George M. Carman

The Saccharomyces cerevisiae Lipin Homolog Is a Mg2+-dependent Phosphatidate Phosphatase Enzyme

Mg2+-dependent phosphatidate (PA) phosphatase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) catalyzes the dephosphorylation of PA to yield diacylglycerol and Pi. In this work, we identified the Saccharomyces cerevisiae PAH1 (previously known as SMP2) gene that encodes Mg2+-dependent PA phosphatase using amino acid sequence information derived from a purified preparation of the enzyme (Lin, Y.-P., and Carman, G. M. (1989) J. Biol. Chem. 264, 8641–8645). Overexpression of PAH1 in S. cerevisiae directed elevated levels of Mg2+-dependent PA phosphatase activity, whereas the pah1Δ mutation caused reduced levels of enzyme activity. Heterologous expression of PAH1 in Escherichia coli confirmed that Pah1p is a Mg2+-dependent PA phosphatase enzyme and showed that its enzymological properties were very similar to those of the enzyme purified from S. cerevisiae. The PAH1-encoded enzyme activity was associated with both the membrane and cytosolic fractions of the cell, and the membrane-bound form of the enzyme was salt-extractable. Lipid analysis showed that mutants lacking PAH1 accumulated PA and had reduced amounts of diacylglycerolanditsderivativetriacylglycerol.ThePAH1-encoded Mg2+-dependent PA phosphatase shows homology to mammalian lipin, a fat-regulating protein whose molecular function is unknown. Heterologous expression of human LPIN1 in E. coli showed that lipin 1 is also a Mg2+-dependent PA phosphatase enzyme.

Phospholipid biosynthesis in the yeast Saccharomyces cerevisiae and interrelationship with other metabolic processes.

In this review, we have discussed recent progress in the study of the regulation that controls phospholipid metabolism in S. cerevisiae. This regulation occurs on multiple levels and is tightly integrated with a large number of other cellular processes and related metabolic and signal transduction pathways. Progress in deciphering this complex regulation has been very rapid in the last few years, aided by the availability of the sequence of the entire Saccharomyces genome. The assignment of functions to the remaining unassigned open reading frames, as well as ascertainment of remaining gene-enzyme relationships in phospholipid biosynthesis in yeast, promises to provide detailed understanding of the genetic regulation of a crucial area of metabolism in a key eukaryotic model system. Since the processes of lipid metabolism, secretion, and signal transduction show fundamental similarities in all eukaryotes, the dissection of this regulation in yeast promises to have wide application to our understanding of metabolic control in all eukaryotes.

Metabolism and Regulation of Glycerolipids in the Yeast Saccharomyces cerevisiae

Due to its genetic tractability and increasing wealth of accessible data, the yeast Saccharomyces cerevisiae is a model system of choice for the study of the genetics, biochemistry, and cell biology of eukaryotic lipid metabolism. Glycerolipids (e.g., phospholipids and triacylglycerol) and their precursors are synthesized and metabolized by enzymes associated with the cytosol and membranous organelles, including endoplasmic reticulum, mitochondria, and lipid droplets. Genetic and biochemical analyses have revealed that glycerolipids play important roles in cell signaling, membrane trafficking, and anchoring of membrane proteins in addition to membrane structure. The expression of glycerolipid enzymes is controlled by a variety of conditions including growth stage and nutrient availability. Much of this regulation occurs at the transcriptional level and involves the Ino2–Ino4 activation complex and the Opi1 repressor, which interacts with Ino2 to attenuate transcriptional activation of UASINO-containing glycerolipid biosynthetic genes. Cellular levels of phosphatidic acid, precursor to all membrane phospholipids and the storage lipid triacylglycerol, regulates transcription of UASINO-containing genes by tethering Opi1 to the nuclear/endoplasmic reticulum membrane and controlling its translocation into the nucleus, a mechanism largely controlled by inositol availability. The transcriptional activator Zap1 controls the expression of some phospholipid synthesis genes in response to zinc availability. Regulatory mechanisms also include control of catalytic activity of glycerolipid enzymes by water-soluble precursors, products and lipids, and covalent modification of phosphorylation, while in vivo function of some enzymes is governed by their subcellular location. Genome-wide genetic analysis indicates coordinate regulation between glycerolipid metabolism and a broad spectrum of metabolic pathways.

