenously supplied nicotinamide riboside is imported into yeast cells by a dedicated transporter, and it extends replicative lifespan on high glucose medium. Here, we show that nicotinamide riboside and nicotinic acid riboside are authentic intracellular metabolites in yeast. Secreted nicotinamide riboside was detected with a biological assay, and intracellular levels of nicotinamide riboside, nicotinic acid riboside, and other NAD⁺ metabolites were determined by a liquid chromatography-mass spectrometry method. A biochemical genomic screen indicated that three yeast enzymes possess nicotinamide mononucleotide 5′-nucleotidase activity in vitro. Metabolic profiling of knock-out mutants established that Isn1 and Sdt1 are responsible for production of nicotinamide riboside and nicotinic acid riboside in cells. Isn1, initially classified as an IMP-specific 5′-nucleotidase, and Sdt1, initially classified as a pyrimidine 5′-nucleotidase, are additionally responsible for dephosphorylation of pyridine mononucleotides. Sdt1 overexpression is growth-inhibitory to cells in a manner that depends on its active site and correlates with reduced cellular NAD⁺. Expression of Isn1 protein is positively regulated by the availability of nicotinic acid and glucose. These results reveal unanticipated and highly regulated steps in NAD⁺ metabolism.     

The protein kinase Hal5p is the high-copy suppressor of lithium-sensitive mutations of genes involved in the sporulation and meiosis as well as the ergosterol biosynthesis in Saccharomyces cerevisiae

From a genome-scale genetic screen, we have identified 114 lithium-sensitive and 6 lithium-tolerant gene mutations in Saccharomyces cerevisiae. Twenty-five of these identified lithium-sensitive mutations are of genes previously reported to be involved in sporulation and meiosis, whereas thirty-six of them are of genes involved in the vacuolar protein sorting (VPS) pathway, mainly functioning in the membrane docking and fusion. Accordingly, the lithium-sensitive phenotypes for one third of identified VPS mutants well correlate to their intracellular lithium contents in response to lithium stress. This indicates the integrity of the VPS pathway is critic for the ion homeostasis in yeast cells. The halotolerant protein kinase Hal5p, a regulator of the potassium transporter Trk1p, is shown to be the high-copy suppressor of nearly one third of identified lithium-sensitive mutations of genes involved in the sporulation and meiosis as well as in the biosynthesis of ergosterol. These results suggest that Hal5p-mediated ion homeostasis is important for these two biological processes.     

Physiological response of central metabolism in Escherichia coli to deletion of pyruvate oxidase and introduction of heterologous pyruvate carboxylase

We studied the physiological response of Escherichia coli central metabolism to the expression of heterologous pyruvate carboxylase (PYC) in the presence and absence of pyruvate oxidase (POX). These studies were complemented with expression analysis of central and intermediary metabolic genes and conventional in vitro enzyme assays to evaluate glucose metabolism at steady-state growth conditions (chemostats). The absence of POX activity reduced nongrowth-related energy metabolism (maintenance coefficient) and increased the maximum specific rate of oxygen consumption. The presence of PYC activity (i.e., with POX activity) increased the biomass yield coefficient and reduced the maximum specific oxygen consumption rate compared to the wildtype. The presence of PYC in a poxB mutant resulted in a 42% lower maintenance coefficient and a 42% greater biomass yield compared to the wildtype. Providing E. coli with PYC or removing POX increased the threshold specific growth rate at which acetate accumulation began, with an 80% reduction in acetate accumulation observed at a specific growth rate of 0.4 h-1 in the poxB-pyc+ strain. Gene expression analysis suggests utilization of energetically less favorable glucose metabolism via glucokinase and the Entner-Doudoroff pathway in the absence of functional POX, while the upregulation of the phosphotransferase glucose uptake system and several amino acid biosynthetic pathways occurs in the presence of PYC. The physiological and expression changes resulting from these genetic perturbations demonstrate the importance of the pyruvate node in respiration and its impact on acetate overflow during aerobic growth.     

Effect of carbohydrates in the culture medium on amino acid composition of a cephalosporin C producer

The intracellular and extracellular amino acid composition of an auxotrophic methionine-deficient strain of Acremonium chrysogenum was studied in respect of the content of various carbohydrates in the fermentation medium. In the presence of glucose, an intensive involvement of exogenous DL-methionine into the cell metabolism was observed at earlier stages than in the presence of dextran or succrose. The total content of intracellular amino acids was lower in the cells grown on the medium with glucose. The production of cephalosporin C depended on the intracellular content of methionine, glutamic acid and lysine.     

Hepatic histidase and muscle branched chain aminotransferase gene expression in experimental nephrosis

Protein and amino acid metabolism is altered during nephrotic syndrome. However, the expression of the amino acid degrading enzymes has not been well studied. The objective of this work was to assess the expression of hepatic histidase (Hal) and skeletal muscle mitochondrial branched chain amino transferase (BCATm) in rats with experimental nephrotic syndrome induced by a single injection of puromycin aminonucleoside (150 mg/kg). Six days after the injection rats were killed and hepatic Hal and skeletal muscle BCATm activities were measured. Also, total mRNA from both tissues was isolated and Hal and BCATm mRNA expression were analyzed by Northern blot. Rats with NS showed a reduction in food intake with respect to the control group. Hepatic Hal activity increased significantly in nephrotic and pair fed rats by 59% compared to control group. This change in activity was associated with a corresponding increase in Hal mRNA abundance. On the other hand, skeletal muscle BCATm activity and mRNA abundance were similar in the three groups studied. These results suggest that the increase in Hal expression was associated with the reduced food intake and not to the NS. However, BCAT expression did not change indicating the importance of BCAA in body nitrogen conservation.