Coordination of Storage Lipid Synthesis and Membrane Biogenesis
2010; Elsevier BV; Volume: 286; Issue: 3 Linguagem: Inglês
10.1074/jbc.m110.172296
ISSN1083-351X
AutoresMaría L. Gaspar, Harald F. Hofbauer, Sepp D. Kohlwein, Susan A. Henry,
Tópico(s)Cellular transport and secretion
ResumoDespite the importance of triacylglycerols (TAG) and steryl esters (SE) in phospholipid synthesis in cells transitioning from stationary-phase into active growth, there is no direct evidence for their requirement in synthesis of phosphatidylinositol (PI) or other membrane phospholipids in logarithmically growing yeast cells. We report that the dga1Δlro1Δare1Δare2Δ strain, which lacks the ability to synthesize both TAG and SE, is not able to sustain normal growth in the absence of inositol (Ino− phenotype) at 37 °C especially when choline is present. Unlike many other strains exhibiting an Ino− phenotype, the dga1Δlro1Δare1Δare2Δ strain does not display a defect in INO1 expression. However, the mutant exhibits slow recovery of PI content compared with wild type cells upon reintroduction of inositol into logarithmically growing cultures. The tgl3Δtgl4Δtgl5Δ strain, which is able to synthesize TAG but unable to mobilize it, also exhibits attenuated PI formation under these conditions. However, unlike dga1Δlro1Δare1Δare2Δ, the tgl3Δtgl4Δtgl5Δ strain does not display an Ino− phenotype, indicating that failure to mobilize TAG is not fully responsible for the growth defect of the dga1Δlro1Δare1Δare2Δ strain in the absence of inositol. Moreover, synthesis of phospholipids, especially PI, is dramatically reduced in the dga1Δlro1Δare1Δare2Δ strain even when it is grown continuously in the presence of inositol. The mutant also utilizes a greater proportion of newly synthesized PI than wild type for the synthesis of inositol-containing sphingolipids, especially in the absence of inositol. Thus, we conclude that storage lipid synthesis actively influences membrane phospholipid metabolism in logarithmically growing cells. Despite the importance of triacylglycerols (TAG) and steryl esters (SE) in phospholipid synthesis in cells transitioning from stationary-phase into active growth, there is no direct evidence for their requirement in synthesis of phosphatidylinositol (PI) or other membrane phospholipids in logarithmically growing yeast cells. We report that the dga1Δlro1Δare1Δare2Δ strain, which lacks the ability to synthesize both TAG and SE, is not able to sustain normal growth in the absence of inositol (Ino− phenotype) at 37 °C especially when choline is present. Unlike many other strains exhibiting an Ino− phenotype, the dga1Δlro1Δare1Δare2Δ strain does not display a defect in INO1 expression. However, the mutant exhibits slow recovery of PI content compared with wild type cells upon reintroduction of inositol into logarithmically growing cultures. The tgl3Δtgl4Δtgl5Δ strain, which is able to synthesize TAG but unable to mobilize it, also exhibits attenuated PI formation under these conditions. However, unlike dga1Δlro1Δare1Δare2Δ, the tgl3Δtgl4Δtgl5Δ strain does not display an Ino− phenotype, indicating that failure to mobilize TAG is not fully responsible for the growth defect of the dga1Δlro1Δare1Δare2Δ strain in the absence of inositol. Moreover, synthesis of phospholipids, especially PI, is dramatically reduced in the dga1Δlro1Δare1Δare2Δ strain even when it is grown continuously in the presence of inositol. The mutant also utilizes a greater proportion of newly synthesized PI than wild type for the synthesis of inositol-containing sphingolipids, especially in the absence of inositol. Thus, we conclude that storage lipid synthesis actively influences membrane phospholipid metabolism in logarithmically growing cells. IntroductionEukaryotic organisms store excess energy as triacylglycerols (TAG) 3The abbreviations used are: TAGtriacylglycerol(s)PIphosphatidylinositolPAphosphatidic acidERendoplasmic reticulumCDP-DAGCDP-diacylglycerolDAGdiacylglycerolPSphosphatidylserinePCphosphatidylcholineSEsteryl ester(s)SLtotal sphingolipidsIPCinositolphosphorylceramideMIPCmannosyl-inositol-phosphorylceramideM(IP)2Cmannosyl-diinositol-phosphorylceramideIinositolCcholine. and steryl esters (SE) for later use during times of deprivation. TAG and SE are hydrophobic compounds separated from the aqueous cellular environment of the cytoplasm in specialized structures called lipid droplets (1Martin S. Parton R.G. Nat. Rev. Mol. Cell Biol. 2006; 7: 373-378Crossref PubMed Scopus (898) Google Scholar). Lipid droplets are dynamic organelles that play important roles in the biosynthesis, mobilization, and trafficking of intracellular neutral lipids. They function in close apposition with other organelles, particularly the endoplasmic reticulum (ER), endosomes, mitochondria, and