Anna Szkopińska

Enhanced levels of Pis1p (phosphatidylinositol synthase) improve the growth of Saccharomyces cerevisiae cells deficient in Rsp5 ubiquitin ligase

The Rsp5 ubiquitin ligase plays a role in many cellular processes including the biosynthesis of unsaturated fatty acids. The PIS1 (phosphatidylinositol synthase gene) encoding the enzyme Pis1p which catalyses the synthesis of phosphatidylinositol from CDP-diacyglycerol and inositol, was isolated in a screen for multicopy suppressors of the rsp5 temperature sensitivity phenotype. Suppression was allele non-specific. Interestingly, expression of PIS1 was 2-fold higher in the rsp5 mutant than in wild-type yeast, whereas the introduction of PIS1 in a multicopy plasmid increased the level of Pis1p 6-fold in both backgrounds. We demonstrate concomitantly that the expression of INO1 (inositol phosphate synthase gene) was also elevated approx. 2-fold in the rsp5 mutant as compared with the wild-type, and that inositol added to the medium improved growth of rsp5 mutants at a restrictive temperature. These results suggest that enhanced phosphatidylinositol synthesis may account for PIS1 suppression of rsp5 defects. Analysis of lipid extracts revealed the accumulation of saturated fatty acids in the rsp5 mutant, as a consequence of the prevention of unsaturated fatty acid synthesis. Overexpression of PIS1 did not correct the cellular fatty acid content; however, saturated fatty acids (C(16:0)) accumulated preferentially in phosphatidylinositol, and (wild-type)-like fatty acid composition in phosphatidylethanolamine was restored.

Effect of squalene synthase gene disruption on synthesis of polyprenols in Saccharomyces cerevisiae

Biosynthesis of polyprenols was investigated in a wild‐type strain of Saccharomyces cerevisiae and a squalene synthase deficient strain auxotrophic for ergosterol. The quantitative data showed that disruption of squalene synthase gene caused a 6‐fold increase in the synthesis of polyprenols in vitro in comparison with the wild‐type strain. Microsomal preparation from the deleted strain only slightly reacted to the additional exogenous FPP, while that from the wild‐type strain presented a 4‐fold increase of polyprenol synthesis. Restoration of ergosterol synthesis, by introducing ERG9 functional allele into the deleted strain resulted in a significant lowering of polyprenol synthesis, indicating the immediate shift of the common substrate (FPP) to the sterol pathway. The role of squalene synthase in the regulation of polyprenol synthesis and ‘flow diversion hypothesis’ is discussed.

Products of S cerevisiae cis-prenyltransferase activity in vitro

Products of cis-prenyltransferase activity, the first committed enzyme of the dolichol biosynthetic pathway, have been characterized in Saccharomyces cerevisiae. The evidence based on the results of ion exchange, HPTLC chromatography and acid phosphatase digestion has been presented indicating that the final product of the enzyme action in vitro is free polyprenol and not polyprenol mono- or diphosphate. On the other hand, the results of HPLC analysis confirmed that in vivo yeast accumulate alpha-saturated polyprenols (dolichols). Phosphorylation of endogenous dolichols by cytidine triphosphate (CTP)-dependent kinase is demonstrated. The hypothesis is put forth that in S cerevisiae free polyprenol is the substrate for the alpha-reductase responsible for its conversion to dolichol which in turn is phosphorylated into its active form: dolichyl phosphate.

Farnesyl diphosphate synthase activity affects ergosterol level and proliferation of yeast Saccharomyces cerevisae

The yeast farnesyl diphosphate synthase (FPPS) gene was engineered so as to construct allelic forms giving various activities of the enzyme. One of the substitutions was F96W in the chain length determination region. The other, K197, conserved within a consensus sequence found in the majority of FPP and GGPP synthases, was substituted by R, E and V. An intricate correlation has been found between the FPPS activity, the amount of ergosterol synthesized and cell growth of a mutant strain defective in FPPS. About 40% of wt FPPS activity was sufficient to support normal growth of the mutant. With further decline of FPPS activity (20 down to 3%) the amount of ergosterol remained unchanged at ∼0.16% (vs dry weight), whereas growth yield decreased and lag times increased. We postulate that, in addition to ergosterol initiating and maintaining growth of yeast cells, FPP and/or its derivatives participate in these processes.

