Lipid synthesis and membrane contact sites: a crossroads for cellular physiology

Membrane contact sites (MCSs) are regions of close apposition between different organelles that contribute to the functional integration of compartmentalized cellular processes. In recent years, we have gained insight into the molecular architecture of several contact sites, as well as into the regulatory mechanisms that underlie their roles in cell physiology. We provide an overview of two selected topics where lipid metabolism intersects with MCSs and organelle dynamics. First, the role of phosphatidic acid phosphatase, Pah1, the yeast homolog of metazoan lipin, toward the synthesis of triacylglycerol is outlined in connection with the seipin complex, Fld1/Ldb16, and lipid droplet formation. Second, we recapitulate the different contact sites connecting mitochondria and the endomembrane system and emphasize their contribution to phospholipid synthesis and their coordinated regulation. A comprehensive view is emerging where the multiplicity of contact sites connecting different cellular compartments together with lipid transfer proteins functioning at more than one MCS allow for functional redundancy and cross-regulation. Membrane contact sites (MCSs) are regions of close apposition between different organelles that contribute to the functional integration of compartmentalized cellular processes. In recent years, we have gained insight into the molecular architecture of several contact sites, as well as into the regulatory mechanisms that underlie their roles in cell physiology. We provide an overview of two selected topics where lipid metabolism intersects with MCSs and organelle dynamics. First, the role of phosphatidic acid phosphatase, Pah1, the yeast homolog of metazoan lipin, toward the synthesis of triacylglycerol is outlined in connection with the seipin complex, Fld1/Ldb16, and lipid droplet formation. Second, we recapitulate the different contact sites connecting mitochondria and the endomembrane system and emphasize their contribution to phospholipid synthesis and their coordinated regulation. A comprehensive view is emerging where the multiplicity of contact sites connecting different cellular compartments together with lipid transfer proteins functioning at more than one MCS allow for functional redundancy and cross-regulation. The pervasive importance of lipids in cell biology has become evident. From structural roles defining cellular compartments and surfaces with specific biological properties to functional roles in signal transduction processes and modulation of regulatory networks, the involvement of lipids in varied cellular functions is well-established. Concurrently, we have gained a broad understanding about the repertoire of proteins involved in synthesis, degradation, transport, and sensing of lipids, as well as the regulatory mechanisms that interconnect lipid metabolism with other cellular processes. The amenability of Saccharomyces cerevisiae to classical approaches of molecular genetics, and to high throughput analyses, makes this yeast a powerful organism to unveil the intricacies of this field. The yeast S. cerevisiae has played a foundational role in the field of molecular and cellular biology of lipids, including the identification of membrane contact sites (MCSs) and how they link lipid metabolism with organelle dynamics. We present advances in two topics where extensive and outstanding contributions have recently been made where their relevance transcends the yeast lipid field. We first review new information about the phosphatidic acid (PA) phosphatase, Pah1, the yeast homolog of human lipin, and its role in directing lipid flux into membrane synthesis or storage. We review the contribution of Pah1 to lipid droplet (LD) formation and its interaction with the