peroxisomes (2Goodman J.M. J. Biol. Chem. 2008; 283: 28005-28009Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 3Murphy S. Martin S. Parton R.G. Biochim. Biophys. Acta. 2009; 1791: 441-447Crossref PubMed Scopus (210) Google Scholar).In the budding yeast, Saccharomyces cerevisiae, the formation of lipid droplets is tightly linked to the synthesis of TAG and SE. The diacylglycerol acyltransferases encoded by the DGA1 and LRO1 genes (Fig. 1) are the main enzymes involved in the biosynthesis of TAG (4Oelkers P. Cromley D. Padamsee M. Billheimer J.T. Sturley S.L. J. Biol. Chem. 2002; 277: 8877-8881Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 5Oelkers P. Tinkelenberg A. Erdeniz N. Cromley D. Billheimer J.T. Sturley S.L. J. Biol. Chem. 2000; 275: 15609-15612Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 6Sorger D. Daum G. J. Bacteriol. 2002; 184: 519-524Crossref PubMed Scopus (175) Google Scholar), whereas ARE1 and ARE2 (Fig. 1) encode the enzymes that primarily mediate the esterification of ergosterol and its precursors leading to SE (7Jensen-Pergakes K. Guo Z. Giattina M. Sturley S.L. Bard M. J. Bacteriol. 2001; 183: 4950-4957Crossref PubMed Scopus (46) Google Scholar, 8Sandager L. Dahlqvist A. Banaś A. Ståhl U. Lenman M. Gustavsson M. Stymne S. Biochem. Soc. Trans. 2000; 28: 700-702Crossref PubMed Scopus (26) Google Scholar). These four enzymes account for all TAG and SE biosynthesis in yeast, which begins during exponential growth and reaches its peak as cells enter stationary phase (9Müllner H. Daum G. Acta Biochim. Pol. 2004; 51: 323-347Crossref PubMed Scopus (67) Google Scholar). During times of energy scarcity or upon recovery from stationary phase when exposed to glucose, TAG degradation occurs via the activity of lipid hydrolases encoded by the TGL3, TGL4, and TGL5 genes (10Athenstaedt K. Daum G. J. Biol. Chem. 2005; 280: 37301-37309Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 11Kohlwein S.D. J. Biol. Chem. 2010; 285: 15663-15667Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 12Kurat C.F. Wolinski H. Petschnigg J. Kaluarachchi S. Andrews B. Natter K. Kohlwein S.D. Mol. Cell. 2009; 33: 53-63Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). The products of TAG degradation, diacylglycerols (DAG) and free fatty acids, also serve as precursors for membrane lipid synthesis (13Taylor F.R. Parks L.W. Biochim. Biophys. Acta. 1979; 575: 204-214Crossref PubMed Scopus (53) Google Scholar) as well as for energy production when free fatty acids are the only carbon source available in the growth medium (14Rajakumari S. Grillitsch K. Daum G. Prog. Lipid Res. 2008; 47: 157-171Crossref PubMed Scopus (114) Google Scholar). At the cellular level, TAG degradation is up-regulated by Cdc28p/Cdk1p-dependent phosphorylation of the Tgl4p lipase (12Kurat C.F. Wolinski H. Petschnigg J. Kaluarachchi S. Andrews B. Natter K. Kohlwein S.D. Mol. Cell. 2009; 33: 53-63Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Lipolysis contributes to bud formation, presumably by providing precursors for synthesis of lipids involved in membrane biogenesis or signaling (12Kurat C.F. Wolinski H. Petschnigg J. Kaluarachchi S. Andrews B. Natter K. Kohlwein S.D. Mol. Cell. 2009; 33: 53-63Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Conversely, impairment in membrane trafficking leads to a block in phospholipid synthesis and concomitant TAG accumulation (15Gaspar M.L. Jesch S.A. Viswanatha R. Antosh A.L. Brown W.J. Kohlwein S.D. Henry S.A. J. Biol. Chem. 2008; 283: 25735-25751Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar).However, a yeast quadruple mutant strain (dga1Δlro1Δare1Δare2Δ) lacking the ability to store fatty acids in either TAG or SE is viable (4Oelkers P. Cromley D. Padamsee M. Billheimer J.T. Sturley S.L. J. Biol. Chem. 2002; 277: 8877-8881Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 16Sandager L. Gustavsson M.H. Ståhl U. Dahlqvist A. Wiberg E. Banas A. Lenman M. Ronne H. Stymne S. J. Biol. Chem. 2002; 277: 6478-6482Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar). The sole growth phenotypes relative to wild type that have been reported for this strain are unsaturated fatty acid-induced toxicity and a prolonged lag phase after transfer to fresh YPD media (17Garbarino J. Padamsee M. Wilcox L. Oelkers P.M. D'Ambrosio D. Ruggles K.V. Ramsey N. Jabado O. Turkish A. Sturley S.L. J. Biol. Chem. 2009; 284: 30994-31005Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 18Petschnigg J. Wolinski H. Kolb D. Zellnig G. Kurat C.F. Natter K. Kohlwein S.D. J. Biol. Chem. 2009; 284: 30981-30993Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). No significant change in growth of this strain was observed during exponential or stationary phase (4Oelkers P. Cromley D. Padamsee M. Billheimer J.T. Sturley