Protein-protein interactions among C-4 demethylation enzymes involved in yeast sterol biosynthesis.

A Saccharomyces cerevisae microarray expression study indicated that an ORF, YER044C, now designated ERG28, was strongly coregulated with ergosterol biosynthesis. Disruption of the ERG28 gene results in slow growth and accumulation of sterol intermediates similar to those observed in erg26 and erg27 null strains, suggesting that the Erg28p may interact with Erg26p and/or Erg27p. In this study, a peptide from human hemagglutinin protein (HA) epitope tag was added to ERG26 and ERG27 genes, and a Myc tag was added to the ERG28 gene to detect interactions between Erg28p and Erg26p/Erg27p. Differential centrifugation showed that Erg26p, Erg27p, and Erg28p are all membrane-associated proteins. Green fluorescent protein-fusion protein localization studies showed that Erg26p, Erg27p, and Erg28p are all located in the endoplasmic reticulum. Solubilized membrane protein coimmunoprecipitation studies using rabbit anti-Erg25p indicated that Erg25p coimmunoprecipitates with both Erg27p and Erg28p. Erg28p was also shown to reciprocally coimmunoprecipitate with Erg27p. However, no coimmunoprecipitation was observed with Erg26p, most likely because of the poor solubilization of this protein. Sucrose gradient ultracentrifugation studies suggested that Erg25p/Erg26p/Erg27p/Erg28p, along with other proteins in sterol biosynthesis, might form a complex between 66 and 200 kDa. Using an anti-HA column with Erg27p-HA and Erg26p-HA as target proteins, a complex containing Erg25p/Erg26p/Erg27p/Erg28p was identified. Thus, we suggest that Erg28p works as a transmembrane scaffold to tether Erg27p and possibly other C-4 demethylation proteins (Erg25p, Erg26p), forming a demethylation complex in the endoplasmic reticulum.

The effect of the erg26-1 mutation on the regulation of lipid metabolism in Saccharomyces cerevisiae.

A temperature-sensitive Saccharomyces cerevisiae mutant harboring a lesion in the ERG26 gene has been isolated. ERG26 encodes 4alpha-carboxysterol-C3 dehydrogenase, one of three enzymatic activities required for the conversion of 4,4-dimethylzymosterol to zymosterol. Gas chromatography/mass spectrometry analyses of sterols in this mutant, designated erg26-1, revealed the aberrant accumulation of a 4-methyl-4-carboxy zymosterol intermediate, as well as a novel 4-carboxysterol. Neutral lipid radiolabeling studies showed that erg26-1 cells also harbored defects in the rate of biosynthesis and steady-state levels of mono-, di-, and triglycerides. Phospholipid radiolabeling studies showed defects in the rate of biosynthesis of both phosphatidic acid and phosphatidylinositol. Biochemical studies revealed that microsomes isolated from erg26-1 cells contained greatly reduced 4alpha-carboxysterol-C3 dehydrogenase activity when compared with microsomes from wild type cells. Previous studies have shown that loss of function mutations in either of the fatty acid elongase genes SUR4/ELO3 or FEN1/GNS1/ELO2 can "bypass" the essentiality of certain ERG genes (Ladeveze, V., Marcireau, C., Delourme, D., and Karst, F. (1993) Lipids 28, 907-912; Silve, S., Leplatois, P., Josse, A., Dupuy, P. H., Lanau, C., Kaghad, M., Dhers, C., Picard, C., Rahier, A., Taton, M., Le Fur, G., Caput, D., Ferrara, P., and Loison, G. (1996) Mol. Cell. Biol. 16, 2719-2727). Studies presented here have shown that this sphingolipid-dependent "bypass" mechanism did not suppress the essential requirement for zymosterol biosynthesis. However, studies aimed at understanding the underlying physiology behind the temperature-sensitive growth defect of erg26-1 cells showed that the addition of several antifungal compounds to the growth media of erg26-1 cells could suppress the temperature-sensitive growth defect. Fluorescence microscopic analysis showed that GFP-Erg26p and GFP-Erg27p fusion proteins were localized to the endoplasmic reticulum. Two-hybrid analysis indicated that Erg25p, Erg26p, and Erg27p, which are required for the biosynthesis of zymosterol, form a complex within the cell.

Upregulation of ERG genes in Candida species by azoles and other sterol biosynthesis inhibitors.