The role of ERG20 gene (encoding yeast farnesyl diphosphate synthase) mutation in long dolichol formation. Molecular modeling of FPP synthase.

The yeast Saccharomyces cerevisiae strain LB332 bearing a mutation in the ERG20 gene encoding farnesyl diphosphate synthase (FPPS) synthesizes significantly longer dolichols than the wild type strain FL100 (14-31 and 14-19 isoprene units, respectively). The measurement of the short chain prenyl alcohols excreted into the medium shows that increased amounts of geraniol, dimethylallyl and isopentenyl alcohols but not farnesol are synthesized by the mutant strain. The wild type FPPS synthesizes farnesyl diphosphate (FPP) as the only product. The K197E substitution, as opposed to F112A/F113S in avian FPPS, does not change product specificity. Consequently, the possibility that mutated yeast FPPS synthesizes longer polyprenols is unlikely. This is supported by additional evidence such as in vitro analysis of the mutated FPPS products and molecular modeling. We suggest that formation of longer dolichols in vivo is the result of a change in the isopentenyl diphosphate/farnesyl diphosphate ratio caused by the erg20 mutation which in turn affects the activity of cis-prenyltransferase.

CTP-dependent lipid kinases of yeast

Membrane fractions from yeast Saccharomyces cerevisiae catalyzed a transfer of gamma-phosphate from [gamma-32P]CTP into membranous lipids. Phosphorylated compounds were identified as phosphatidic acid and dolichyl phosphate (DolP). The membrane fraction also catalyzed phosphorylation of the exogenous dolichol. The activity of the phosphorylating enzymes could be modified by the yeast growing conditions; i.e., the enzyme from yeast grown aerobically favored the synthesis of phosphatidate over dolichyl phosphate in the ratio of 3:1, whereas the membrane fraction from anaerobically grown yeast synthesized PA and DolP in the ratio of 0.5:1. The activity of the phosphorylating enzymes could also be modified by divalent cations and the concentration of detergents. Phosphorylation of lipids does not occur in the presence of [gamma-32P]ATP and is not influenced by the presence of UTP or GTP. This result points to the specific role of CTP as a gamma-phosphate donor for the synthesis of phosphatidate and dolichyl phosphates in the yeast system.

The effects of statins on the mevalonic acid pathway in recombinant yeast strains expressing human HMG-CoA reductase.

BackgroundThe yeast Saccharomyces cerevisiae can be a useful model for studying cellular mechanisms related to sterol synthesis in humans due to the high similarity of the mevalonate pathway between these organisms. This metabolic pathway plays a key role in multiple cellular processes by synthesizing sterol and nonsterol isoprenoids. Statins are well-known inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the key enzyme of the cholesterol synthesis pathway. However, the effects of statins extend beyond their cholesterol-lowering action, since inhibition of HMGR decreases the synthesis of all products downstream in the mevalonate pathway. Using transgenic yeast expressing human HMGR or either yeast HMGR isoenzyme we studied the effects of simvastatin, atorvastatin, fluvastatin and rosuvastatin on the cell metabolism.ResultsStatins decreased sterol pools, prominently reducing sterol precursors content while only moderately lowering ergosterol level. Expression of genes encoding enzymes involved in sterol biosynthesis was induced, while genes from nonsterol isoprenoid pathways, such as coenzyme Q and dolichol biosynthesis or protein prenylation, were diversely affected by statin treatment. Statins increased the level of human HMGR protein substantially and only slightly affected the levels of Rer2 and Coq3 proteins involved in non-sterol isoprenoid biosynthesis.ConclusionStatins influence the sterol pool, gene expression and protein levels of enzymes from the sterol and nonsterol isoprenoid biosynthesis branches and this effect depends on the type of statin administered. Our model system is a cheap and convenient tool for characterizing individual statins or screening for novel ones, and could also be helpful in individualized selection of the most efficient HMGR inhibitors leading to the best response and minimizing serious side effects.