S.L. J. Biol. Chem. 2002; 277: 8877-8881Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 18Petschnigg J. Wolinski H. Kolb D. Zellnig G. Kurat C.F. Natter K. Kohlwein S.D. J. Biol. Chem. 2009; 284: 30981-30993Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). These data suggest that the reserve of TAG and SE is not essential to sustain growth in yeast, at least when cells grow in rich media.Certain mutants, defective in membrane trafficking (Sec−), were shown to exhibit increased TAG synthesis at the expense of phospholipid synthesis when shifted to a temperature that restricted membrane trafficking and cell growth (15Gaspar M.L. Jesch S.A. Viswanatha R. Antosh A.L. Brown W.J. Kohlwein S.D. Henry S.A. J. Biol. Chem. 2008; 283: 25735-25751Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). That study specifically showed that deleting the DAG acyltransferases, Dga1p and Lro1p, in the sec13-1 mutant, defective in membrane and protein transport from the ER, led to a lowering of the temperature at which the mutant could grow (i.e. its restrictive temperature). The lowering of the restrictive temperature in the sec13-1dga1Δlro1Δ strain, was especially pronounced when it was grown in the absence of the phospholipid precursor, inositol. In wild type cells, lack of inositol supplementation results in a substantial reduction in the synthesis of phosphatidylinositol (PI) (19Gaspar M.L. Aregullin M.A. Jesch S.A. Henry S.A. J. Biol. Chem. 2006; 281: 22773-22785Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 20Kelley M.J. Bailis A.M. Henry S.A. Carman G.M. J. Biol. Chem. 1988; 263: 18078-18085Abstract Full Text PDF PubMed Google Scholar).The above-summarized evidence suggests that the cell coordinates the synthesis and breakdown of storage lipids with its demand for membrane lipid synthesis. Consistent with this idea, we showed in a previous study that the fatty acids required for the rapid burst of PI synthesis after inositol supplementation to cells deprived of inositol are derived in part from both de novo fatty acid synthesis and phosphatidylcholine (PC) turnover (19Gaspar M.L. Aregullin M.A. Jesch S.A. Henry S.A. J. Biol. Chem. 2006; 281: 22773-22785Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 21Loewen C.J. Gaspar M.L. Jesch S.A. Delon C. Ktistakis N.T. Henry S.A. Levine T.P. Science. 2004; 304: 1644-1647Crossref PubMed Scopus (364) Google Scholar). However, these two sources of fatty acids do not fully account for the burst in PI synthesis (19Gaspar M.L. Aregullin M.A. Jesch S.A. Henry S.A. J. Biol. Chem. 2006; 281: 22773-22785Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), suggesting that additional fatty acids might be derived from hydrolysis of TAG. In the current study we tested the ability of the cells to grow in the absence of inositol and to rapidly restore PI content in response to inositol reintroduction when they are unable to mobilize TAG. We report that upon inositol reintroduction, the dga1Δlro1Δare1Δare2Δ strain, unable to synthesize TAG or SE, and the tgl3Δtgl4Δtgl5Δ strain, lacking the TAG lipases, both exhibit slow recovery of PI content in comparison to wild type cells. However, only the dga1Δlro1Δare1Δare2Δ strain failed to grow in the absence of inositol. Thus, failure to hydrolyze TAG does not fully explain the Ino− phenotype of the dga1Δlro1Δare1Δare2Δ strain. We present evidence that this mutant exhibits reduced synthesis of PI even when grown continuously in the presence of exogenous inositol. The mutant also devotes a larger percentage of newly synthesized PI to the synthesis of inositol-containing sphingolipids for which PI serves as a precursor.DISCUSSIONThe Ino− phenotype is classically associated with a deficiency in expression of INO1, the structural gene for the inositol-3-phosphate synthase (23Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (261) Google Scholar, 39Greenberg M.L. Lopes J.M. Microbiol. Rev. 1996; 60: 1-20Crossref PubMed Google Scholar). However, we report that the dga1Δlro1Δare1Δare2Δ mutant, which totally lacks lipid droplets (4Oelkers P. Cromley D. Padamsee M. Billheimer J.T. Sturley S.L. J. Biol. Chem. 2002; 277: 8877-8881Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 16Sandager L. Gustavsson M.H. Ståhl U. Dahlqvist A. Wiberg E. Banas A. Lenman M. Ronne H. Stymne S. J. Biol. Chem. 2002; 277: 6478-6482Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar), exhibited no significant defect in INO1 expression at 37 °C in I−C− medium compared with wild type. Nevertheless, its ability to grow on agar plates under these conditions was greatly reduced (Fig. 2). Immediately after a shift of actively growing cells to I−C+ medium at 37 °C, the mutant actually exhibited higher INO1 expression than the wild type strain (Fig. 5), but within a few hours