Mitochondrial transporters involved in oleic acid utilization and glutamate metabolism in yeast

Utilization of fatty acids such as oleic acid as sole carbon source by the yeast Saccharomyces cerevisiae requires coordinated function of peroxisomes, where the fatty acids are degraded, and the mitochondria, where oxidation is completed. We identified two mitochondrial oxodicarboxylate transporters, Odc1p and Odc2p, as important in efficient utilization of oleic acid in yeast [Tibbetts et al., Arch. Biochem. Biophys. 406 (2002) 96-104]. Yet, the growth phenotype of odc1delta odc2delta strains indicated that additional transporter(s) were also involved. Here, we identify two putative transporter genes, YMC1 and YMC2, as able to suppress the odc1delta odc2delta growth phenotype. The mRNA levels for both are elevated in the presence of glycerol or oleic acid, as compared to glucose. Ymc1p and Ymc2p are localized to the mitochondria in oleic acid-grown cells. Deletion of all four transporters (quad mutant) prevents growth on oleic acid as sole carbon source, while growth on acetate is retained. It is known that the glutamate-sensitive retrograde signaling pathway is important for upregulation of peroxisomal function in response to oleic acid and the oxodicarboxylate alpha-ketoglutarate is transported out of the mitochondria for synthesis of glutamate. So, citric acid cycle function and glutamate synthesis were examined in transporter mutants. The quad mutant has significantly decreased citrate synthase activity and whole cell alpha-ketoglutarate levels, while isocitrate dehydrogenase activity is unaffected and glutamate dehydrogenase activity is increased 10-fold. Strains carrying only two or three transporter deletions exhibit intermediate affects. 13C NMR metabolic enrichment experiments confirm a defect in glutamate biosynthesis in the quad mutant and, in double and triple mutants, suggest increased cycling of the glutamate backbone in the mitochondria before export. Taken together these studies indicate that these four transporters have overlapping activity, and are important not only for utilization of oleic acid, but also for glutamate biosynthesis.     

Activity of Pichia pastoris alternative cis-prenyltransferase is correlated with proliferation of peroxisomes

We have investigated dolichol synthesis in yeast Pichia pastoris. Growth of these cells on methanol causes peroxisome proliferation and induction of peroxisomal enzymes. Twenty-four hours methanol treatment was sufficient for the appearance of longer-chain dolichols. Less specific oleic acid induction needed 48 h for the synthesis of longer dolichol family with typical one still present. Cells cultured in non-inducing conditions for 48 h did not reveal the presence of additional dolichol family. Peroxisomes purified from oleic acid treated cells synthesize in vitro polyprenols longer by two isoprene residues than those synthesized by microsomal fraction from glucose culture. These observations lead us to suggest that chain length of dolichols synthesized in yeast cell may depend on the carbon and energy source supply which mobilizes metabolic pathways localized to different cellular compartments.     

Lipid composition of peroxisomes from the yeast Pichia pastoris grown on different carbon sources

Highly purified peroxisomes from the yeast Pichia pastoris grown on methanol or oleic acid, respectively, were used to characterize the lipid composition of this organelle. For this purpose, an isolation procedure had to be adapted which yielded highly purified P. pastoris peroxisomes. When peroxisome proliferation was induced by growth on methanol, alcohol oxidase was the predominant peroxisomal protein. Cultivation of P. pastoris on oleic acid led to induction of a family of peroxisomal enzymes catalyzing fatty acid beta-oxidation, whose most prominent members were identified by mass spectrometry. On either carbon source, phosphatidylcholine and phosphatidylethanolamine were the major peroxisomal phospholipids, and cardiolipin was present in peroxisomal membranes at a substantial amount, indicating that this phospholipid is a true peroxisomal component. Ergosterol was the most abundant sterol of P. pastoris peroxisomal membranes irrespective of the culture conditions. The fatty acid composition of whole cells and peroxisomes was highly affected by cultivation of P. pastoris on oleic acid. Under these conditions, oleic acid became the predominant fatty acid in phospholipids from total cell and peroxisomal extracts. Thus, oleic acid was not only utilized as an appropriate carbon source but also as a building block for complex membrane lipids. In summary, our data provide first insight into biochemical properties of P. pastoris peroxisomal membranes, which may become important for the biotechnological use of this yeast.     

