Triphosphopyridine nucleotide: cytochrome C reductase of Saccharomyces Cerevisiae a “microsomal” enzyme

Schatz, Gottfried; Klima, Jörg

doi:10.1016/0926-6569(64)90130-0

Cited by 39 publications

(21 citation statements)

References 34 publications

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“…It was therQfore of interest to determine whether this enzyme, as many other mitochondrial enzymes of yeast, is subject to glucose repression. Enzyme activity assayed in promitochondria isolated from glucose-grown cells was only slightly tess (28 nkat/mg of mitochondrial protein) than that in fully derepressed mitochondria (37 nkat/mg of mitochondrial protein) which we.re isolated from cells cultivated on lactate.…”

Section: Resultsmentioning

confidence: 99%

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Subcellular and submitochondrial localization of phospholipid-synthesizing enzymes in Saccharomyces cerevisiae

Kuchler¹,

Daum²,

Paltauf³

1986

J Bacteriol

198

143

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Using highly enriched membrane preparations from lactate-grown Saccharomyces cerevisiae cells, the subcellular and submitochondrial location of eight enzymes involved in the biosynthesis of phospholipids was determined. Phosphatidylserine decarboxylase and phosphatidylglycerolphosphate synthase were localized exclusively in the inner mitochondrial membrane, while phosphatidylethanolamine methyltransferase activity was confined to microsomal fractions. The other five enzymes tested in this study were common both to the outer mitochondrial membrane and to microsomes. The transmembrane orientation of the mitochondrial enzymes was investigated by protease digestion of intact mitochondria and of outside-out sealed vesicles of the outer mitochondrial membrane. Glycerolphosphate acyltransferase, phosphatidylinositol synthase, and phosphatidylserine synthase were exposed at the cytosolic surface of the outer mitochondrial membrane. Cholinephosphotransferase was apparentiy located at the inner aspect or within the outer mitochondrial membrane. Phosphatidate cytidylyltransferase was localized in the endoplasmic reticulum, on the cytoplasmic side of the outer mitochondrial membrane, and in the inner mitochondrial membrane. Inner membrane activity of this enzyme constituted 80% of total mitochondrial activity; inactivation by trypsin digestion was observed only after preincubation of membranes with detergent (0.1% Triton X-100). Total activity of those enzymes that are common to mitochondria and the endoplasmic reticulum was about equally distributed between the two organelles. Data concerning susceptibility to various inhibitors, heat sensitivity, and the pH optima indicate that there is a close similarity of the mitochondrial and microsomal enzymes that catalyze the same reaction.In eucaryotic cells, phospholipids required for the biogenesis and maintenance of membranes are synthesized in distinct subcellular compartments. In mammalian cells, the bulk of cellular phospholipids is synthesized in the endoplasmic reticulum (13), with specific contributions from mitochondria (phosphatidylglycerol and cardiolipin [18]; phosphatidylethanolamine via decarboxylation of phosphatidylserine [13]) and peroxisomes (acyldihydroxyacetonephosphate [25] and its alkyl analog [15]). In contrast to mammalian cells, in Saccharomyces cerevisiae most of the reactions involved in phospholipid biosynthesis have been reported to occur both in mitochondria and in the endoplasmic reticulum (9).The present study on the subcellular and submitochondrial lpcalization of phospholipid synthesizing enzymes in S. cerevisiae was motivated mainly by our interest in the mechanism(s) underlying intracellular phospholipid transport. Knowledge of the origin of the various phospholipids is a prerequisite for an understanding of the process of their transfer across and between subcellular membranes.There are only few reports dealing with the subcellular localization of phospholipid-synthesizing enzymes in S. cerevisiae. Some are at variance, e.g., with respect to ...