after the shift to I−C+ medium at 37 °C, INO1 expression was similar to that in the wild type strain. The dga1Δlro1Δare1Δare2Δ strain is not the first mutant reported to exhibit an Ino− phenotype and yet to be able to express INO1. The sac1Δ mutant (40Rivas M.P. Kearns B.G. Xie Z. Guo S. Sekar M.C. Hosaka K. Kagiwada S. York J.D. Bankaitis V.A. Mol. Biol. Cell. 1999; 10: 2235-2250Crossref PubMed Scopus (115) Google Scholar) and mutants defective in the protein kinase C (PKC) stress response pathway also have Ino− phenotypes and yet express INO1 (30Nunez L.R. Jesch S.A. Gaspar M.L. Almaguer C. Villa-Garcia M. Ruiz-Noriega M. Patton-Vogt J. Henry S.A. J. Biol. Chem. 2008; 283: 34204-34217Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Moreover, PKC signaling is triggered both by growth in the absence of inositol and by growth at high temperature (30Nunez L.R. Jesch S.A. Gaspar M.L. Almaguer C. Villa-Garcia M. Ruiz-Noriega M. Patton-Vogt J. Henry S.A. J. Biol. Chem. 2008; 283: 34204-34217Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 41Levin D.E. Microbiol. Mol. Biol. Rev. 2005; 69: 262-291Crossref PubMed Scopus (871) Google Scholar, 42Levin D.E. Bartlett-Heubusch E. J. Cell Biol. 1992; 116: 1221-1229Crossref PubMed Scopus (302) Google Scholar). The Ino− phenotypes of mutants defective in the PKC pathway, like that of the dga1Δlro1Δare1Δare2Δ mutant, are strengthened in the presence of choline at 37 °C (30Nunez L.R. Jesch S.A. Gaspar M.L. Almaguer C. Villa-Garcia M. Ruiz-Noriega M. Patton-Vogt J. Henry S.A. J. Biol. Chem. 2008; 283: 34204-34217Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Growth in the absence of inositol activates a number of stress response pathways in addition to PKC (43Jesch S.A. Liu P. Zhao X. Wells M.T. Henry S.A. J. Biol. Chem. 2006; 281: 24070-24083Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The phenotype of the dga1Δlro1Δare1Δare2Δ strain suggests that the absence of storage lipid synthesis adds to the stress-associated with growth in I−C+ medium at 37 °C. The fact that the mutant devotes a higher proportion of newly synthesized PI to sphingolipid synthesis than the wild type strain under these conditions is consistent with this hypothesis, as sphingolipids have been implicated in several stress response pathways including PKC signaling (44Dickson R.C. J. Lipid Res. 2008; 49: 909-921Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar).However, the mutant also displayed alterations in phospholipid biosynthesis including reduced synthesis of PI even when grown continuously in I+C+ medium (Fig. 6), a growth condition under which inositol was completely derived from the medium. Labeling of PA, CDP-DAG, and PS were also lower in the mutant in comparison to wild type in the presence of inositol, suggesting that the mutant synthesizes PA, the immediate precursor of CDP-DAG, PI, and PS at a reduced rate. Because the INO1 gene was completely repressed in the mutant as well as in the wild type strain in medium containing inositol (Fig. 5), the decrease in PI synthesis in the mutant under these conditions (Fig. 6) is clearly independent from its ability to derepress the INO1 gene or its gene product, inositol-3-phosphate synthase. Moreover, in the mutant, in comparison to wild type, newly synthesized PI was also utilized to a greater extent in the synthesis of inositol-containing sphingolipids, especially when it was growing in the absence of inositol, a topic that will be discussed below. Based on these observations, we conclude that the storage lipid synthesis actively influences the metabolism of membrane lipids, both phospholipids and sphingolipids, in logarithmically growing cells.Lack of TAG Hydrolysis in Logarithmically Growing Cells Attenuates the Burst in PI Synthesis in Response to Inositol ReintroductionIn studies on stationary-phase cells reentering active growth, Taylor and Parks in 1979 (13Taylor F.R. Parks L.W. Biochim. Biophys. Acta. 1979; 575: 204-214Crossref PubMed Scopus (53) Google Scholar) proposed that fatty acids derived from TAG are utilized for the synthesis of phospholipids upon resumption of growth. Most recently, the lipid hydrolases that catalyze TAG degradation were identified (10Athenstaedt K. Daum G. J. Biol. Chem. 2005; 280: 37301-37309Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 12Kurat C.F. Wolinski H. Petschnigg J. Kaluarachchi S. Andrews B. Natter K. Kohlwein S.D. Mol. Cell. 2009; 33: 53-63Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 32Athenstaedt K. Daum G. J. Biol. Chem. 2003; 278: 23317-23323Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 33Kurat C.F. Natter K. Petschnigg J. Wolinski H. Scheuringer K. Scholz H. Zimmermann R. Leber R. Zechner R. Kohlwein S.D. J. Biol. Chem. 2006; 281: 491-500Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). In