Regulation of phosphatidylinositol 4-kinase from the yeast Saccharomyces cerevisiae by CDP-diacylglycerol

Regulation of the 45- and 55-kDa forms of Saccharomyces cerevisiae membrane-associated phosphatidylinositol (PI) 4-kinase (ATP:phosphatidylinositol 4-phosphotransferase) by phospholipids was examined using Triton X-100/phospholipid-mixed micelles. CDP-diacylglycerol and phosphatidylglycerol inhibited 45-kDa PI 4-kinase activity in a dose-dependent manner. Kinetic analyses of the 45-kDa PI 4-kinase showed that phosphatidylglycerol was a competitive inhibitor with respect to PI (Ki = 2 mol %), and CDP-diacylglycerol was a mixed type of inhibitor with respect to PI (Ki = 4 mol %) and MgATP (Ki = 5 mol %). 55-kDa PI 4-kinase activity was not significantly affected by phospholipids. The physiological relevance of CDP-diacylglycerol inhibition of 45-kDa PI 4-kinase activity was examined using plasma membranes from inositol auxotrophic (ino1) cells. Immunoblot analysis showed that 45-kDa PI 4-kinase expression in plasma membranes was not affected by inositol starvation of ino1 cells. However, both 45-kDa PI 4-kinase activity and its product PI 4-phosphate were reduced in plasma membranes from inositol-starved ino1 cells. The CDP-diacylglycerol concentration (9.6 mol %) in plasma membranes of inositol-starved ino1 cells was 12-fold higher than its concentration (0.8 mol %) in plasma membranes of inositol-supplemented cells. Plasma membranes of inositol-starved ino1 cells also had increased levels of phosphatidate, phosphatidylserine, phosphatidylethanolamine, and cardiolipin. However, these phospholipids did not affect pure 45-kDa PI 4-kinase activity. The concentration of CDP-diacylglycerol in plasma membranes of inositol-starved ino1 cells was in the range of the inhibitor constants determined for CDP-diacylglycerol by kinetic analyses using pure 45-kDa PI 4-kinase. These results raised the suggestion that 45-kDa PI 4-kinase activity may be regulated in vivo by CDP-diacylglycerol.     

Glutathione depletion in the yeast Saccharomyces cerevisiae

The effect of chosen compounds on the total glutathione (GSH) level in stationary cultures of S. cerevisiae was compared. 1-Chloro-2,4-dinitrobenzene, 1-fluoro-2,4-dinitrobenzene, maleimide, iodacetamide and allyl alcohol (1 mM), and menadione (0.5 mM) caused an almost complete GSH depletion during several minutes. Bromobenzoic acid and chloramine T (I mM), and daunomycin (60 mu M) induced a slower GSH decrease, down to 30-70% after 60 min. Paraquat (1 mM), CuSO(4) (0.5 mM) and cadmium acetate (1 mM) decreased glutathione level down to ca 70%. Diamide (0.5 mM), phenazine methosulphate, phenylhydrazine, acetylphenylhydrazine and H(2)O(2) (1 mM), and t-butyl hydroperoxide (2 mM) did not affect total GSH during 60-min exposure. There was no clear-cut dependence between the ability of various chemicals to deplete cellular GSH and their increased toxicity to a glutathione-poor mutant.     

Regulation of phosphatidate phosphatase activity from the yeast Saccharomyces cerevisiae by sphingoid bases

The regulation of Saccharomyces cerevisiae membrane-associated phosphatidate phosphatase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) activity by sphingoid bases was examined using Triton X-100/lipid-mixed micelles. Sphingosine, phytosphingosine, and sphinganine inhibited purified preparations of the 104- and 45-kDa forms of phosphatidate phosphatase in a dose-dependent manner. The structural requirements for the sphingoid base inhibition of phosphatidate phosphatase activity were a free amino group and a long chain hydrocarbon. A detailed kinetic analysis was performed to determine the mechanism of phosphatidate phosphatase inhibition by sphingoid bases. The phosphatidate phosphatase dependence on phosphatidate was cooperative (Hill numbers of approximately 2) in the absence and presence of sphingoid bases. Sphingosine, phytosphingosine, and sphinganine were parabolic competitive inhibitors of phosphatidate phosphatase activity. This indicated that more than one inhibitor molecule contributed to the exclusion of phosphatidate from the enzyme. The aKi values (inhibitor constants) for sphingosine, phytosphingosine, and sphinganine were 1.5, 0.4, and 0.2 mol %, respectively, and the Km value for phosphatidate was 2.2 mol %. The cellular concentrations of free phytosphingosine and sphinganine were 0.16 and 0.53 mol %, respectively, relative to the total phospholipids in S. cerevisiae. The cellular concentrations of phytosphingosine and sphinganine were in the range of the aKi values for these sphingoid bases. These results raised the suggestion that phosphatidate phosphatase activity may be regulated in vivo by sphingoid bases.     