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Section: Resultsmentioning

confidence: 99%

“…Previously published procedures were used to assay the mitochondrial (succinate dehydrogenase [1], kynurenine hydroxylase [39], cytochrome b2 [26], fumarase [34]) and the microsomal (NADPH-cytochrome c reductase [37]) marker enzymes.…”

mentioning

confidence: 99%

Subcellular and submitochondrial localization of phospholipid-synthesizing enzymes in Saccharomyces cerevisiae

Kuchler¹,

Daum²,

Paltauf³

1986

J Bacteriol

198

143

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“…In yeast membrane fractions marker enzyme proteins were detected by immunoblot analysis. The distribution of ER membranes was assayed by determination of NADPH-cytochrome c oxidoreductase activity (41). SDS-PAGE and Western blotting were performed as described (18) using a monoclonal antibody raised against the 100-kDa subunit of the vacuolar proton ATPase VPH1, a monoclonal antibody against the late Golgi protein vps10p, and a monoclonal antibody against the endosomal/prevacuolar protein pep12p (Molecular Probes).…”

Section: Methodsmentioning

confidence: 99%

A Novel Intracellular K+/H+ Antiporter Related to Na+/H+ Antiporters Is Important for K+ Ion Homeostasis in Plants

Venema

Belver

Marín-Manzano

et al. 2003

Journal of Biological Chemistry

137

117

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With concentrations between 0.1 and 0.2 M, potassium is the most abundant cation in plant cells. The main pool of potassium inside the plant cell is in the vacuole. The function of potassium in this organelle is thought to be purely biophysical; to generate cell turgor to drive cell expansion (1). Under conditions that limit K ϩ availability, the role of K ϩ in the vacuole can be replaced by other ions like Na ϩ , as has been reported to occur under conditions of salt stress (2). Vacuolar concentrations thus vary from high (200 mM) to low (20 mM), suggesting the existence of active K ϩ import and export mechanisms at the vacuolar membrane (3).The role and concentration of K ϩ in other endomembrane organelles in plants are largely unknown. Apart from a specific K ϩ requirement, secondary ion transporters might rely on K ϩ for pH regulation in these organelles. It was shown that apart from the vacuole, V-type H ϩ -transporting ATPase is found in various membranes of the secretory system where vesicle acidification is important for ligand-receptor binding and protein modification, trafficking, and sorting (4, 5). In animal cells, the pH gradient from neutral to acidic along organelles from both the secretory and endocytic pathways is suggested to be under tight control by the operation of secondary ion transporters providing proton leak pathways (6,7,8). Second, solute uptake energized by the pH gradient might be required to generate the osmotic pressure needed for vesicle fusion (5). In view of the abundance of K ϩ in the cell, K ϩ /H ϩ exchangers are likely candidates to be involved in pH and osmoregulation of intracellular compartments as well as active uptake of K ϩ into vacuoles (3) although the biochemical evidence for the existence of such antiporters is scarce (9, 10).In contrast to the limited information available on K ϩ /H ϩ antiporters, many reports have indicated the existence of vacuolar Na ϩ /H ϩ antiporters in plants (11). The first vacuolar Na ϩ /H ϩ exchanger AtNHX1 was identified recently (12), and it was shown that overexpression of this gene in plants enhances salt tolerance (13,14,15). A family of six genes was identified in Arabidopsis (AtNHX1 to AtNHX6) that shows sequence homology to mammalian and yeast NHE or NHX exchangers (16,17). It was demonstrated however that AtNHX1 could catalyze both Na ϩ /H ϩ and K ϩ /H ϩ exchange (14, 18). A similar ion specificity was reported for the human NHE7 isoform. It was shown that this isoform is expressed in the trans-Golgi network, indicating that regulation of pH and ionic composition of intracellular compartments by K ϩ /H ϩ or Na ϩ /H ϩ exchange is an important task of these antiporters (7). This notion was strengthened by the observation that the NHX1 protein is essential for osmotolerance and endosomal protein trafficking in yeast (19,20). Indeed, plant NHX genes were shown not only to be involved in salinity tolerance, but also in vacuolar pH regulation (21,22), and to be induced by NaCl, KCl, and osmotic stress (12,16,(23)(24)(25)(26), or even h...