Phosphatidic Acid Plays a Central Role in the Transcriptional Regulation of Glycerophospholipid Synthesis in Saccharomyces cerevisiae

In eukaryotic cells, PA2 is a central precursor for the synthesis of major glycerophospholipids, DAG and TAG, as well as a major signaling lipid. In mammalian cells, PA is implicated as an activator of cell growth and proliferation, vesicular trafficking, secretion, and endocytosis (1–7). In plants, PA is implicated in seed germination and response to stress induced by drought, salinity, and low temperature (3, 4). The signaling roles of PA in the yeast Saccharomyces cerevisiae have not received as much attention as they have in higher eukaryotic cells. However, it is known that PA production via phospholipase D-mediated turnover of PC is necessary for suppression of growth and membrane trafficking defects in mutants defective in Sec14p, an essential PI/PC-binding protein (8–10). In addition, PA production via phospholipase D is implicated in Spo20p-mediated fusion of vesicles with the pre-spore membrane during sporogenesis (11, 12). The best studied regulatory function of PA in S. cerevisiae is its role as a signaling molecule in the transcriptional regulation of glycerophospholipid synthesis itself, the major topic of this review. We will focus on the pathways generating and utilizing PA in yeast and the evidence that PA plays a central role in the transcriptional regulation of glycerophospholipid synthesis.

Regulation of Phospholipid Biosynthesis in the Yeast Saccharomyces cerevisiae

Phospholipids are key molecules that contribute to the structural definition of cells and that participate in the regulation of cellular processes. Phospholipid metabolism is a major activity that cells engage in throughout their growth. The yeast, Saccharomyces cerevisiae, serves as a model system in which to study the regulation of phospholipid synthesis and its regulation in eucaryotes. Its membranous organelles, the lipids that comprise these membranes, and the phospholipid biosynthetic pathways that generate these membranes typify eucaryotic cells (1, 2). Many of the structural genes encoding for the phospholipid biosynthetic enzymes have been cloned and characterized (Table I) (3–25), and a number of mutations in these genes have been isolated (3, 7–9, 11–13, 17, 23, 26–33). In addition, a number of the phospholipid biosynthetic enzymes have been purified and studied (Table I) (34–44). The characterization of the wild-type and mutant genes, as well as the gene products encoded by these alleles, has significantly advanced our understanding both of phospholipid biosynthesis and of its regulation. Results from these genetic, molecular, and biochemical studies have shown that the regulation of phospholipid synthesis is a complex, highly coordinated process. The mechanisms that govern this regulation mediate the mRNA and protein levels of the biosynthetic enzymes as well as their activity and localization (1, 2, 45). This review summarizes our current understanding of the regulation of phospholipid metabolism in S. cerevisiae with a particular focus on the regulation of the activity of the biosynthetic enzymes. For more comprehensive reviews, the reader is directed to recent articles by Paltauf et al. (2) and Greenberg and Lopes (45).

Control of phospholipid synthesis by phosphorylation of the yeast lipin Pah1p/Smp2p Mg2+-dependent phosphatidate phosphatase.

Phosphorylation of the conserved lipin Pah1p/Smp2p in Saccharomyces cerevisiae was previously shown to control transcription of phospholipid biosynthetic genes and nuclear structure by regulating the amount of membrane present at the nuclear envelope (Santos-Rosa, H., Leung, J., Grimsey, N., Peak-Chew, S., and Siniossoglou, S. (2005) EMBO J. 24, 1931-1941). A recent report identified Pah1p as a Mg2+-dependent phosphatidate (PA) phosphatase that regulates de novo lipid synthesis (Han G.-S., Wu, W. I., and Carman, G. M. (2006) J. Biol. Chem. 281, 9210-9218). In this work we use a combination of mass spectrometry and systematic mutagenesis to identify seven Ser/Thr-Pro motifs within Pah1p that are phosphorylated in vivo. We show that phosphorylation on these sites is required for the efficient transcriptional derepression of key enzymes involved in phospholipid biosynthesis. The phosphorylation-deficient Pah1p exhibits higher PA phosphatase-specific activity than the wild-type Pah1p, indicating that phosphorylation of Pah1p controls PA production. Opi1p is a transcriptional repressor of phospholipid biosynthetic genes, responding to PA levels. Genetic analysis suggests that Pah1p regulates transcription of these genes through both Opi1p-dependent and -independent mechanisms. We also provide evidence that derepression of phospholipid biosynthetic genes is not sufficient to induce the nuclear membrane expansion shown in the pah1Δ cells.