MAP kinase pathways in the yeast Saccharomyces cerevisiae

A cascade of three protein kinases known as a mitogen-activated protein kinase (MAPK) cascade is commonly found as part of the signaling pathways in eukaryotic cells. Almost two decades of genetic and biochemical experimentation plus the recently completed DNA sequence of the Saccharomyces cerevisiae genome have revealed just five functionally distinct MAPK cascades in this yeast. Sexual conjugation, cell growth, and adaptation to stress, for example, all require MAPK-mediated cellular responses. A primary function of these cascades appears to be the regulation of gene expression in response to extracellular signals or as part of specific developmental processes. In addition, the MAPK cascades often appear to regulate the cell cycle and vice versa. Despite the success of the gene hunter era in revealing these pathways, there are still many significant gaps in our knowledge of the molecular mechanisms for activation of these cascades and how the cascades regulate cell function. For example, comparison of different yeast signaling pathways reveals a surprising variety of different types of upstream signaling proteins that function to activate a MAPK cascade, yet how the upstream proteins actually activate the cascade remains unclear. We also know that the yeast MAPK pathways regulate each other and interact with other signaling pathways to produce a coordinated pattern of gene expression, but the molecular mechanisms of this cross talk are poorly understood. This review is therefore an attempt to present the current knowledge of MAPK pathways in yeast and some directions for future research in this area.     

Cascades of mammalian caspase activation in the yeast Saccharomyces cerevisiae

Caspases (aspartate-specific cysteine proteases) play a critical role in the execution of the mammalian apoptotic program. To address the regulation of human caspase activation, we used the yeast Saccharomyces cerevisiae, which is devoid of endogenous caspases. The apical procaspases, -8beta and -10, were efficiently processed and activated in yeast. Although protease activity, per se, was insufficient to drive cell death, caspase-10 activity had little effect on cell viability, whereas expression of caspase-8beta was cytotoxic. This lethal phenotype was abrogated by co-expression of the pan-caspase inhibitor, baculovirus p35, and by mutation of the active site cysteine of procaspase-8beta. In contrast, autoactivation of the executioner caspase-3 and -6 zymogens was not detected. Procaspase-3 activation required co-expression of procaspase-8 or -10. Surprisingly, activation of procaspase-6 required proteolytic activities other than caspase-8, -10, or -3. Caspase-8beta or -10 activity was insufficient to catalyze the maturation of procaspase-6. Moreover, a constitutively active caspase-3, although cytotoxic in its own right, was unable to induce the processing of wild-type procaspase-6 and vice versa. These results distinguish sequential modes of activation for different caspases in vivo and establish a yeast model system to examine the regulation of caspase cascades. Moreover, the distinct terminal phenotypes induced by various caspases attest to differences in the cellular targets of these apoptotic proteases, which may be defined using this system.     

Role of the RAD57 gene in the repair of double-stranded DNA gaps in the yeast Saccharomyces cerevisiae

The linearized plasmid with complementary (cohesive) ends was shown to restore the circular form in cells of the rad57 mutant with a lower efficiency than in Rad+ cells. This process proved to be cold-sensitive in mutant cells, in contrast to wild-type cells. When mutant cells were shifted from 23 up to 36 degrees C, the repair efficiency increased approximately 1.5 times. In most cases examined, the repair was not accompanied by the doublestrand gap repair within the break site and did not depend on temperature. Homology between chromosomal and plasmid DNA sequences in the break region and the presence of cohesive ends were shown to be essential for the repair of linearized plasmids with a double-strand gap in cells of the rad57 mutant. Degradation of cohesive ends of the linearized plasmid during its repair in rad57 cells is insignificant. Possible mechanisms of linearized plasmid repair in the rad57 mutant are proposed.     