seipin complex, Fld1/Ldb16, to regulate endoplasmic reticulum (ER)-LD contact site dynamics. Second, we recapitulate recent developments revealing MCS connections between the ER and mitochondria with other cellular compartments, and how these intersect with the maintenance of lipid homeostasis. Furthermore, we explore MCS redundancies that allow cells to cope with changing metabolic demand. PA is the common precursor for the synthesis of membrane phospholipids (PLs) and the major component of LDs, such as triacylglycerol (TAG) (Fig. 1). The de novo biosynthesis of PA, as well as its consumption for PL and TAG synthesis, takes place in the ER (1Athenstaedt K. Daum G. Phosphatidic acid, a key intermediate in lipid metabolism.Eur. J. Biochem. 1999; 266: 1-16Crossref PubMed Scopus (249) Google Scholar, 2Henry S.A. Kohlwein S.D. Carman G.M. Metabolism and regulation of glycerolipids in the yeast Saccharomyces cerevisiae.Genetics. 2012; 190: 317-349Crossref PubMed Scopus (302) Google Scholar). The size of the pool of PA localized at the ER impacts the expression of the genes involved in PL synthesis, as a transcriptional regulatory module directly connects PA availability at the ER with gene expression of PL biosynthetic genes in the nucleus (3Henry S.A. Patton-Vogt J.L. Genetic regulation of phospholipid metabolism: yeast as a model eukaryote.Prog. Nucleic Acid Res. Mol. Biol. 1998; 61: 133-179Crossref PubMed Google Scholar, 4Carman G.M. Henry S.A. Phosphatidic acid plays a central role in the transcriptional regulation of glycerophospholipid synthesis in Saccharomyces cerevisiae.J. Biol. 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Weissman J.S. et al.Comprehensive characterization of genes required for protein folding in the endoplasmic reticulum.Science. 2009; 323: 1693-1697Crossref PubMed Scopus (452) Google Scholar, 8Schuck S. Prinz W.A. Thorn K.S. Voss C. Walter P. Membrane expansion alleviates endoplasmic reticulum stress independently of the unfolded protein response.J. Cell Biol. 2009; 187: 525-536Crossref PubMed Scopus (286) Google Scholar). The combined functional proficiency of these cellular processes is required for normal growth. The activity of Pah1, the yeast homolog of human lipin, has a prominent role, as this enzyme diverts the flux of PA from PL synthesis into TAG synthesis for accumulation into LDs based on the cellular need of membrane expansion and cell growth versus cessation of growth and lipid storage (9Han G-S. Wu W-I. Carman G.M. The Saccharomyces cerevisiae Lipin homolog is a Mg2+-dependent phosphatidate phosphatase enzyme.J. Biol. 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Phosphorylation of phosphatidate phosphatase regulates its membrane association and physiological functions in Saccharomyces cerevisiae: identification of SER(602), THR(723), and SER(744) as the sites phosphorylated by CDC28 (CDK1)-encoded cyclin-dependen.J. Biol. Chem. 2011; 286: 1486-1498Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 13Choi H-S. Su W-M. Han G-S. Plote D. Xu Z. Carman G.M. Pho85p-Pho80p phosphorylation of yeast Pah1p phosphatidate phosphatase regulates its activity, location, abundance, and function in lipid metabolism.J. Biol. Chem. 2012; 287: 11290-11301Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Upon dephosphorylation by the conserved ER-localized protein phosphatase, Nem1/Spo7, Pah1 becomes membrane bound and active, generating diacylglycerol (DAG) for TAG synthesis (14Santos-Rosa H. Leung J. Grimsey N. Peak-Chew S. Siniossoglou S. The yeast lipin Smp2 couples phospholipid biosynthesis to nuclear membrane growth.EMBO J. 2005; 24: 1931-1941Crossref PubMed Scopus (264) Google Scholar, 15Su W-M. Han G-S. Carman G.M. Yeast Nem1-Spo7 protein phosphatase activity on Pah1 phosphatidate phosphatase is specific for the Pho85-Pho80 protein