Vesicular transport of extracellular acid phosphatases in yeast Saccharomyces cerevisiae.

A method for isolation of secretory vesicles from the yeast Saccharomyces cerevisiae based on the disintegration of protoplasts by osmotic shock followed by separation of the vesicles by centrifugation in a density gradient of Urografin was developed in this study. Two populations of the secretory vesicles that differ in density and shape were separated. Acid phosphatases (EC 3.1.3.2) were used as markers of the secretory vesicles. It was shown that the constitutive acid phosphatase (PHO3 gene product) is mainly transported to the cell surface by a lower density population of vesicles, while the repressible acid phosphatase (a heteromer encoded by PHO5, PHO10, and PHO11 genes) by a vesicle population of higher density. These data provide evidence that at least two pathways of transport of yeast secretory proteins from the place of their synthesis and maturation to the cell surface may exist. To reveal the probable reasons for transport of Pho3p and Pho5p/Pho10p/Pho11p enzymes by two different kinds of vesicles, we isolated vesicles from strains that synthesize the homomeric forms of the repressible acid phosphatase. It was demonstrated that glycoproteins encoded by the PHO10 and/or PHO11 genes could be responsible for the choice of one of the alternative transport pathways of the repressible acid phosphatase. A high correlation coefficient between bud formation and secretion of Pho5p phosphatase and the absence of correlation between bud formation and secretion of minor phosphatases Pho10p and Pho11p suggests different functional roles of the polypeptides that constitute the native repressible acid phosphatase.

[Mutual effect of invertase and acid phosphatase from the yeast Saccharomyces cerevisiae on their secretion into culture media]. / Vzaimnoe vliianie invertazy i kisloi fosfatazy drozhzhei Saccharomyces cerevisiae na ikh sekretsiiu v sredu kul'tivirovaniia.

The hypothesis that various extracellular enzymes produced by the yeast Saccharomyces cerevisiae exert a mutual influence on their secretion into the culture medium was tested experimentally. The statistically processed results indicate that extracellular invertase affects the secretion of acid phosphatase, and acid phosphatase affects the secretion of invertase. In addition, the secretion of each of these enzymes was shown to be subject to autoregulation.

Biochemical properties and excretion behavior of repressible acid phosphatases with altered subunit composition.

Yeast repressible acid phosphatase (rAP) is the oligomeric extracellular enzyme encoded by the three structural genes PH05 (p60), PHO10 (p58) and PHO11 (p56). We examined the ability of acid phosphatases formed by various subunit combinations to be excreted into the medium. Plasmids with repressible acid phosphatase structural genes under control of the yeast glyceraldehyde-phosphate dehydrogenase (GAP) promoter were constructed to obtain constitutive expression of acid phosphatase, and yeast strains with disruptions in PHO5, PHO10 and PHO11, respectively, were used to generate mutants expressing single genes or specific gene combinations. EndoF treatment of acid phosphatases, produced by these strains, followed by SDS-electrophoresis in combination with densitometry techniques revealed that the ratio p60/(p56 + p58) among structural polypeptides in extracellular enzyme is constant and equals to 6.0. A study of acid phosphatases formed by single type subunits was undertaken. Expression products of PHO5, PHO10 and PHO11 genes were isolated from the culture medium. The specific activities of the enzymes were found to be 33, 2 and 2 mM x mg-1 x min-1, respectively. The values of Mr estimated by HPLC chromatography for the enzymes encoded for by the genes PHO5, PHO10 and PHO11 and SDS-polyacrilamide gel electrophoresis data suggested an oligomeric organisation of the enzymes. Isoelectric focusing in polyacrylamide gel with immobilised pH gradient followed by activity staining yielded numerous sharp bands of homopolymeric acid phosphatases forms being different in their pI. The kinetic characterisation of the enzymes revealed differences in Km values, sensitivity to temperature inactivation, inhibition by orthophosphate and the effect of pH on the enzyme activity.

[Analysis of the quaternary structure of secreted repressible acid phosphatase from the yeast Saccharomyces cerevisiae]. / Analiz chetvertichnoi struktury sekretiruemoi repressibel'noi kisloi fosfatazy drozhzhei Saccharomyces cerevisiae.

The structural organization of extracellular repressible acid phosphatase from S. cerevisiae has been studied. The existence of multiple acid phosphatase forms with isoelectric points at pH 4.1-4.8 has been confirmed by isoelectrofocusing. The molecular masses of three acid phosphatase isoforms (56, 57-59, and 60 kDa) obtained after enzymatic deglycosylation correlate with the data obtained previously during the analysis of translation products in cell-free systems. Electron microscopic studies revealed that the acid phosphatase molecule has a square shape and is made up of four identical subunits with molecular masses of about 125 kDa.

[Possible existence of alternative secretion routes of acid phosphatase in the yeast Saccharomyces cerevisiae]. / O vozmozhnosti sushchestvovaniia razlichnykh putei sekretsii kisloi fosfatazy u drozhzhei Saccharomyces cerevisiae.

The secretion of different acid phosphatase forms into the growth medium was studied in the yeast Saccharomyces cerevisiae. The secretion of constitutive acid phosphatase highly correlated with the process of bud formation. This exoenzyme might be involved in the construction of the bud cell wall. Analysis of the rate, at which repressible acid phosphatase was excreted into the growth medium, as well as a negative correlation between its secretion and the process of bud formation were indicative of an additional route via which the repressible form of acid phosphatase was secreted. It is possible that yeast cells contain vesicles similar to "safe" vesicles in higher eukaryotic cells.

[Formation of secreted acid phosphatase during the growth of Saccharomyces cerevisiae yeasts on different sources of carbon and nitrogen nutrition]. / Obazovanie sekretiruemoi kisloi fosfatazay pri roste drozhzhei Saccharomyces cerevisiae na razlichnykh istochnikakh uglerodnogo i azotnogo pitaniia.

The effect of certain components in the growth medium on the secretion of acid phosphatase was studied with Saccharomyces cerevisiae. The presence of phosphate at a concentration of 10 mM in the medium inhibited the formation of repressible forms of this enzyme. The synthesis of the secreted enzyme depended on the sources of carbon and nitrogen nutrition. The enzyme yield was highest in a medium with sucrose as a carbon source and ammonium chloride as a nitrogen source. The secretion of acid phosphatase is stimulated by an increase in the sugar content and a deficiency of the nitrogen source in the medium.

[Isolation and properties of tripolyphosphatase from Neurospora crassa]. / Vydelenie i svoistova tripolifosfatazy griba Neurospora crassa

Homogenous preparation of tripolyphosphatase from Neurospora crassa is obtained. The enzyme is found to consist of two equal subunits with molecular weight of 40 000 and to have pH optimum 7.0 and temperature optimum 50 degrees C. Bivalent metal ions are required for its catalytical activity, the hest activators being Co2+, Mg2+ and Mn2+. Strict specificity of the enzyme to tripolyphosphate is demonstrated, Km being 5.9-10(-4) M. The enzyme hydrolyses tripolyphosphate to equimolar mixture of ortho- and pyrophosphate. The enzyme activity depends on orthophosphate and pyrophosphate concentrations in the incubation medium.