The YBR056W-A and Its Ortholog YDR034W-B of S. cerevisiae Belonging to CYSTM Family Participate in Manganese Stress Overcoming.

The CYSTM (cysteine-rich transmembrane module) protein family comprises small molecular cysteine-rich tail-anchored membrane proteins found in many eukaryotes. The Saccharomyces cerevisiae strains carrying the CYSTM genes YDRO34W-B and YBR056W-A (MNC1) fused with GFP were used to test the expression of these genes under different stresses. The YBR056W-A (MNC1) and YDR034W-B genes are expressed under stress conditions caused by the toxic concentrations of heavy metal ions, such as manganese, cobalt, nickel, zinc, cuprum, and 2.4-dinitrophenol uncoupler. The expression level of YDR034W-B was higher than that of YBR056W-A under alkali and cadmium stresses. The Ydr034w-b-GFP and Ybr056w-a-GFP proteins differ in the cellular localization: Ydr034w-b-GFP was mainly observed in the plasma membrane and vacuolar membrane, while Ybr056w-a-GFP was observed in the cytoplasm, probably in intracellular membranes. The null-mutants in both genes demonstrated decreased cell concentration and lytic phenotype when cultivated in the presence of excess manganese. This allows for speculations about the involvement of Mnc1 and Ydr034w-b proteins in manganese stress overcoming.

Enzymes of Polyphosphate Metabolism in Yeast: Properties, Functions, Practical Significance.

Inorganic polyphosphates (polyP) are the linear polymers of orthophosphoric acid varying in the number of phosphate residues linked by the energy-rich phosphoanhydride bonds. PolyP is an essential component in living cells. Knowledge of polyP metabolizing enzymes in eukaryotes is necessary for understanding molecular mechanisms of polyP metabolism in humans and development of new approaches for treating bone and cardiovascular diseases associated with impaired mineral phosphorus metabolism. Yeast cells represent a rational experimental model for this research due to availability of the methods for studying phosphorus metabolism and construction of knockout mutants and strains overexpressing target proteins. Multicomponent system of polyP metabolism in Saccharomyces cerevisiae cells is presented in this review discussing properties, functioning, and practical significance of the enzymes involved in the synthesis and degradation of this important metabolite.

Ppn2 endopolyphosphatase overexpressed in Saccharomyces cerevisiae: Comparison with Ppn1, Ppx1, and Ddp1 polyphosphatases.

Saccharomyces cerevisiae has high level of inorganic polyphosphate and a multicomponent system of its metabolism, including polyphosphatases Ppx1, Ppn1, Ddp1, and Ppn2. The aim of the study was to construct the yeast strain overexpressing Ppn2 and to compare the properties of Ppn2, Ppx1, Ppn1, and Ddp1 purified from overexpressing strains of S. cerevisiae. We overexpressed Ppn2 in S. cerevisiae under a strong constitutive promoter of the yeast glyceraldehyde-3-phosphate dehydrogenase-encoding gene and suggested biochemical criteria for distinguishing among yeast polyphosphatases, which is important for their identification and understanding of their functions. Ppn2, Ppn1, and Ddp1 had endopolyphosphatase activities, whereas Ppx1 did not. Ppx1 and Ppn1 exhibited high and Ddp1 and Ppn2 low exopolyphosphatase activity: 240, 500, 0.05 and 0.1 U/mg protein, respectively. The enzymes had distinct patterns of exopolyphosphatase activities stimulation by divalent metal ions. Ppn2, Ppn1 and Ddp1 displayed endopolyphosphatase activity in the presence of 1 mM Mg2+. The endopolyphosphatase activities of Ppn2 and Ppn1 were induced by 0.01 mM of Co2+ or Zn2+, whereas that of Ddp1 required 0.1 mM of these cations. The endopolyphosphatase activity of Ppn1 was inhibited by 0.01 mg mL-1 of heparin, while endopolphosphatase activity of Ppn2 was weakly sensitive to 0.25 mg mL-1 of heparin. The Ppx1 and Ppn1 activity with guanosine tetraphosphate was nearly 80% of activity with long-chain polyphosphates. The Ppn1 hydrolyzed dATP, while Ppx1 did not. The differences in the mode of polyphosphate hydrolysis, substrate specificity, metal ion dependence and cell localization suggest distinct roles of these enzymes in yeast.