Ubiquinone biosynthesis in Saccharomyces cerevisiae. Isolation and sequence of COQ3, the 3,4-dihydroxy-5-hexaprenylbenzoate methyltransferase gene

Ubiquinone (or coenzyme Q) is a lipid component of the respiratory chain in the inner mitochondrial membrane, in which it functions in electron transport. Recent reports show that ubiquinone and ubiquinone biosynthetic enzymes are present in both mitochondrial and nonmitochondrial membranes of cells (Kalen, A., Appelkvist, E.-L., Chojnacki, T., and Dallner, G. (1990) J. Biol. Chem. 265, 1158-1164) although the functions that ubiquinone may play outside of the mitochondrion are not understood. To study coenzyme Q synthesis and function we cloned the 3,4-dihydroxy-5-hexaprenylbenzoate (DHHB) methyltransferase gene by functional complementation of a yeast coenzyme Q mutant strain, defective in the COQ3 gene (Tzagoloff, A., and Dieckmann, C. L. (1990) Microbiol. Rev. 54, 211-225). This gene restores both coenzyme Q synthesis in the mutant strain and the ability to grow on media containing glycerol, a nonfermentable substrate. A one-step in situ gene replacement with the cloned DHHB methyltransferase DNA directs integration to the yeast COQ3 locus on chromosome XV of Saccharomyces cerevisiae, establishing that the COQ3 locus encodes the DHHB methyltransferase structural gene. The predicted amino acid sequence of the yeast DHHB methyltransferase contains a methyltransferase consensus sequence and shows a 40% identity with an open reading frame of Escherichia coli, the gyrA5' hypothetical protein. This open reading frame is adjacent to the gyrA gene and close to the mapped location of the ubiG gene at 48 min on the E. coli chromosome. These results suggest that the E. coli gyrA5' open reading frame encodes a methyltransferase and may correspond to the ubiG gene, which is required for ubiquinone biosynthesis.     

基于响应面设计脂肪酶Novo435催化合成甘油二酯的工艺优化

以甘油、油酸为原料,优化在无溶剂体系中以固定化脂肪酶Novo435催化合成甘油二酯（diglyceride,DAG）的工艺。系统考察底物摩尔比（油酸／甘油）、反应温度、时间和加酶量等因素对油酸转化率和甘油二酯含量影响的基础上,利用响应面试验设计优化各主效因子,并经回归分析获得最优的工艺条件。所得最优条件：油酸与甘油底物摩尔比2．27、反应温度48．14℃、反应时间6．3h、加酶量1．68％。在此条件下,实验测得油酸转化率为45．42％,甘油二酯质量分数为70．01％,与响应面模型预测值吻合。     

Suppression of respiratory growth defect of mutant deficient in mitochondrial phospholipase A1 by overexpression of genes involved in coenzyme Q synthesis in Saccharomyces cerevisiae

DDL1 encodes a mitochondrial phospholipase A₁ involved in acyl chain remodeling of mitochondrial phospholipids and degradation of cardiolipin in Saccharomyces cerevisiae. The deletion of DDL1 leads to respiratory growth defects. To elucidate the physiological role of DDL1, we screened for genes that, when overexpressed, suppress the respiratory growth defect of the DDL1 deletion mutant. Introduction of COQ8, COQ9, or COQ5, which are involved in coenzyme Q (CoQ) synthesis, using a multicopy vector suppressed the respiratory growth defect of the DDL1 deletion mutant. In contrast, introduction of COQ8 using a multicopy vector did not accelerate the growth of the deletion mutants of TAZ1 or CLD1, which encode an acyltransferase or phospholipase A₂, respectively, involved in the remodeling of cardiolipin. These results suggest genetic interactions between the mitochondrial phospholipase A₁ gene and the genes involved in CoQ synthesis.     

A short nucleotide sequence required for regulation of HIS4 by the general control system of yeast

We have made deletions of the HIS4 5' noncoding region in vitro and inserted these deletions into the yeast genome by transformation. Deletions that extend from -588 to -235 have no detectable effects on either promoter or regulatory functions. Deletions that extend to -138 affect promoter function, but are still regulated by the general control of amino acid biosynthesis. A deletion that extends to -136 cannot derepress HIS4 mRNA in response to the general control. This deletion removes all copies of the sequence 5'-TGACTC-3', which appears at positions -194, -182 and -138 in strains without the deletion. The importance of at least one copy of this repeat for regulation of HIS4 is shown by the reappearance of this sequence in revertants of the -136 deletion that have regained the regulatory response. The fact that deletion of this sequence leads to the inability to derepress suggests that HIS4 is under positive control.     

