Remodelling of the nuclear membrane is essential for the dynamic changes of nuclear architecture at different stages of the cell cycle and during cell differentiation. The molecular mechanism underlying the regulation of nuclear membrane biogenesis is not known. Here we show that Smp2, the yeast homologue of mammalian lipin, is a key regulator of nuclear membrane growth during the cell cycle. Smp2 is phosphorylated by Cdc28/ Cdk1 and dephosphorylated by a nuclear/endoplasmic reticulum (ER) membrane-localized CPD phosphatase complex consisting of Nem1 and Spo7. Loss of either SMP2 or its dephosphorylated form causes transcriptional upregulation of key enzymes involved in lipid biosynthesis concurrent with a massive expansion of the nucleus. Conversely, constitutive dephosphorylation of Smp2 inhibits cell division. We show that Smp2 associates with the promoters of phospholipid biosynthetic enzymes in a Nem1-Spo7-dependent manner. Our data suggest that Smp2 is a critical factor in coordinating phospholipid biosynthesis at the nuclear/ER membrane with nuclear growth during the cell cycle.

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 pah1⌬ cells.Over the years there has been significant progress in understanding the mechanisms by which proteins are targeted and assembled into the various intracellular compartments. Despite this, little is still known about how eukaryotic cells regulate the growth of membrane-bound organelles. Homeostatic mechanisms must be in place to ensure that organelles grow in size or in number before cell division. Similar mechanisms must operate during development when certain organelles undergo dramatic morphological changes to perform their specialized function in differentiated tissues (1, 2).A defining organelle of eukaryotic cells is the nucleus. The intranuclear compartment is delimited by the nuclear envelope and consists of a double lipid bilayer, the outer and the inner nuclear membrane (3). The outer nuclear membrane is physically and functionally linked to the endoplasmic reticulum (ER), 2 whereas the inner nuclear membrane faces the nucleoplasm and in metazoans is covered by the nuclear lamina. Dynamic changes in the structure of the nuclear envelope are essential for the proper execution of nuclear division in all eukaryotes. In metazoan cells the nuclear envelope breaks down during mitosis, whereas yeast undergo "closed mitosis," where the spindle separates the chromosomes within the confines of an intact nucleus that partitions between mother and daughter cell (4).How the membrane is targeted and incorporated into the nuclear envelope and how the nucleus expands to accommodate changes in chromatin condensation and content during the cell cycle are fascinating but still unanswered questions. Cell-free assays suggest that nuclear membrane expansion and nuclear growth takes place via homotypic fusion of vesicles with the outer nuclear membrane (3, 5-7). It is not clear however whether nuclear growth in vivo depends on vesicle fusion. Because the outer nuclear membrane is continuous with the ER, the major site of phospholipid biosynthesis in eukaryotic cells, an alternative possibility is that nuclear growth results from lateral flow of ER membranes into the nuclear envelope.Phospholipid homeostasis in yeast is regulated primarily by the concen...

contributed equally to this work Two membrane proteins were identified through their genetic interaction with the nucleoporin Nup84p and shown to participate in nuclear envelope morphogenesis in yeast. One component is a known sporulation factor Spo7p, and the other, Nem1p, a novel protein whose C-terminal domain is conserved during eukaryotic evolution. Spo7p and Nem1p localize to the nuclear/ER membrane and behave biochemically as integral membrane proteins. Nem1p binds to Spo7p via its conserved C-terminal domain. Although cells without Spo7p or Nem1p are viable, they exhibit a drastically altered nuclear morphology with long, porecontaining double nuclear membrane extensions. These protrusions emanate from a core nucleus which contains the DNA, and penetrate deeply into the cytoplasm. Interestingly, not only Spo7 -and Nem1 -, but also several nucleoporin mutants are defective in sporulation. Thus, Spo7p and Nem1p, which exhibit a strong genetic link to nucleoporins of the Nup84p complex, fulfil an essential role in formation of a spherical nucleus and meiotic division.