this study we report that both the dga1Δlro1Δare1Δare2Δ mutant, which lacks the ability to produce TAG (4Oelkers P. Cromley D. Padamsee M. Billheimer J.T. Sturley S.L. J. Biol. Chem. 2002; 277: 8877-8881Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 16Sandager L. Gustavsson M.H. Ståhl U. Dahlqvist A. Wiberg E. Banas A. Lenman M. Ronne H. Stymne S. J. Biol. Chem. 2002; 277: 6478-6482Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar) and the tgl3Δtgl4Δtgl5Δ mutant, which is unable to mobilize TAG (10Athenstaedt K. Daum G. J. Biol. Chem. 2005; 280: 37301-37309Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 12Kurat C.F. Wolinski H. Petschnigg J. Kaluarachchi S. Andrews B. Natter K. Kohlwein S.D. Mol. Cell. 2009; 33: 53-63Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 33Kurat C.F. Natter K. Petschnigg J. Wolinski H. Scheuringer K. Scholz H. Zimmermann R. Leber R. Zechner R. Kohlwein S.D. J. Biol. Chem. 2006; 281: 491-500Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar), are compromised in rebuilding PI content to the levels seen in wild type cells immediately after the addition of inositol. Consistent with these results, TAG is consumed in proliferating wild type cells upon inositol reintroduction coincident with the rebuilding of PI content. We propose a new metabolic role for TAG as donor of fatty acids for the synthesis of PI in logarithmically growing yeast cells, particularly when inositol is reintroduced to cultures previously lacking this lipid precursor.However, the dga1Δlro1Δare1Δare2Δ strain also exhibits impaired growth in medium lacking inositol. This growth defect is not simply due to the inability of the dga1Δlro1Δare1Δare2Δ mutant to mobilize TAG for PI synthesis, as the tgl3Δtgl4Δtgl5Δ strain is able to sustain growth in I− medium at 37 °C in the presence of choline. As we discuss below, the ability to synthesize TAG was shown to be protective under conditions of impaired membrane trafficking and declining phospholipid synthesis (15Gaspar M.L. Jesch S.A. Viswanatha R. Antosh A.L. Brown W.J. Kohlwein S.D. Henry S.A. J. Biol. Chem. 2008; 283: 25735-25751Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). In addition, the reduced recovery of PI content immediately after inositol reintroduction displayed by both mutants in comparison to wild type suggests that a pool of fatty acids derived from TAG degradation may be used for rapid restoration of PI after inositol reintroduction. The data presented here also confirm the combined contribution of de novo fatty acid synthesis and PC turnover for the burst of PI content after inositol reintroduction, as previously reported (19Gaspar M.L. Aregullin M.A. Jesch S.A. Henry S.A. J. Biol. Chem. 2006; 281: 22773-22785Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Thus, we conclude that there are at least three different metabolic pathways capable of providing fatty acids for the dramatic increase in PI upon inositol reintroduction in wild type cells.The Absence of Lipid Droplets Affects Synthesis of Both PI and Inositol-containing SphingolipidsThe Ino− phenotype of the dga1Δlro1Δare1Δare2Δ strain is clearly not due simply to its inability to mobilize TAG, and thus, the inability to store fatty acids in TAG and SE must contribute to its phenotype. Despite the fact that the dga1Δlro1Δare1Δare2Δ mutant is not able to store fatty acids in either TAG or SE, it is viable under the culture conditions employed in the studies by Oelkers et al. (4Oelkers P. Cromley D. Padamsee M. Billheimer J.T. Sturley S.L. J. Biol. Chem. 2002; 277: 8877-8881Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar) and Sandager et al. (16Sandager L. Gustavsson M.H. Ståhl U. Dahlqvist A. Wiberg E. Banas A. Lenman M. Ronne H. Stymne S. J. Biol. Chem. 2002; 277: 6478-6482Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar). These two studies showed that storage lipids are not essential to sustain active logarithmic growth in yeast. However, TAG hydrolysis does play an important role in the recovery of cells from stationary phase into active growth (12Kurat C.F. Wolinski H. Petschnigg J. Kaluarachchi S. Andrews B. Natter K. Kohlwein S.D. Mol. Cell. 2009; 33: 53-63Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 13Taylor F.R. Parks L.W. Biochim. Biophys. Acta. 1979; 575: 204-214Crossref PubMed Scopus (53) Google Scholar). Moreover, active TAG biosynthesis was shown to be protective under conditions of secretory stress (15Gaspar M.L. Jesch S.A. Viswanatha R. Antosh A.L. Brown W.J. Kohlwein S.D. Henry S.A. J. Biol. Chem. 2008; 283: 25735-25751Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The sec13-1 strain, which has a defect in COPII vesicle trafficking from the ER when shifted to its restrictive temperature of 37 °C, experienced a