kinase phosphorylation sites.J. Biol. Chem. 2014; 289: 34699-34708Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Remarkably, the active form of Pah1 is unstable and rapidly degraded, resulting in a tightly controlled system for regulation of membrane versus storage lipid synthesis (16Pascual F. Hsieh L-S. Soto-Cardalda A.I. Carman G.M. Yeast Pah1p phosphatidate phosphatase is regulated by proteasome-mediated degradation.J. Biol. Chem. 2014; 289: 9811-9822Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 17Hsieh L-S. Su W-M. Han G-S. Carman G.M. Phosphorylation regulates the ubiquitin-independent degradation of yeast Pah1 phosphatidate phosphatase by the 20S proteasome.J. Biol. Chem. 2015; 290: 11467-11478Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Interestingly, LDs are tightly associated with the ER at MCSs (18Szymanski K.M. Binns D. Bartz R. Grishin N.V. Li W-P. Agarwal A.K. Garg A. Anderson R.G.W. Goodman J.M. The lipodystrophy protein seipin is found at endoplasmic reticulum lipid droplet junctions and is important for droplet morphology.Proc. Natl. Acad. Sci. USA. 2007; 104: 20890-20895Crossref PubMed Scopus (390) Google Scholar, 19Jacquier N. Choudhary V. Mari M. Toulmay A. Reggiori F. Schneiter R. Lipid droplets are functionally connected to the endoplasmic reticulum in Saccharomyces cerevisiae.J. Cell Sci. 2011; 124: 2424-2437Crossref PubMed Scopus (225) Google Scholar, 20Wolinski H. Kolb D. Hermann S. Koning R.I. Kohlwein S.D. A role for seipin in lipid droplet dynamics and inheritance in yeast.J. Cell Sci. 2011; 124: 3894-3904Crossref PubMed Scopus (100) Google Scholar, 21Grippa A. Buxó L. Mora G. Funaya C. Idrissi F-Z. Mancuso F. Gomez R. Muntanyà J. Sabidó E. Carvalho P. The seipin complex Fld1/Ldb16 stabilizes ER-lipid droplet contact sites.J. Cell Biol. 2015; 211: 829-844Crossref PubMed Google Scholar), and the activation and membrane recruitment of Pah1 takes place in the vicinity of LDs, suggesting a channeling mechanism of DAG delivery for TAG synthesis and sorting into LDs (22Adeyo O. Horn P.J. Lee S. Binns D.D. Chandrahas A. Chapman K.D. Goodman J.M. The yeast lipin orthologue Pah1p is important for biogenesis of lipid droplets.J. Cell Biol. 2011; 192: 1043-1055Crossref PubMed Scopus (166) Google Scholar, 23Karanasios E. Barbosa A.D. Sembongi H. Mari M. Han G-S. Reggiori F. Carman G.M. Siniossoglou S. 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Metabolism and regulation of glycerolipids in the yeast Saccharomyces cerevisiae.Genetics. 2012; 190: 317-349Crossref PubMed Scopus (302) Google Scholar, 10Pascual F. Carman G.M. Phosphatidate phosphatase, a key regulator of lipid homeostasis.Biochim. Biophys. Acta. 2013; 1831: 514-522Crossref PubMed Scopus (99) Google Scholar, 26Kohlwein S.D. Veenhuis M. van der Klei I.J. Lipid droplets and peroxisomes: key players in cellular lipid homeostasis or a matter of fat–store 'em up or burn 'em down.Genetics. 2013; 193: 1-50Crossref PubMed Scopus (138) Google Scholar, 27Wang C-W. Lipid droplet dynamics in budding yeast.Cell. Mol. Life Sci. 2015; 72: 2677-2695Crossref PubMed Scopus (37) Google Scholar), to enable facile insight into the relevance of the research we review here. PA is synthesized in the ER where it is activated to CDP-DAG for subsequent synthesis of membrane lipids by the ER resident enzyme, Cds1 (Fig. 1). The high energy CDP-DAG is consumed at the ER for the synthesis of phosphatidylinositol (PI) catalyzed by the PI synthase, Pis1, and phosphatidylserine (PS) catalyzed by the PS synthase, Cho1. PS, in turn, generates phosphatidylethanolamine (PE) by decarboxylation catalyzed primarily by mitochondrially-localized Psd1, with a small contribution from Golgi/endosome localized Psd2. PE is subsequently converted into phosphatidylcholine (PC) by three successive methylations that take place in the ER. The first methylation is carried out by Cho2 and the second and third by Opi3. PA is also transported to the mitochondria where it is converted to CDP-DAG by the activity of the mitochondrial CDP-DAG synthase, Tam41 (28Tamura Y. Harada Y. Nishikawa S. Yamano K. Kamiya M. Shiota T. Kuroda T. Kuge O. Sesaki H. Imai K. et al.Tam41 is a CDP-diacylglycerol synthase required for cardiolipin biosynthesis in mitochondria.Cell Metab. 