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“…Succinate dehydrogenase (1), invertase (18), NADPHcytochrome c reductase (34), and a-D-mannosidase (33) were assayed according to published procedures.…”

Section: Downloaded Frommentioning

confidence: 99%

Phospholipid synthesis and lipid composition of subcellular membranes in the unicellular eukaryote Saccharomyces cerevisiae

et al. 1991

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improved procedures and analyzed for their lipid composition and their capacity to synthesize phospholipids and to catalyze sterol A24-methylation. The microsomal fraction is heterogeneous in terms of density and classical microsomal marker proteins and also with respect to the distribution of phospholipid-synthesizing enzymes. The specific activity of phosphatidylserine synthase was highest in a microsomal subfraction which was distinct from heavier microsomes harboring phosphatidylinositol synthase and the phospholipid N-methyltransferases. The exclusive location of phosphatidylserine decarboxylase in mitochondria was confirmed. CDP-diacylglycerol synthase activity was found both in mitochondria and in microsomal membranes. Highest specific activities of glycerol-3-phosphate acyltransferase and sterol A24-methyltransferase were observed in the lipid particle fraction. Nuclear and plasma membranes, vacuoles, and peroxisomes contain only marginal activities of the lipid-synthesizing enzymes analyzed. The plasma membrane and secretory vesicles are enriched in ergosterol and in phosphatidylserine. Lipid particles are characterized by their high content of ergosteryl esters. The rigidity of the plasma membrane and of secretory vesicles, determined by measuring fluorescence anisotropy by using trimethylammonium diphenylhexatriene as a probe, can be attributed to the high content of ergosterol.Most of the enzymes involved in cellular phospholipid biosynthesis are membrane associated. In mammalian cells, the majority of phospholipids is synthesized in the endoplasmic reticulum (14). Phospholipids specifically required for mitochondrial function (cardiolipin and its precursor phosphatidylglycerol) as well as phosphatidylethanolamine (via decarboxylation of phosphatidylserine) are synthesized in mitochondrial membranes (11).In previous studies, several enzymes of phospholipid biosynthesis of the yeast Saccharomyces cerevisiae (10,26), namely glycerol-3-phosphate acyltransferase, CDP-diacylglycerol synthase, phosphatidylserine synthase, and phosphatidylinositol synthase, were detected both in the microsomal fraction and in the outer mitochondrial membrane. These observations were based mainly on the separation of subcellular membranes by differential centrifugation and on commonly used marker enzymes for the respective fractions. Motivated by our interest in the mechanisms of lipid flow and membrane assembly in yeasts and by conflicting data concerning the subcellular targeting of phosphatidylserine synthase (38), we reinvestigated the subcellular distribution of lipid-synthesizing enzymes by employing recently developed or improved fractionation procedures for mitochondrial and microsomal membranes, the nuclear membrane (24), the plasma membrane (37) Yeast subcellular membranes were also characterized with respect to their protein-to-lipid ratio, their content of ergosterol and ergosteryl esters, and their pattern of individual glycerophospholipids. Measurements of fluorescence anisotropy revealed significant difference...

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Triphosphopyridine nucleotide: cytochrome C reductase of Saccharomyces Cerevisiae a “microsomal” enzyme

Cited by 39 publications

References 34 publications

Subcellular and submitochondrial localization of phospholipid-synthesizing enzymes in Saccharomyces cerevisiae

Subcellular and submitochondrial localization of phospholipid-synthesizing enzymes in Saccharomyces cerevisiae

A Novel Intracellular K+/H+ Antiporter Related to Na+/H+ Antiporters Is Important for K+ Ion Homeostasis in Plants

Phospholipid synthesis and lipid composition of subcellular membranes in the unicellular eukaryote Saccharomyces cerevisiae

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