Regulation of phosphatidylglycerophosphate synthase levels in Saccharomyces cerevisiae

The PGS1 gene of Saccharomyces cerevisiae encodes phosphatidylglycerophosphate (PG-P) synthase. PG-P synthase activity is regulated by factors affecting mitochondrial development and through cross-pathway control by inositol. The molecular mechanism of this regulation was examined by using a reporter gene under control of the PGS1 gene promoter (PPGS1-lacZ). Gene expression subject to carbon source regulation was monitored both at steady-state level and during the switch between different carbon sources. Cells grown in a non-fermentable carbon source had beta-galactosidase levels 3-fold higher than those grown in glucose. A shift from glucose to lactate rapidly raised the level of gene expression, whereas a shift back to glucose had the opposite effect. In either a pgs1 null mutant or a rho mutant grown in glucose, PPGS1-lacZ expression was 30-50% of the level in wild type cells. Addition of inositol to the growth medium resulted in a 2-3-fold reduction in gene expression in wild type cells. In ino2 and ino4 mutants, gene expression was greatly reduced and was not subject to inositol regulation consistent with inositol repression being dependent on the INO2 and INO4 regulatory genes. PPGS1-lacZ expression was elevated in a cds1 null mutant in the presence or absence of inositol, indicating that the capacity to synthesize CDP-diacylglycerol affects gene expression. Lack of cardiolipin synthesis (cls1 null mutant) had no effect on reporter gene expression.     

Calcium ion influx during sporulation in the yeast Saccharomyces cerevisiae

The changes in intra- and (or) extra-cellular concentrations of Ca2+, Mg2+, K+, and Na+ during sporulation of a MATa/MAT alpha diploid yeast of Saccharomyces cerevisiae were examined in a nutrition-deprived medium with potassium acetate. Among these, Ca2+ in external medium was preferentially incorporated into cells, and sporulation was induced when the magnitude of free Ca2+ gradient between cytosol [Ca2+]i and external medium [Ca2+]o reached more than 3 x 10(3) ([Ca2+]i/[Ca2+]o = 3.5 x 10(3)). The result indicated that the meiosis and (or) sporulation signal of the yeast S. cerevisiae was generated through elevated Ca2+ influx rather than release from the internal Ca2+ stores.     

Overproduction and affinity purification of Saccharomyces cerevisiae replication factor C

Yeast replication factor C (RF-C) is a heteropentamer encoded by the RFC1-5 genes. RF-C activity in yeast extracts was overproduced about 80-fold after induction of a strain containing all five genes on a single plasmid, with expression of each gene placed under control of the galactose-inducible GAL1-10 promoter. This strongly indicates that overexpression of the five known RFC genes is sufficient for overproduction of RF-C. Overexpression of all five genes was also necessary to achieve overproduction of RF-C as omission of any single gene from the plasmid gave uninduced, i.e. normal cellular levels of RF-C. The interaction between RF-C and proliferating cell nuclear antigen (PCNA) was studied with PCNA-agarose beads. Binding of RF-C to PCNA-agarose beads is negligible in buffers containing 0.3 M NaCl. However, addition of Mg-ATP to the binding buffer caused strong binding of RF-C to the beads even at 0.8 M NaCl. Binding of ATP, but not its hydrolysis, was required for the strong binding mode as nonhydrolyzable analogs were also effective. The existence of two distinct binding modes between PCNA and RF-C was used as the key step in a greatly improved procedure for the purification of RF-C. RF-C from the overproduction strain purified by this procedure was essentially homogeneous and had a severalfold higher specific activity than RF-C preparations that had previously been purified through multicolumn procedures.     