2013; 17: 709-718Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), for subsequent synthesis of the mitochondrial-specific lipids, phosphatidylglycerol (PG) by Pgs1/Gep4 and cardiolipin (CL) by Crd1 (Fig. 1) (29Horvath S.E. Daum G. Lipids of mitochondria.Prog. Lipid Res. 2013; 52: 590-614Crossref PubMed Scopus (398) Google Scholar, 30Scharwey M. Tatsuta T. Langer T. Mitochondrial lipid transport at a glance.J. Cell Sci. 2013; 126: 5317-5323Crossref PubMed Scopus (38) Google Scholar, 31Tamura Y. Sesaki H. Endo T. Phospholipid transport via mitochondria.Traffic. 2014; 15: 933-945Crossref PubMed Scopus (47) Google Scholar). The PL, PA functions at a critical branching point in lipid metabolism where lipid biosynthetic flux can be directed into membrane PL synthesis concomitant with membrane proliferation, or into TAG synthesis and LD formation (Fig. 1). To generate TAG, PA is dephosphorylated at the ER to form DAG by the PA phosphatase, Pah1 (9Han G-S. Wu W-I. Carman G.M. The Saccharomyces cerevisiae Lipin homolog is a Mg2+-dependent phosphatidate phosphatase enzyme.J. Biol. Chem. 2006; 281: 9210-9218Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar). DAG is acylated by the acyltransferases, Dga1 and Lro1, to produce TAG for accumulation in LDs (32Oelkers P. Tinkelenberg A. Erdeniz N. Cromley D. Billheimer J.T. Sturley S.L. A lecithin cholesterol acyltransferase-like gene mediates diacylglycerol esterification in yeast.J. Biol. Chem. 2000; 275: 15609-15612Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 33Oelkers P. Cromley D. Padamsee M. Billheimer J.T. Sturley S.L. The DGA1 gene determines a second triglyceride synthetic pathway in yeast.J. Biol. Chem. 2002; 277: 8877-8881Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 34Sorger D. Daum G. Synthesis of triacylglycerols by the acyl-coenzyme A:diacyl-glycerol acyltransferase Dga1p in lipid particles of the yeast Saccharomyces cerevisiae.J. Bacteriol. 2002; 184: 519-524Crossref PubMed Google Scholar, 35Sorger D. Daum G. Triacylglycerol biosynthesis in yeast.Appl. Microbiol. Biotechnol. 2003; 61: 289-299Crossref PubMed Google Scholar). DAG can also be phosphorylated back to PA by the ER-localized lipid kinase, Dgk1 (36Han G-S. O'Hara L. Carman G.M. Siniossoglou S. An unconventional diacylglycerol kinase that regulates phospholipid synthesis and nuclear membrane growth.J. Biol. Chem. 2008; 283: 20433-20442Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 37Han G-S. O'Hara L. Siniossoglou S. Carman G.M. Characterization of the yeast DGK1-encoded CTP-dependent diacylglycerol kinase.J. Biol. Chem. 2008; 283: 20443-20453Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). There are three other PA phosphatases in yeast, Dpp1, Lpp1, and App1, which, together with Pah1, constitute the whole measurable PA phosphatase complement in yeast extracts (38Chae M. Han G-S. Carman G.M. The Saccharomyces cerevisiae actin patch protein App1p is a phosphatidate phosphatase enzyme.J. Biol. Chem. 2012; 287: 40186-40196Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Dpp1, Lpp1, and App1 are not involved in diverting PA from PL synthesis to generate DAG for TAG synthesis, as the DAG, TAG, and PL relative composition of the triple dpp1Δ lpp1Δ app1Δ mutant do not differ appreciably from wild-type cells (38Chae M. Han G-S. Carman G.M. The Saccharomyces cerevisiae actin patch protein App1p is a