Involvement of glutathione peroxidase 1 in growth and peroxisome formation in Saccharomyces cerevisiae in oleic acid medium

Saccharomyces cerevisiae is able to use some fatty acids, such as oleic acid, as a sole source of carbon. β-oxidation, which occurs in a single membrane-enveloped organelle or peroxisome, is responsible for the assimilation of fatty acids. In S. cerevisiae, β-oxidation occurs only in peroxisomes, and H(2)O(2) is generated during this fatty acid-metabolizing pathway. S. cerevisiae has three GPX genes (GPX1, GPX2, and GPX3) encoding atypical 2-Cys peroxiredoxins. Here we show that expression of GPX1 was induced in medium containing oleic acid as a carbon source in an Msn2/Msn4-dependent manner. We found that Gpx1 was located in the peroxisomal matrix. The peroxisomal Gpx1 showed peroxidase activity using thioredoxin or glutathione as a reducing power. Peroxisome biogenesis was induced when cells were cultured with oleic acid. Peroxisome biogenesis was impaired in gpx1? cells, and subsequently, the growth of gpx1? cells was lowered in oleic acid-containing medium. Gpx1 contains six cysteine residues. Of the cysteine-substituted mutants of Gpx1, Gpx1(C36S) was not able to restore growth and peroxisome formation in oleic acid-containing medium, therefore, redox regulation of Gpx1 seems to be involved in the mechanism of peroxisome formation.     

Characterization of the promoter region of the human peroxisomal multifunctional enzyme type 2 gene

Peroxisomal multifunctional enzyme type 2 (perMFE-2) catalyzes conversion of (24E)-3alpha,7alpha, 12alpha-trihydroxy-5beta-cholest-24-enoyl-CoA to (24-keto)-3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoyl-CoA, which are physiological intermediates in cholic acid synthesis. In contrast to long chain fatty acid oxidizing enzymes clofibrate does not induce peroxisomal enzymes metabolizing bile acid intermediates. We proposed the existence of PPAR-independent regulation of cholesterol side chain oxidation in the process of bile acid synthesis. In the present study, we characterized the promoter region of the human perMFE-2 gene. The promoter contains the Sp1/AP2 binding site (-151/-142) within 197 base pairs upstream of the translation start site. Mutation of the Sp1/AP2 binding site decreases the promoter activity. Analysis by the luciferase assay revealed that the activity of the promoter region is strong in HepG2 and HeLa cell lines, although the activity in HepG2 cells was five- to sixfold higher than that in HeLa cells. Transient transfection assays have confirmed that AP2alpha and AP2gamma were able to transactivate the perMFE-2 promoter/luciferase chimeric gene. Cotransfections with Sp1 expression plasmid decreased the promoter activity. We suggest that perMFE-2 promoter activity is the result of both the abundance of AP2 and Sp1 family members and their relative ratios.     

Preliminary characterization of Yor180Cp: identification of a novel peroxisomal protein of saccharomyces cerevisiae involved in fatty acid metabolism.

Here we report the preliminary characterization of Yor180Cp, a novel peroxisomal protein involved in fatty acid metabolism in the yeast Saccharomyces cerevisiae. A computer-based screen identified Yor180Cp as a putative peroxisomal protein, and Yor180Cp targeted GFP to peroxisomes in a PEX8-dependent manner. Yor180Cp was also detected by mass spectrometric analysis of an HPLC-separated extract of yeast peroxisomal matrix proteins. YOR180C is upregulated during growth on oleic acid, and deletion of YOR180C from the yeast genome resulted in a mild but significant growth defect on oleic acid, indicating a role for Yor180Cp in fatty acid metabolism. In addition, we observed that yor180cDelta cells fail to efficiently import the enzyme Delta3,Delta2-enoyl-CoA isomerase (Eci1p) to peroxisomes. This result suggested that Yor180Cp might associate with Eci1p in vivo, and a Yor180Cp-Eci1p interaction was detected using the yeast two-hybrid system. Potential roles for Yor180Cp in peroxisomal fatty acid metabolism are discussed.