Regulation of Phospholipid Synthesis in the Yeast Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae, with its full complement of organelles, synthesizes membrane phospholipids by pathways that are generally common to those found in higher eukaryotes. Phospholipid synthesis in yeast is regulated in response to a variety of growth conditions (e.g., inositol supplementation, zinc depletion, and growth stage) by a coordination of genetic (e.g., transcriptional activation and repression) and biochemical (e.g., activity modulation and localization) mechanisms. Phosphatidate (PA), whose cellular levels are controlled by the activities of key phospholipid synthesis enzymes, plays a central role in the transcriptional regulation of phospholipid synthesis genes. In addition to the regulation of gene expression, phosphorylation of key phospholipid synthesis catalytic and regulatory proteins controls the metabolism of phospholipid precursors and products.

Phosphatidic Acid Phosphatase, a Key Enzyme in the Regulation of Lipid Synthesis

PA2 phosphatase (PAP3; 3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) catalyzes the Mg2+-dependent dephosphorylation of PA, yielding DAG and Pi (Fig. 1) (1–3). In de novo lipid synthesis, the DAG generated from the PAP reaction is utilized for the synthesis of the phospholipids PE and PC via the Kennedy pathway and for the synthesis of TAG (Fig. 1) (3). The enzyme substrate PA is also used for phospholipid synthesis via CDP-DAG (Fig. 1) (1–3). In lipid signaling, PAP generates a pool of DAG used for protein kinase C activation, and by the nature of its reaction, it attenuates the signaling functions of PA (Fig. 1) (4–8). Thus, the regulation of PAP activity may govern the pathways by which phospholipids are synthesized and control the cellular contents of important signaling lipids. Recent genetic and biochemical studies in yeast and mammalian cells have revealed key roles that PAP plays in lipid metabolism and cell physiology. FIGURE 1. Roles of PAP in lipid synthesis and signaling in yeast. The reaction catalyzed by PAH1-encoded PAP (highlighted in yellow) is shown. Regulation of PAP activity plays a role in governing whether cells synthesize membrane phospholipids via CDP-DAG or ...

The Cellular Functions of the Yeast Lipin Homolog Pah1p Are Dependent on Its Phosphatidate Phosphatase Activity

The Saccharomyces cerevisiae PAH1-encoded Mg2+-dependent phosphatidate phosphatase (PAP1, 3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) catalyzes the dephosphorylation of phosphatidate to yield diacylglycerol and Pi. This enzyme plays a major role in the synthesis of triacylglycerols and phospholipids in S. cerevisiae. PAP1 contains the DXDX(T/V) catalytic motif (DIDGT at residues 398-402) that is shared by the mammalian fat-regulating protein lipin 1 and the superfamily of haloacid dehalogenase-like proteins. The yeast enzyme also contains a conserved glycine residue (Gly80) that is essential for the fat-regulating function of lipin 1 in a mouse model. In this study, we examined the roles of the putative catalytic motif and the conserved glycine for PAP1 activity by a mutational analysis. The PAP1 activities of the D398E and D400E mutant enzymes were reduced by >99.9%, and the activity of the G80R mutant enzyme was reduced by 98%. The mutant PAH1 alleles whose products lacked PAP1 activity were nonfunctional in vivo and failed to complement the pah1Δ mutant phenotypes of temperature sensitivity, respiratory deficiency, nuclear/endoplasmic reticulum membrane expansion, derepression of INO1 expression, and alterations in lipid composition. These results demonstrated that the PAP1 activity of the PAH1 gene product is essential for its roles in lipid metabolism and cell physiology.

A phosphorylation-regulated amphipathic helix controls the membrane translocation and function of the yeast phosphatidate phosphatase

Regulation of membrane lipid composition is crucial for many aspects of cell growth and development. Lipins, a novel family of phosphatidate (PA) phosphatases that generate diacylglycerol (DAG) from PA, are emerging as essential regulators of fat metabolism, adipogenesis, and organelle biogenesis. The mechanisms that govern lipin translocation onto membranes are largely unknown. Here we show that recruitment of the yeast lipin (Pah1p) is regulated by PA levels onto the nuclear/endoplasmic reticulum (ER) membrane. Recruitment requires the transmembrane protein phosphatase complex Nem1p-Spo7p. Once dephosphorylated, Pah1p can bind to the nuclear/ER membrane independently of Nem1p-Spo7p via a short amino-terminal amphipathic helix. Dephosphorylation enhances the activity of Pah1p, both in vitro and in vivo, but only in the presence of a functional helix. The helix is required for both phospholipid and triacylglycerol biosynthesis. Our data suggest that dephosphorylation of Pah1p by the Nem1p-Spo7p complex enables the amphipathic helix to anchor Pah1p onto the nuclear/ER membrane allowing the production of DAG for lipid biosynthesis.