phosphatidate phosphatase enzyme.J. Biol. Chem. 2012; 287: 40186-40196Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Instead, Dpp1, Lpp1, and App1 are thought to be involved in lipid signaling or membrane structural/curvature changes associated with vesicular trafficking (10Pascual F. Carman G.M. Phosphatidate phosphatase, a key regulator of lipid homeostasis.Biochim. Biophys. Acta. 2013; 1831: 514-522Crossref PubMed Scopus (99) Google Scholar, 38Chae M. Han G-S. Carman G.M. The Saccharomyces cerevisiae actin patch protein App1p is a phosphatidate phosphatase enzyme.J. Biol. Chem. 2012; 287: 40186-40196Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). The physiological relevance of Pah1 has been uncovered by studying pah1Δ yeast strains (9Han G-S. Wu W-I. Carman G.M. The Saccharomyces cerevisiae Lipin homolog is a Mg2+-dependent phosphatidate phosphatase enzyme.J. Biol. Chem. 2006; 281: 9210-9218Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar, 14Santos-Rosa H. Leung J. Grimsey N. Peak-Chew S. Siniossoglou S. The yeast lipin Smp2 couples phospholipid biosynthesis to nuclear membrane growth.EMBO J. 2005; 24: 1931-1941Crossref PubMed Scopus (264) Google Scholar, 22Adeyo O. Horn P.J. Lee S. Binns D.D. Chandrahas A. Chapman K.D. Goodman J.M. The yeast lipin orthologue Pah1p is important for biogenesis of lipid droplets.J. Cell Biol. 2011; 192: 1043-1055Crossref PubMed Scopus (166) Google Scholar, 39Fakas S. Qiu Y. Dixon J.L. Han G.S. Ruggles K.V. Garbarino J. Sturley S.L. Carman G.M. Phosphatidate phosphatase activity plays key role in protection against fatty acid-induced toxicity in yeast.J. Biol. Chem. 2011; 286: 29074-29085Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 40Sasser T. Qiu Q.S. Karunakaran S. Padolina M. Reyes A. Flood B. Smith S. Gonzales C. Fratti R.a. Yeast lipin 1 orthologue Pah1p regulates vacuole homeostasis and membrane fusion.J. Biol. Chem. 2012; 287: 2221-2236Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 41Park Y. Han G-S. Mileykovskaya E. Garrett T.A. Carman G.M. Altered lipid synthesis by lack of yeast Pah1 phosphatidate phosphatase reduces chronological life span.J. Biol. Chem. 2015; 290: 25382-25394Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). The absence of Pah1 leads to elevated levels of PA and other PLs, which is thought to cause the observed hyperproliferation of the nuclear ER (10Pascual F. Carman G.M. Phosphatidate phosphatase, a key regulator of lipid homeostasis.Biochim. Biophys. Acta. 2013; 1831: 514-522Crossref PubMed Scopus (99) Google Scholar, 11O'Hara L. Han G-S. Peak-Chew S. Grimsey N. Carman G.M. Siniossoglou S. Control of phospholipid synthesis by phosphorylation of the yeast lipin Pah1p/Smp2p Mg2+-dependent phosphatidate phosphatase.J. Biol. Chem. 2006; 281: 34537-34548Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 36Han G-S. O'Hara L. Carman G.M. Siniossoglou S. An unconventional diacylglycerol kinase that regulates phospholipid synthesis and nuclear membrane growth.J. Biol. Chem. 2008; 283: 20433-20442Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). This metabolic block also leads to reduced amounts of DAG and TAG, a reduced number of LDs, and lipotoxicity, as the reduced level of DAG observed in pah1Δ cells hampers TAG synthesis and the ability to cope with excess exogenous fatty acids (9Han G-S. Wu W-I. Carman G.M. The Saccharomyces cerevisiae Lipin homolog is a Mg2+-dependent phosphatidate phosphatase enzyme.J. Biol. Chem. 2006; 281: 9210-9218Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar, 22Adeyo O. Horn P.J. Lee S. Binns D.D. Chandrahas A. Chapman K.D. Goodman J.M. The yeast lipin orthologue Pah1p is important for biogenesis of lipid droplets.J. Cell Biol. 2011; 192: 1043-1055Crossref PubMed Scopus (166) Google Scholar, 39Fakas S. Qiu Y. Dixon J.L. Han G.S. Ruggles