Inorganic polyphosphate in methylotrophic yeasts.

Inorganic polyphosphate (polyP) is a significant regulatory and metabolic compound in yeast cells. We compared polyP content and localization, polyphosphatase activities, and transcriptional profile of polyP-related genes in industrially important methylotrophic yeasts, Hansenula polymorpha and Pichia pastoris. The increased need for phosphate, the decrease of long-chain polyP level, the accumulation of short-chain polyP, and enhanced endopolyphosphatase activity in the crude membrane fraction were observed in methanol-grown cells compared with glucose-grown cells of both species. Transcriptome analysis revealed notable differences in the expression patterns of key genes encoding proteins related to polyP metabolism. In methanol-grown cells, the genes encoding endopolyphosphatases and phosphate transporters were upregulated. The changes in polyP metabolism are probably related to the peculiarities of bioenergetics of methanol-grown cells.

Cell wall canals formed upon growth of Candida maltosa in the presence of hexadecane are associated with polyphosphates.

Canals are supramolecular complexes observed in the cell wall of Candida maltosa grown in the presence of hexadecane as a sole carbon source. Such structures were not observed in glucose-grown cells. Microscopic observations of cells stained with diaminobenzidine revealed the presence of oxidative enzymes in the canals. 4΄,6΄-diamino-2-phenylindole staining revealed that a substantial part of cellular polyphosphate was present in the cell wall of cells grown on hexadecane in condition of phosphate limitation. The content and chain length of polyphosphates were higher in hexadecane-grown cells than in glucose grown ones. The treatment of cells with yeast polyphosphatase PPX1 resulted in the decrease of the canal size. These data clearly indicated that polyphosphates are constituents of canals; they might play an important role in the canal structure and functioning.

Manganese tolerance in yeasts involves polyphosphate, magnesium, and vacuolar alterations.

Basidiomycetous and ascomycetous yeast species were tested for manganese tolerance. Basidiomycetous Cryptococcus humicola, Cryptococcus terricola, Cryptococcus curvatus and ascomycetous Candida maltosa, Kluyveromyces marxianus, Kuraishia capsulata, Lindnera fabianii and Sacharomyces cerevisiae were able to grow at manganese excess (2.5 mmol/L), while the growth of basidiomycetous Rhodotorula bogoriensis was completely suppressed. The lag phase duration increased and the exponential growth rate decreased at manganese excess. The increase of cell size and enlargement of vacuoles were characteristics for the cells grown at manganese excess. The alterations in inorganic polyphosphate content and cellular localization were studied. L. fabianii, K. capsulata, C. maltosa, and Cr. humicola accumulated the higher amounts of inorganic polyphosphates, while Cr. terricola and Cr. curvatus demonstrated no such accumulation. The polyphosphate content in the cell wall tested by DAPI staining increased in all species under the study; however, this effect was more pronounced in Cr. terricola and Cr. curvatus. The accumulation of Mg(2+) in the cell wall under Mn(2+) excess was observed in Cr. humicola, Cr. curvatus and Cr. terricola. The accumulation of polyphosphate and magnesium in the cell wall was supposed to be a factor of manganese tolerance in yeasts.

Cytoplasmic inorganic polyphosphate participates in the heavy metal tolerance of Cryptococcus humicola.