dramatic decrease in phospholipid synthesis relative to the wild type control, whereas synthesis of TAG increased. Thus, it appears that when membrane trafficking pathways are compromised, synthesis of phospholipids required for membrane proliferation declines in a fashion that is coordinated with the slowing of membrane trafficking. Under these conditions, excess fatty acids are channeled into TAG (15Gaspar M.L. Jesch S.A. Viswanatha R. Antosh A.L. Brown W.J. Kohlwein S.D. Henry S.A. J. Biol. Chem. 2008; 283: 25735-25751Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Moreover, when the genes encoding the DAG acyltransferases, Dga1p and Lro1p, were deleted in the sec13-1 mutant, the restrictive temperature of the sec13-1 dga1Δ lro1Δ strain decreased by two degrees compared with the parental strain (sec13-1 DGA1 LRO1) in media lacking inositol (15Gaspar M.L. Jesch S.A. Viswanatha R. Antosh A.L. Brown W.J. Kohlwein S.D. Henry S.A. J. Biol. Chem. 2008; 283: 25735-25751Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Thus, the increased synthesis of TAG appears to serve under these conditions to absorb the excess fatty acids not being used in membrane proliferation, providing a degree of protection under conditions of secretory stress (15Gaspar M.L. Jesch S.A. Viswanatha R. Antosh A.L. Brown W.J. Kohlwein S.D. Henry S.A. J. Biol. Chem. 2008; 283: 25735-25751Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar).The above evidence suggests that both TAG synthesis and breakdown are interdependent with ongoing membrane lipid synthesis. In agreement with these reports, we found that the lack of storage lipids in the dga1Δlro1Δare1Δare2Δ mutant has a profound impact on the synthesis of membrane lipids. The inability of the mutant to synthesize TAG and SE affects the synthesis of all the phospholipids derived directly from PA (Fig. 6), and this effect is not specific to PI synthesis alone. These results indicate that the storage lipids in lipid droplets are required to maintain normal rates of phospholipid synthesis in proliferating cells. Furthermore, the dga1Δlro1Δare1Δare2Δ mutant utilizes more of its newly synthesized PI in the formation of inositol-containing sphingolipids than the wild type strain. In contrast, the tgl3Δtgl4Δtgl5Δ mutant was reported to exhibit decreased formation of sphingolipids in comparison to wild type (45Rajakumari S. Rajasekharan R. Daum G. Biochim. Biophys. Acta. 2010; 1801: 1314-1322Crossref PubMed Scopus (36) Google Scholar).Sphingolipids together with sterols are highly enriched in the plasma membrane (46Hechtberger P. Zinser E. Saf R. Hummel K. Paltauf F. Daum G. Eur. J. Biochem. 1994; 225: 641-649Crossref PubMed Scopus (103) Google Scholar, 47Lange Y. Swaisgood M.H. Ramos B.V. Steck T.L. J. Biol. Chem. 1989; 264: 3786-3793Abstract Full Text PDF PubMed Google Scholar, 48Patton J.L. Lester R.L. J. Bacteriol. 1991; 173: 3101-3108Crossref PubMed Google Scholar, 49Schneiter R. Brügger B. Sandhoff R. Zellnig G. Leber A. Lampl M. Athenstaedt K. Hrastnik C. Eder S. Daum G. Paltauf F. Wieland F.T. Kohlwein S.D. J. 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Because this mode of transport is not possible in the dga1Δlro1Δare1Δare2Δ mutant, the delivery of free sterols must occur by routes such as membrane transport or non-vesicular transport pathways. There is also evidence indicating a role for sterols and sphingolipids in the generation of lipid gradients along the secretory pathway (53Klemm R.W. Ejsing C.S. Surma M.A. Kaiser H.J. Gerl M.J. Sampaio J.L. de Robillard Q. Ferguson C. Proszynski T.J. Shevchenko A. Simons K. J. Cell Biol. 2009; 185: 601-612Crossref PubMed Scopus (298) Google Scholar). Thus, the active production of complex sphingolipids in the dga1Δlro1Δare1Δare2Δ mutant in the absence of lipid droplets might also be required to facilitate the transport of the excess free sterols produced in the mutant from the ER to the plasma membrane by means of the membrane transport pathways.In summary, we have found that the dga1Δlro1Δare1Δare2Δ strain is not able to sustain growth in the absence of inositol at 37 °C. This is not due to a defect in INO1 expression but may be due in part to the absence of a pool of storage lipids from which to draw fatty acids as precursors to sustain optimal synthesis of PI and other membrane phospholipids in logarithmically growing yeast cells. The need for a higher proportion of newly synthesized PI in producing sphingolipids may also be a factor contributing to the growth defect experienced by the mutant in the absence of inositol. Together, these results suggest that synthesis of storage lipids and membrane-forming lipids, in particular PI and inositol-containing sphingolipids, are interdependent and operate in a coordinated fashion to maintain lipid