K.V. Garbarino J. Sturley S.L. Carman G.M. Phosphatidate phosphatase activity plays key role in protection against fatty acid-induced toxicity in yeast.J. Biol. Chem. 2011; 286: 29074-29085Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 42Han G-S. Siniossoglou S. Carman G.M. The cellular functions of the yeast lipin homolog PAH1p are dependent on its phosphatidate phosphatase activity.J. Biol. Chem. 2007; 282: 37026-37035Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Additional phenotypes of pah1Δ cells are slow growth, thermosensitivity, and an impairment of pah1Δ cells to grow in nonfermentable carbon sources. The inability to grow on nonfermentable carbon sources is not believed to be due to a respiratory defect, instead a reduction of ATP level observed in pah1Δ cells as a consequence of deregulated PL synthesis (which consumes ATP) was proposed as the limiting factor for growth (41Park Y. Han G-S. Mileykovskaya E. Garrett T.A. Carman G.M. Altered lipid synthesis by lack of yeast Pah1 phosphatidate phosphatase reduces chronological life span.J. Biol. Chem. 2015; 290: 25382-25394Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). In addition, pah1Δ cells exhibited an increased level of mitochondrial superoxides and a decreased tolerance to oxidative stress resulting in reduced chronological life span (41Park Y. Han G-S. Mileykovskaya E. Garrett T.A. Carman G.M. Altered lipid synthesis by lack of yeast Pah1 phosphatidate phosphatase reduces chronological life span.J. Biol. Chem. 2015; 290: 25382-25394Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Cells defective in Pah1 activity also displayed vacuolar fragmentation and decreased vacuolar fusion consistent with the observed reduction in vacuolar recruitment of several protein factors implicated in the vacuolar fusion process (40Sasser T. Qiu Q.S. Karunakaran S. Padolina M. Reyes A. Flood B. Smith S. Gonzales C. Fratti R.a. Yeast lipin 1 orthologue Pah1p regulates vacuole homeostasis and membrane fusion.J. Biol. Chem. 2012; 287: 2221-2236Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Of interest, Pah1 has also been associated with the vacuolar membrane and the conversion from PA to DAG was proposed as a critical event leading to vacuole fusion (40Sasser T. Qiu Q.S. Karunakaran S. Padolina M. Reyes A. Flood B. Smith S. Gonzales C. Fratti R.a. Yeast lipin 1 orthologue Pah1p regulates vacuole homeostasis and membrane fusion.J. Biol. Chem. 2012; 287: 2221-2236Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). PA can also be synthesized by Dgk1, an atypical DAG kinase that utilizes CTP for the synthesis of PA (37Han G-S. O'Hara L. Siniossoglou S. Carman G.M. Characterization of the yeast DGK1-encoded CTP-dependent diacylglycerol kinase.J. Biol. Chem. 2008; 283: 20443-20453Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) (Fig. 1). Dgk1 is an integral membrane protein localized at the ER/nuclear membrane. Dgk1 antagonizes Pah1 activity, and they constitute a counteracting pair that controls the levels of PA and DAG at the nuclear ER. Indeed, Dgk1 was identified in two screens for multicopy suppressors of the lethality that arises from deregulated Pah1 activity (36Han G-S. O'Hara L. Carman G.M. Siniossoglou S. An unconventional diacylglycerol kinase that regulates phospholipid synthesis and nuclear membrane growth.J. Biol. Chem. 2008; 283: 20433-20442Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Dgk1 overexpression leads to an increased level of PA, derepression of UASINO-containing genes (see subsequent section), and nuclear membrane proliferation, phenotypes also observed in pah1Δ cells. Inactivation of the DGK1 gene suppresses phenotypes elicited by Pah1 deficiency, including elevated PA level, abnormal nuclear/ER morphology, and derepressed UASINO gene transcription (36Han G-S. O'Hara