Infections due to Candida albicans are usually treated with azole antifungals such as fluconazole, but treatment failure is not uncommon especially in immunocompromised individuals. Relatedly, in vitro studies demonstrate that azoles are nonfungicidal, with continued growth at strain-dependent rates even at high azole concentrations. We hypothesized that upregulation of ERG11, which encodes the azole target enzyme lanosterol demethylase, contributes to this azole tolerance in Candida species. RNA analysis revealed that ERG11 expression in C. albicans is maximal during logarithmic-phase growth and decreases as the cells approach stationary phase. Incubation with fluconazole, however, resulted in a two- to fivefold increase in ERG11 RNA levels within 2 to 3 h, and this increase was followed by resumption of culture growth. ERG11 upregulation also occurred following treatment with other azoles (itraconazole, ketoconazole, clotrimazole, and miconazole) and was not dependent on the specific medium or pH. Within 1 h of drug removal ERG11 upregulation was reversed. Azole-dependent upregulation was not limited to ERG11: five of five ERG genes tested whose products function upstream and downstream of lanosterol demethylase in the sterol biosynthetic pathway were also upregulated. Similarly, ERG11 upregulation occurred following treatment of C. albicans cultures with terbinafine and fenpropimorph, which target other enzymes in the pathway. These data suggest a common mechanism for global ERG upregulation, e.g., in response to ergosterol depletion. Finally, azole-dependent ERG11 upregulation was demonstrated in three additional Candida species (C. tropicalis, C. glabrata, and C. krusei), indicating a conserved response to sterol biosynthesis inhibitors in opportunistic yeasts.

A ceramide-activated protein phosphatase mediates ceramide-induced G1 arrest of Saccharomyces cerevisiae.

Certain mammalian growth modulators, such as tumor necrosis factor alpha, interleukin-1beta, and gamma-interferon, induce an antiproliferative response-terminal differentiation, apoptosis, or cell cycle arrest-through a novel signal transduction pathway mediated by the lipid ceramide as a second messenger. Both a ceramide-activated protein phosphatase and a ceramide-activated protein kinase have been implicated in transmitting the signals elicited by ceramide. We have determined that ceramide addition to the yeast Saccharomyces causes a similar antiproliferative response, resulting in arrest of cells in the G1 phase of the cell cycle. We have also determined that yeast cells contain a ceramide-activated protein phosphatase composed of regulatory subunits encoded by TPD3 and CDC55 and a catalytic subunit encoded by SIT4. Because mutation of any one of these three genes renders strains resistant to ceramide inhibition, we conclude that the G1 effects of ceramide are mediated at least in part by the yeast ceramide-activated protein phosphatase. These results highlight the conservation of signaling systems in yeast and mammalian cells and provide a novel approach to dissecting this ubiquitous signal transduction pathway.

Regulation of lipid biosynthesis in Saccharomyces cerevisiae by fumonisin B1.

The regulation of lipid biosynthesis in the yeast Saccharomyces cerevisiae by fumonisin B1 was examined. Fumonisin B1 inhibited the growth of yeast cells. Cells supplemented with fumonisin B1 accumulated free sphinganine and phytosphingosine in a dose-dependent manner. The cellular concentration of ceramide was reduced in fumonisin B1-supplemented cells. Ceramide synthase activity was found in yeast cell membranes and was inhibited by fumonisin B1. Fumonisin B1 inhibited the synthesis of the inositol-containing sphingolipids inositol phosphorylceramide, mannosylinositol phosphorylceramide, and mannosyldiinositol phosphorylceramide. Fumonisin B1 also caused a decrease in the synthesis of the major phospholipids synthesized via the CDP-diacylglycerol-dependent pathway and the synthesis of neutral lipids. The effects of fumonisin B1 and sphingoid bases on the activities of enzymes in the pathways leading to the synthesis of sphingolipids, phospholipids, and neutral lipids were also examined. Other than ceramide synthase, fumonisin B1 did not affect the activities of any of the enzymes examined. However, sphinganine and phytosphingosine inhibited the activities of inositol phosphorylceramide synthase, phosphatidylserine synthase, and phosphatidate phosphatase. These are key enzymes responsible for the synthesis of lipids in yeast. The data reported here indicated that the biosynthesis of sphingolipids, phospholipids and neutral lipids was coordinately regulated by fumonisin B1 through the regulation of lipid biosynthetic enzymes by sphingoid bases.

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.

Photoaffinity labeling of the 45-kDa and 55-kDa forms of phosphatidylinositol 4-kinase from the yeast Saccharomyces cerevisiae.