homeostasis in actively growing yeast cells. IntroductionEukaryotic organisms store excess energy as triacylglycerols (TAG) 3The abbreviations used are: TAGtriacylglycerol(s)PIphosphatidylinositolPAphosphatidic acidERendoplasmic reticulumCDP-DAGCDP-diacylglycerolDAGdiacylglycerolPSphosphatidylserinePCphosphatidylcholineSEsteryl ester(s)SLtotal sphingolipidsIPCinositolphosphorylceramideMIPCmannosyl-inositol-phosphorylceramideM(IP)2Cmannosyl-diinositol-phosphorylceramideIinositolCcholine. and steryl esters (SE) for later use during times of deprivation. TAG and SE are hydrophobic compounds separated from the aqueous cellular environment of the cytoplasm in specialized structures called lipid droplets (1Martin S. Parton R.G. Nat. Rev. Mol. Cell Biol. 2006; 7: 373-378Crossref PubMed Scopus (898) Google Scholar). Lipid droplets are dynamic organelles that play important roles in the biosynthesis, mobilization, and trafficking of intracellular neutral lipids. They function in close apposition with other organelles, particularly the endoplasmic reticulum (ER), endosomes, mitochondria, and peroxisomes (2Goodman J.M. J. Biol. Chem. 2008; 283: 28005-28009Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 3Murphy S. Martin S. Parton R.G. Biochim. Biophys. Acta. 2009; 1791: 441-447Crossref PubMed Scopus (210) Google Scholar).In the budding yeast, Saccharomyces cerevisiae, the formation of lipid droplets is tightly linked to the synthesis of TAG and SE. The diacylglycerol acyltransferases encoded by the DGA1 and LRO1 genes (Fig. 1) are the main enzymes involved in the biosynthesis of TAG (4Oelkers P. Cromley D. Padamsee M. Billheimer J.T. Sturley S.L. J. Biol. Chem. 2002; 277: 8877-8881Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 5Oelkers P. Tinkelenberg A. Erdeniz N. Cromley D. Billheimer J.T. Sturley S.L. J. Biol. Chem. 2000; 275: 15609-15612Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 6Sorger D. Daum G. J. Bacteriol. 2002; 184: 519-524Crossref PubMed Scopus (175) Google Scholar), whereas ARE1 and ARE2 (Fig. 1) encode the enzymes that primarily mediate the esterification of ergosterol and its precursors leading to SE (7Jensen-Pergakes K. Guo Z. Giattina M. Sturley S.L. Bard M. J. Bacteriol. 2001; 183: 4950-4957Crossref PubMed Scopus (46) Google Scholar, 8Sandager L. Dahlqvist A. Banaś A. Ståhl U. Lenman M. Gustavsson M. Stymne S. Biochem. Soc. Trans. 2000; 28: 700-702Crossref PubMed Scopus (26) Google Scholar). These four enzymes account for all TAG and SE biosynthesis in yeast, which begins during exponential growth and reaches its peak as cells enter stationary phase (9Müllner H. Daum G. Acta Biochim. Pol. 2004; 51: 323-347Crossref PubMed Scopus (67) Google Scholar). During times of energy scarcity or upon recovery from stationary phase when exposed to glucose, TAG degradation occurs via the activity of lipid hydrolases encoded by the TGL3, TGL4, and TGL5 genes (10Athenstaedt K. Daum G. J. Biol. Chem. 2005; 280: 37301-37309Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 11Kohlwein S.D. J. Biol. Chem. 2010; 285: 15663-15667Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 12Kurat C.F. Wolinski H. Petschnigg J. Kaluarachchi S. Andrews B. Natter K. Kohlwein S.D. Mol. Cell. 2009; 33: 53-63Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). The products of TAG degradation, diacylglycerols (DAG) and free fatty acids, also serve as precursors for membrane lipid synthesis (13Taylor F.R. Parks L.W. Biochim. Biophys. Acta. 1979; 575: 204-214Crossref PubMed Scopus (53) Google Scholar) as well as for energy production when free fatty acids are the only carbon source available in the growth medium (14Rajakumari S. Grillitsch K. Daum G. Prog. Lipid Res. 2008; 47: 157-171Crossref PubMed Scopus (114) Google Scholar). At the cellular level, TAG degradation is up-regulated by Cdc28p/Cdk1p-dependent phosphorylation of the Tgl4p lipase (12Kurat C.F. Wolinski H. Petschnigg J. Kaluarachchi S. Andrews B. Natter K. Kohlwein S.D. Mol. Cell. 2009; 33: 53-63Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Lipolysis contributes to bud formation, presumably by providing precursors for synthesis of lipids involved in membrane biogenesis or signaling (12Kurat C.F. Wolinski H. Petschnigg J. Kaluarachchi S. Andrews B. Natter K. Kohlwein S.D. Mol. Cell. 2009; 33: 53-63Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Conversely, impairment in membrane trafficking leads to a block in phospholipid synthesis and concomitant TAG accumulation (15Gaspar M.L. Jesch S.A. Viswanatha R. Antosh A.L. Brown W.J. Kohlwein S.D. Henry S.A. J. Biol. Chem. 2008; 283: 25735-25751Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar).However, a yeast quadruple mutant strain (dga1Δlro1Δare1Δare2Δ) lacking the ability to store fatty acids in either TAG or