Investigating the effects of statins on cellular lipid metabolism using a yeast expression system.

In humans, defects in lipid metabolism are associated with a number of severe diseases such as atherosclerosis, obesity and type II diabetes. Hypercholesterolemia is a primary risk factor for coronary artery disease, the major cause of premature deaths in developed countries. Statins are inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the key enzyme of the sterol synthesis pathway. Since yeast Saccharomyces cerevisiae harbours many counterparts of mammalian enzymes involved in lipid-synthesizing pathways, conclusions drawn from research with this single cell eukaryotic organism can be readily applied to higher eukaryotes. Using a yeast strain with deletions of both HMG1 and HMG2 genes (i.e. completely devoid of HMGR activity) with introduced wild-type or mutant form of human HMGR (hHMGR) gene we investigated the effects of statins on the lipid metabolism of the cell. The relative quantification of mRNA demonstrated a different effect of simvastatin on the expression of the wild-type and mutated hHMGR gene. GC/MS analyses showed a significant decrease of sterols and enhanced conversion of squalene and sterol precursors into ergosterol. This was accompanied by the mobilization of ergosterol precursors localized in lipid particles in the form of steryl esters visualized by confocal microscopy. Changes in the level of ergosterol and its precursors in cells treated with simvastatin depend on the mutation in the hHMGR gene. HPLC/MS analyses indicated a reduced level of phospholipids not connected with the mevalonic acid pathway. We detected two significant phenomena. First, cells treated with simvastatin develop an adaptive response compensating the lower activity of HMGR. This includes enhanced conversion of sterol precursors into ergosterol, mobilization of steryl esters and increased expression of the hHMGR gene. Second, statins cause a substantial drop in the level of glycerophospholipids.

The enzymic formation of dolichyl phosphate mannose from C-3 enantiomeric dolichyl phosphates

Abstract The racemic, optically inactive mixture of C-3 enantiomeric forms of C 95 -dolichyl phosphate can be prepared chemically from fully unsaturated, di trans -polycis-C 95 -prenol isolated from leaves of Sorbus suecica . Purified mannosyl transferase from yeast was capable of catalysing the mannosylation of over 75% of this 1:1 R / S mixture, demonstrating that both enantiomers could function as acceptors. However, the R -form appears to accept mannose more slowly than the S -form. Both R - and S -forms of C 75 -dolichyl phosphate and C 55 -dolichyl phosphate prepared from the respective plant tri trans -poly cis -prenol were also active as lipid acceptors of mannose from GDPmannose.

Rsp5p ubiquitin ligase and the transcriptional activators Spt23p and Mga2p are involved in co-regulation of biosynthesis of end products of the mevalonate pathway and triacylglycerol in yeast Saccharomyces cerevisiae

Rsp5p, a yeast S. cerevisiae ubiquitin ligase, is essential for regulation of unsaturated fatty acid synthesis via activation of the transcriptional activators Spt23p and Mga2p. Here we show that the conditional mutant rsp5-19 produces decreased levels of the end products of mevalonate pathway, such as ergosterol, ubiquinone and of dolichols, especially those with 19-24 isoprene units. The mechanism of Rsp5p involvement in the control of these lipid synthesis pathways was addressed by overproduction of Rsp5p-independent Spt23p or Mga2p. Expression of constitutively active forms of these transactivators resulted in excess production of ergosterol, but did not restore a wild-type level of dolichols. Moreover, synthesis of long chain dolichols was decreased in the wild-type and a rsp5-19 background. Finally, overproduction of active Spt23p or Mga2p was accompanied by the appearance of large lipid particles in the wild-type and rsp5-19 strains as observed by Nile Red staining, due to accumulation of unsaturated triacylglycerol. Thus, we conclude that Rsp5p, Spt23p and Mga2p may participate in the control of the homeostasis of lipids and lipid particles.