The membrane-associated 45- and 55-kDa forms of phosphatidylinositol (PI) 4-kinase (ATP:phosphatidylinositol 4-phosphotransferase, EC 2.7.1.67) from Saccharomyces cerevisiae are inhibited by ADP by a competitive mechanism with respect to ATP. We initiated studies toward defining the ATP and ADP sites on the PI 4-kinases using azidonucleotide photoaffinity labeling probes. The photoprobe 8-azido-ATP fulfilled the criteria of a specific photoaffinity label for the 45- and 55-kDa PI 4-kinases. 8-Azido-ATP was a substrate and a competitive inhibitor of the PI 4-kinases with Ki values similar to the Km for ATP. 8-Azido-ATP photoinactivated the enzymes and was photoincorporated into the enzymes in a dose-dependent manner at concentrations similar to the Ki values for the photoprobe. ATP, the true substrate, provided specific protection against photoinactivation and photoincorporation of the PI 4-kinases with 8-azido-ATP, whereas GTP, a nonspecific nucleotide, provided no protection against photoinactivation and photoincorporation. Photoaffinity labeling of the PI 4-kinases with 8-azido-ATP was specifically prevented with ADP. The photoprobe 8-azido-ADP also fulfilled the criteria needed to validate its use as a specific photoprobe for the PI 4-kinases. Photoinactivation of the PI 4-kinases with 8-azido-ADP was prevented specifically with ATP. Taken together, these data supported the conclusion that the ATP and ADP sites on the membrane-associated 45- and 55-kDa PI 4-kinases from S. cerevisiae were the same.

Regulation of the 45- and 55-kDa forms of phosphatidylinositol 4-kinase from the yeast Saccharomyces cerevisiae by nucleotides.

The synthesis of phosphatidylinositol (PI) 4-phosphate and PI 4,5-bisphosphate in the yeast Saccharomyces cerevisiae is stimulated by glucose. PI 4-kinase (ATP:phosphatidylinositol 4-phosphotransferase, EC 2.7.1.67) catalyzes the committed step in the synthesis of these phosphoinositides. Previous studies have suggested that the glucose effect on phosphoinositide synthesis is mediated by cellular levels of ATP and ADP and by the RAS/cAMP pathway. Using purified preparations of the membrane-associated 45- and 55-kDa forms of PI 4-kinase, we examined the regulation of these activities by nucleotides and cAMP-dependent protein kinase. MgADP was a potent inhibitor of both forms of the enzyme. Detailed kinetic analyses of the 45- and 55-kDa enzymes using Triton X-100/PI-mixed micelles showed that MgADP was a competitive inhibitor (Ki = 0.14 and 0.25 mM, respectively) with respect to MgATP and a noncompetitive inhibitor (Ki = 1.3 and 0.9 mM, respectively) with respect to PI. The Ki values for MgADP were about 2-fold lower than the Km values the enzymes have for their substrate MgATP and about 2-fold lower than the cellular concentration of ADP. The 45- and 55-kDa forms of PI 4-kinase activity were regulated differentially by CTP, an important nucleotide involved in phospholipid biosynthesis. Whereas the 55-kDa PI 4-kinase was inhibited by CTP, the 45-kDa enzyme was unaffected by CTP. CTP was a mixed type of inhibitor (Ki = 1.5 mM) with respect to MgATP and a noncompetitive inhibitor (Ki = 4 mM) with respect to PI. The Ki value for CTP was 4-fold higher than the Km value for MgATP and 7-fold higher than the cellular concentration of CTP. The 45- and 55-kDa PI 4-kinases were neither phosphorylated nor regulated by cAMP-dependent protein kinase. These results did not support the previous conclusion that PI 4-phosphate synthesis was mediated by the RAS/cAMP pathway. Our kinetic studies supported the conclusion that the glucose effect on the synthesis of PI 4-phosphate was mediated by cellular levels of ATP and ADP through the regulation of membrane-associated PI 4-kinase activity.

The 45- and 104-kDa forms of phosphatidate phosphatase from Saccharomyces cerevisiae are regulated differentially by phosphorylation via cAMP-dependent protein kinase.

Evidence is presented that demonstrated that the 45- and 104-kDa forms of phosphatidate phosphatase from Saccharomyces cerevisiae (Morlock, K. R., McLaughlin, J. J., Lin, Y.-P., and Carman, G. M. (1991) J. Biol. Chem. 266, 3586-3593) were regulated differentially by phosphorylation. Purified 45-kDa phosphatidate phosphatase was phosphorylated by cAMP-dependent protein kinase whereas purified 104-kDa phosphatidate phosphatase was not phosphorylated. cAMP-dependent protein kinase catalyzed the phosphorylation of pure 45-kDa phosphatidate phosphatase at a serine residue which resulted in a stimulation (2.4-fold) of phosphatidate phosphatase activity. Alkaline phosphatase catalyzed the dephosphorylation of pure 45-kDa phosphatidate phosphatase which resulted in an inhibition (1.3-fold) of phosphatidate phosphatase activity. Results of studies using mutants (bcy1 and cyr1) defective in cAMP-dependent protein kinase activity corroborated the results of the phosphorylation studies using pure preparations of phosphatidate phosphatase. The 45-kDa phosphatidate phosphatase phosphorylated in vitro and in vivo had phosphopeptides in common. The activation of the GAL10-RAS2val19 allele in mutant cells resulted in an increase in the synthesis of diacylglycerols and triacylglycerols. These results were consistent with the phosphorylation and activation of 45-kDa phosphatidate phosphatase by cAMP-dependent protein kinase in vivo.