SE is viable (4Oelkers P. Cromley D. Padamsee M. Billheimer J.T. Sturley S.L. J. Biol. Chem. 2002; 277: 8877-8881Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 16Sandager L. Gustavsson M.H. Ståhl U. Dahlqvist A. Wiberg E. Banas A. Lenman M. Ronne H. Stymne S. J. Biol. Chem. 2002; 277: 6478-6482Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar). The sole growth phenotypes relative to wild type that have been reported for this strain are unsaturated fatty acid-induced toxicity and a prolonged lag phase after transfer to fresh YPD media (17Garbarino J. Padamsee M. Wilcox L. Oelkers P.M. D'Ambrosio D. Ruggles K.V. Ramsey N. Jabado O. Turkish A. Sturley S.L. J. Biol. Chem. 2009; 284: 30994-31005Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 18Petschnigg J. Wolinski H. Kolb D. Zellnig G. Kurat C.F. Natter K. Kohlwein S.D. J. Biol. Chem. 2009; 284: 30981-30993Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). No significant change in growth of this strain was observed during exponential or stationary phase (4Oelkers P. Cromley D. Padamsee M. Billheimer J.T. Sturley S.L. J. Biol. Chem. 2002; 277: 8877-8881Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 18Petschnigg J. Wolinski H. Kolb D. Zellnig G. Kurat C.F. Natter K. Kohlwein S.D. J. Biol. Chem. 2009; 284: 30981-30993Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). These data suggest that the reserve of TAG and SE is not essential to sustain growth in yeast, at least when cells grow in rich media.Certain mutants, defective in membrane trafficking (Sec−), were shown to exhibit increased TAG synthesis at the expense of phospholipid synthesis when shifted to a temperature that restricted membrane trafficking and cell growth (15Gaspar M.L. Jesch S.A. Viswanatha R. Antosh A.L. Brown W.J. Kohlwein S.D. Henry S.A. J. Biol. Chem. 2008; 283: 25735-25751Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). That study specifically showed that deleting the DAG acyltransferases, Dga1p and Lro1p, in the sec13-1 mutant, defective in membrane and protein transport from the ER, led to a lowering of the temperature at which the mutant could grow (i.e. its restrictive temperature). The lowering of the restrictive temperature in the sec13-1dga1Δlro1Δ strain, was especially pronounced when it was grown in the absence of the phospholipid precursor, inositol. In wild type cells, lack of inositol supplementation results in a substantial reduction in the synthesis of phosphatidylinositol (PI) (19Gaspar M.L. Aregullin M.A. Jesch S.A. Henry S.A. J. Biol. Chem. 2006; 281: 22773-22785Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 20Kelley M.J. Bailis A.M. Henry S.A. Carman G.M. J. Biol. Chem. 1988; 263: 18078-18085Abstract Full Text PDF PubMed Google Scholar).The above-summarized evidence suggests that the cell coordinates the synthesis and breakdown of storage lipids with its demand for membrane lipid synthesis. Consistent with this idea, we showed in a previous study that the fatty acids required for the rapid burst of PI synthesis after inositol supplementation to cells deprived of inositol are derived in part from both de novo fatty acid synthesis and phosphatidylcholine (PC) turnover (19Gaspar M.L. Aregullin M.A. Jesch S.A. Henry S.A. J. Biol. Chem. 2006; 281: 22773-22785Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 21Loewen C.J. Gaspar M.L. Jesch S.A. Delon C. Ktistakis N.T. Henry S.A. Levine T.P. Science. 2004; 304: 1644-1647Crossref PubMed Scopus (364) Google Scholar). However, these two sources of fatty acids do not fully account for the burst in PI synthesis (19Gaspar M.L. Aregullin M.A. Jesch S.A. Henry S.A. J. Biol. Chem. 2006; 281: 22773-22785Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), suggesting that additional fatty acids might be derived from hydrolysis of TAG. In the current study we tested the ability of the cells to grow in the absence of inositol and to rapidly restore PI content in response to inositol reintroduction when they are unable to mobilize TAG. We report that upon inositol reintroduction, the dga1Δlro1Δare1Δare2Δ strain, unable to synthesize TAG or SE, and the tgl3Δtgl4Δtgl5Δ strain, lacking the TAG lipases, both exhibit slow recovery of PI content in comparison to wild type cells. However, only the dga1Δlro1Δare1Δare2Δ strain failed to grow in the absence of inositol. Thus, failure to hydrolyze TAG does not fully explain the Ino− phenotype of the dga1Δlro1Δare1Δare2Δ strain. We present evidence that this mutant exhibits reduced synthesis of PI even when grown continuously in the presence of exogenous inositol. The mutant also devotes a larger percentage of newly synthesized PI to the synthesis of inositol-containing sphingolipids for which PI serves as a precursor.
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