Adenylosuccinate synthetase of the yeast Saccharomyces cerevisiae: purification and properties

Motor and sensory nerve conduction velocities (MNCV and SNCV) were reduced in the sciatic nerve of rats after 4 weeks of untreated streptozotocin-induced diabetes, and declined further during the following 4 weeks. Treating diabetic rats with the novel peptide HP228 had no effect on the decline of MNCV after the first 4 weeks of diabetes but attenuated the decline in SNCV. HP228 treatment also prevented any further decline in MNCV or SNCV between weeks 4 and 8 of diabetes. Consequently, at the conclusion of the study, the nerve conduction velocities (NCVs) in treated rats were significantly (both P < .001) higher than in untreated diabetic rats. Reduced nerve homogenate Na+,K+-adenosine triphosphatase (ATPase) activity in diabetic rats was significantly (P < .05) increased by HP228 but remained significantly (P < .05) lower than in untreated controls. HP228 treatment also reduced nerve Na+,K+-ATPase activity of control rats compared with untreated controls (P < .05). There was no effect of HP228 on the hyperglycemia, nerve polyol accumulation, myo-inositol depletion, reduced nerve laser Doppler blood flow, thermal hypoalgesia, or reduced mean axonal caliber in diabetic rats or on any of these parameters in control rats. These data demonstrate that a novel peptide may protect against the slowing of nerve conduction in prolonged diabetes and that the mechanism of action is unrelated to aldose reductase inhibition, prevention of nerve ischemia, or axonal atrophy. HP228 may prove a potential therapeutic agent for the treatment of prolonged diabetic neuropathy.     

Molecular cloning of the actin gene from yeast Saccharomyces cerevisiae

Two overlapping DNA fragments from yeast Saccharomyces cerevisiae containing the actin gene have been inserted into pBR322 and cloned in E.coli. Clones were identified by hybridization to complementary RNA from a plasmid containing a copy of Dictyostelium actin mRNA. One recombinant plasmid obtained (pYA102) contains a 3.93-kb Hindlll fragment, the other (pYA208) a 5.1-kb Pstl fragment, both share a common 2.2-kb fragment harboring part of the actin gene. Cloned yeast actin DNA was identified by R-loop formation and translation of the hybridized actin mRNA and by DNA sequence analysis. Cytoplasmic actin mRNA has been estimated to be about 1250 nucleotides long. There is only one type of the actin gene in S.cerevisiae.     

Regulation of acetylcholine synthesis: does cytoplasmic acetylcholine control high affinity choline uptake?

When brain synaptosomes are obtained from animals that have been injected intravenously with [2H4]choline 1 minute before being killed, their high affinity [3H] choline uptake is correlated inversely with their acetylcholine content and directly with the rate at which they synthesize [2H4]acetylcholine. The control of such choline uptake by the cytoplasmic acetylcholine concentration is proposed as a mechanism regulating acetylcholine synthesis in cholinergic nerve terminals.     

Stabilization of microsatellite sequences by variant repeats in the yeast Saccharomyces cerevisiae

We examined the effect of a single variant repeat on the stability of a 51-base pair (bp) microsatellite (poly GT). We found that the insertion stabilizes the microsatellite about fivefold in wild-type strains. The stabilizing effect of the variant base was also observed in strains with mutations in the DNA mismatch repair genes pms1, msh2 and msh3, indicating that this effect does not require a functional DNA mismatch repair system. Most of the microsatellite alterations in the pms1, msh2 and msh3 strains were additions or deletions of single GT repeats, but about half of the alterations in the wild-type and msh6 strains were large (> 8 bp) deletions or additions.     

Purification and characterization of DNA helicase III from the yeast Saccharomyces cerevisiae

Two forms of DNA helicase activity, Rad3 and ATPase III, were previously purified from the yeast Saccharomyces cerevisiae and characterized. Here, we have identified and purified an additional DNA helicase activity from S. cerevisiae to near homogeneity. This helicase differs from those described previously in its chromatographic behavior, molecular weight, enzymatic properties, and genetic properties. Thus, we named it DNA helicase III. Its apparent molecular mass is about 120 kDa as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. DNA helicase III requires a divalent cation Mg2+ or Mn2+, either ATP or dATP, and a single-stranded portion on the duplex substrate. Helicase III moves in the 5'-->3' direction on single-stranded portions of the substrate and unwinds the strand of DNA in the 3'-->5' direction. It also has an intrinsic DNA-dependent ATPase (dATPase) activity that hydrolyzes either ATP or dATP to ADP or dADP and orthophosphate in the presence of DNA. DNA helicase III activity was not affected by either rad3 or radH mutations, suggesting that it is encoded by a gene different from RAD3 and RADH.