L. Carman G.M. Siniossoglou S. An unconventional diacylglycerol kinase that regulates phospholipid synthesis and nuclear membrane growth.J. Biol. Chem. 2008; 283: 20433-20442Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). This interaction suggests that Dgk1 contributes to the PA pool that is a substrate for Pah1. In addition, the absence of Dgk1 restores LD formation to wild-type level in pah1Δ cells (22Adeyo O. Horn P.J. Lee S. Binns D.D. Chandrahas A. Chapman K.D. Goodman J.M. The yeast lipin orthologue Pah1p is important for biogenesis of lipid droplets.J. Cell Biol. 2011; 192: 1043-1055Crossref PubMed Scopus (166) Google Scholar, 39Fakas S. Qiu Y. Dixon J.L. Han G.S. Ruggles K.V. Garbarino J. Sturley S.L. Carman G.M. Phosphatidate phosphatase activity plays key role in protection against fatty acid-induced toxicity in yeast.J. Biol. Chem. 2011; 286: 29074-29085Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). The susceptibility of pah1Δ cells to oxidative stress and their inability to grow in nonfermentable carbon sources were partially suppressed by loss of Dgk1 (41Park Y. Han G-S. Mileykovskaya E. Garrett T.A. Carman G.M. Altered lipid synthesis by lack of yeast Pah1 phosphatidate phosphatase reduces chronological life span.J. Biol. Chem. 2015; 290: 25382-25394Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), while other phenotypes of pah1Δ cells, including decreased TAG synthesis and lipotoxicity, were not suppressed by Dgk1 deficiency (36Han G-S. O'Hara L. Carman G.M. Siniossoglou S. An unconventional diacylglycerol kinase that regulates phospholipid synthesis and nuclear membrane growth.J. Biol. Chem. 2008; 283: 20433-20442Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 39Fakas S. Qiu Y. Dixon J.L. Han G.S. Ruggles K.V. Garbarino J. Sturley S.L. Carman G.M. Phosphatidate phosphatase activity plays key role in protection against fatty acid-induced toxicity in yeast.J. Biol. Chem. 2011; 286: 29074-29085Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). This suggests that only a subset of the PA pool produced by Dgk1 overlaps with the function of that consumed by Pah1. In addition to its function as a central precursor for PL and TAG syntheses, the pool of PA localized at the ER plays a regulatory role in modulating the expression of genes required for PL and fatty acid synthesis primarily through repressing transcription from genes containing an UASINO promoter element (3Henry S.A. Patton-Vogt J.L. Genetic regulation of phospholipid metabolism: yeast as a model eukaryote.Prog. Nucleic Acid Res. Mol. Biol. 1998; 61: 133-179Crossref PubMed Google Scholar, 4Carman G.M. Henry S.A. Phosphatidic acid plays a central role in the transcriptional regulation of glycerophospholipid synthesis in Saccharomyces cerevisiae.J. Biol. Chem. 2007; 282: 37293-37297Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 43Loewen C.J.R. Gaspar M.L. Jesch S.A. Delon C. Ktistakis N.T. Henry S.A. Levine T.P. Phospholipid metabolism regulated by a transcription factor sensing phosphatidic acid.Science. 2004; 304: 1644-1647Crossref PubMed Scopus (329) Google Scholar) (Fig. 2). Opi1 is a transcriptional repressor that senses the level of PA at the ER (43Loewen C.J.R. Gaspar M.L. Jesch S.A. Delon C. Ktistakis N.T. Henry S.A. Levine T.P. Phospholipid metabolism regulated by a transcription factor sensing phosphatidic acid.Science. 2004; 304: 1644-1647Crossref PubMed Scopus (329) Google Scholar), and through the simultaneous interaction of PA and the integral ER protein, Scs2, Opi1 is bound to the ER and is inactive. A drop in PA level allows for Opi1 detachment from the ER membrane and translocation into the nucleus where it acts as a transcriptional repressor. The genes controlled by this PA-Opi1-regulated circuit contain the UASINO element in their promoters where the t