The basidiomycetous yeast Cryptococcus humicola was shown to be tolerant to manganese, cobalt, nickel, zinc, lanthanum, and cadmium cations at a concentration of 2.5 mmol/L, which is toxic for many yeasts. The basidiomycetous yeast Cryptococcus terreus was sensitive to all these ions and did not grow at the above concentration. In the presence of heavy metal cations, С. humicola, as opposed to C. terreus, was characterized by the higher content of acid-soluble inorganic polyphosphates. In vivo 4',6'-diamino-2-phenylindole dihydrochloride staining revealed polyphosphate accumulation in the cell wall and cytoplasmic inclusions of С. humicola in the presence of heavy metals. In C. terreus, polyphosphates in the presence of heavy metals accumulate mainly in vacuoles, which results in morphological changes in these organelles and, probably, disturbance of their function. The role of polyphosphate accumulation and cellular localization as factors of heavy metal tolerance of Cryptococcus humicola is discussed.

Adaptation of Saccharomyces cerevisiae to toxic manganese concentration triggers changes in inorganic polyphosphates.

The ability of Saccharomyces cerevisiae to adapt to toxic Mn(2+) concentration (4 mM) after an unusually long lag phase has been demonstrated for the first time. The mutants lacking exopolyphosphatase PPX1 did not change the adaptation time, whereas the mutants lacking exopolyphosphatase PPN1 reduced the lag period compared with the wild-type strains. The cell populations of WT and ΔPPN1 in the stationary phase at cultivation with Mn(2+) contained a substantial number of enlarged cells with a giant vacuole. The adaptation correlated with the triggering of polyphosphate metabolism: the drastic increase in the rate and chain length of acid-soluble polyphosphate. The share of this fraction, which is believed to be localized in the cytoplasm, increased to 76%. Its average chain length increased to 200 phosphate residues compared with 15 at the cultivation in the absence of manganese. DAPI-stained inclusions in the cytoplasm were accumulated in the lag phase during the cultivation with Mn(2+).

Accumulation of phosphate and polyphosphate by Cryptococcus humicola and Saccharomyces cerevisiae in the absence of nitrogen.

The search for new phosphate-accumulating microorganisms is of interest in connection with the problem of excess phosphate in environment. The ability of some yeast species belonging to ascomycetes and basidiomycetes for phosphate (P (i) ) accumulation in nitrogen-deficient medium was studied. The ascomycetous Saccharomyces cerevisiae and Kuraishia capsulata and basidiomycetous Cryptococcus humicola, Cryptococcus curvatus, and Pseudozyma fusiformata were the best in P (i) removal. The cells of Cryptococcus humicola and S. cerevisiae took up 40% P (i) from the media containing P (i) and glucose (5 and 30 mM, respectively), and up to 80% upon addition of 5 mM MgSO(4) (.) The cells accumulated P (i) mostly in the form of polyphosphate (PolyP). In the presence of Mg(2+) , the content of PolyP with longer average chain length increased in both yeasts; they both had numerous inclusions fluorescing in the yellow region of the spectrum, typical of DAPI-PolyP complexes. Among the yeast species tested, Cryptococcus humicola is a new promising model organisms to study phosphorus removal from the media and biomineralization in microbial cells.

Phosphate accumulation of Acetobacter xylinum.

The cells of Acetobacter xylinum decreased phosphate concentration in the medium from 5 to 2.5 or 0.3 mM during incubation in the presence of Mg(2+) and glucose, or Mg(2+) and casamino acids, respectively. The prevalence of orthophosphate or polyphosphate in the biomass of A. xylinum depends on the medium composition. Under phosphate uptake in the presence of glucose, the content of orthophosphate in the biomass changed little, while that of polyphosphate increased fourfold. At incubation with casamino acids, the content of orthophosphate increased 15 times, while that of polyphosphate increased only 2.5 times. Some part of orthophosphate in this case seems to be bound with the cell surface. The polyphosphate chain length in the cells of A. xylinim increases under phosphate uptake. This increase is more noticeable in the presence of glucose. Casamino acids can be replaced by alpha-ketoglutaric acid in combination with (NH(4))(2)SO(4), or arginine, or glutamine, the catabolism of which results in formation of NH(4) (+) and alpha-ketoglutarate.