Purification, characterization, and kinetic analysis of a 55-kDa form of phosphatidylinositol 4-kinase from Saccharomyces cerevisiae.

A 55-kDa form of membrane-associated phosphatidylinositol 4-kinase (ATP:phosphatidylinositol 4-phosphotransferase, EC 2.7.1.67) was purified 10,166-fold from Saccharomyces cerevisiae. The purification procedure included solubilization of microsome membranes with 1% Triton X-100 followed by chromatography with DE52, hydroxylapatite I, Q-Sepharose, Mono Q, and hydroxylapatite II. The procedure resulted in a nearly homogeneous 55-kDa phosphatidylinositol 4-kinase preparation. The 55-kDa phosphatidylinositol 4-kinase and the previously purified 45-kDa phosphatidylinositol 4-kinase differed with respect to their amino acid composition, isoelectric points, and peptide maps. Furthermore, the two forms of phosphatidylinositol 4-kinase did not show an immunological relationship. Maximum 55-kDa phosphatidylinositol 4-kinase activity was dependent on magnesium (10 mM) or manganese (0.5 mM) ions and Triton X-100 at the pH optimum of 7.0. The activation energy for the reaction was 12 kcal/mol, and the enzyme was labile above 30 degrees C. The enzyme was inhibited by thioreactive agents, MgADP, and calcium ions. A detailed kinetic analysis of the purified enzyme was performed using Triton X-100/phosphatidylinositol-mixed micelles. 55-kDa phosphatidylinositol 4-kinase activity followed saturation kinetics with respect to the bulk and surface concentrations of phosphatidylinositol and followed surface dilution kinetics. The interfacial Michaelis constant (Km) and the dissociation constant (Ks) for phosphatidylinositol in the Triton X-100 micelle surface were 1.3 mol % and 0.035 mM, respectively. The Km for MgATP was 0.36 mM. 55-kDa phosphatidylinositol 4-kinase catalyzed a sequential reaction mechanism as indicated by the results of kinetic and isotopic exchange reactions. The enzyme bound to phosphatidylinositol before ATP and released phosphatidylinositol 4-phosphate before ADP. The enzymological and kinetic properties of the 55-kDa phosphatidylinositol 4-kinase differed significantly from those of the 45-kDa phosphatidylinositol 4-kinase. This may suggest that the two forms of phosphatidylinositol 4-kinase from S. cerevisiae are regulated differentially in vivo.

Phosphatidylinositol 4-kinase from Saccharomyces cerevisiae. Kinetic analysis using Triton X-100/phosphatidylinositol-mixed micelles.

Phosphatidylinositol 4-kinase (ATP:phosphatidylinositol 4-phosphotransferase, EC 2.7.1.67) was purified from Saccharomyces cerevisiae by an improved procedure over that previously reported (Belunis, C.J., Bae-Lee, M., Kelley, M.J., and Carman, G.M. (1988) J. Biol. Chem. 263, 18897-18903) for the enzyme. The molecular mass of the enzyme was 45 kDa. The 35-kDa protein previously identified as PI 4-kinase was a proteolysis product of the 45-kDa protein. A detailed kinetic analysis of the purified enzyme was performed with Triton X-100/phosphatidylinositol-mixed micelles according to the "surface dilution" (Deems, R.A., Eaton, B.R., and Dennis, E.A. (1975) J. Biol. Chem. 250, 9013-9020) and "dual phospholipid" (Hendrickson, H.S., and Dennis, E.A. (1984) J. Biol. Chem. 259, 5734-5739) kinetic models. Phosphatidylinositol 4-kinase activity followed saturation kinetics with respect to the bulk and surface concentrations of phosphatidylinositol at concentrations of phosphatidylinositol below 0.1 mM. Above 0.1 mM activity was only dependent on the surface concentration of phosphatidylinositol. The enzyme more closely followed the dual phospholipid model where the enzyme associated with Triton X-100 micelles when phosphatidylinositol was present. The interfacial Michaelis constant (KmB) for phosphatidylinositol was 0.0036 mol fraction and the dissociation constant (KsA) for phosphatidylinositol in the micelle surface was 0.26 mM. The results of glycerol gradient centrifugation studies showed that the enzyme was physically associated with Triton X-100/phosphatidylinositol micelles.