The Saccharomyces cerevisiae PAH1-encoded Mg 2؉ -dependent phosphatidate phosphatase (PAP1, 3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) catalyzes the dephosphorylation of phosphatidate to yield diacylglycerol and P i . 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 (Gly 80 ) 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 pah1⌬ 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.The PAH1 gene (previously known as SMP2) in the yeast Saccharomyces cerevisiae encodes Mg 2ϩ -dependent PA 2 phosphatase (PAP1, 3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) (1). PAP1 catalyzes the dephosphorylation of PA yielding DAG and P i (2). This enzyme generates the DAG used for the synthesis of TAG (1) and may generate the DAG used for the synthesis of PE and PC via the Kennedy pathway ( Fig. 1) (3). PAH1-encoded PAP1 also controls the cellular concentration of its substrate PA (1), which is the precursor for phospholipids that are synthesized via the CDP-DAG pathway (Fig. 1) (4, 5).The activity of the PAH1-encoded PAP1 enzyme is regulated by lipids (6, 7) and nucleotides (8) and by the covalent modification of phosphorylation (9, 10). PAP1 activity is enhanced by CDP-DAG, PI, and cardiolipin (6), whereas activity is inhibited by the sphingoid bases phytosphingosine and sphinganine (7), and by the nucleotides ATP and CTP (8). Pah1p 3 is phosphorylated by cyclin-dependent Cdc28p kinase and dephosphorylated by the Nem1p-Spo7p phosphatase complex (9). A phosphorylation-deficient Pah1p mutant exhibits elevated PAP1 activity (10) indicating that phosphorylation inhibits PAP1 activity. The regulation of PAP1 activity by these factors is thought to be part of a complex mechanism by which cells coordinate the synthesis of phospholipids via the CDP-DAG and Kennedy pathways and the synthesis of TAG (3, 4).PAH1 is a gene whose mutation results in increased plasmid maintenance and causes slow growth, temperature sensitivity, and respiratory deficiency (11). Pah1p is the yeast homolog of the mammalian fat-regulating protein lipin ...

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.L ipids play multiple key roles in membrane biogenesis, in signaling cascades, or in energy metabolism. These pathways depend largely on the regulated activation and recruitment of enzymes that respond to changes in membrane lipid composition. Lipins define a unique family of phosphatidate phosphatase (PAP) enzymes, conserved from yeasts to mammals, that catalyze a fundamental reaction in lipid and membrane biogenesis: the dephosphorylation of phosphatidate (PA) to diacylglycerol (DAG) (1), which is then acylated to produce triacylglycerol (TAG), the major form of fat stored in lipid droplets. In addition, both PA and DAG are intermediates for the biosynthesis of membrane phospholipids (Fig. 1A) (2, 3).Consistent with these key functions, recent studies have implicated lipins in a variety of processes in different systems. In budding yeast, the single lipin orthologue Pah1p regulates phospholipid and TAG content (1) as well as the transcription of many genes encoding lipid biosynthetic enzymes (4). Mammals express three paralogues called lipin 1, 2, and 3 that exhibit distinct but overlapping expression in many tissues (5). In mice, lipin 1 deficiency causes lipodystrophy, characterized by significant reduction in fat mass and lack of adipocyte differentiation (6), whereas overexpression of lipin 1 promotes obesity (7). Interestingly, lipins are also required for nuclear/endoplasmic reticulum (ER) membrane organization, both in fission and budding yeast (4,8) and Caenorhabditis elegans (9, 10).Surprisingly, in contrast to most other lipid biosynthetic enzymes, lipins lack transmembrane domains and therefore must first translocate onto membranes in order to dephosphorylate PA. How their activity is regulated in response to signals that modulate membrane biogenesis or energy storage is poorly understood. We show here that elevated PA levels recruit the yeast Pah1p onto a nuclear membrane s...

Changes in nuclear size and shape during the cell cycle or during development require coordinated nuclear membrane remodeling, but the underlying molecular events are largely unknown. We have shown previously that the activity of the conserved phosphatidate phosphatase Pah1p/Smp2p regulates nuclear structure in yeast by controlling phospholipid synthesis and membrane biogenesis at the nuclear envelope. Two screens for novel regulators of phosphatidate led to the identification of DGK1. We show that Dgk1p is a unique diacylglycerol kinase that uses CTP, instead of ATP, to generate phosphatidate. DGK1 counteracts the activity of PAH1 at the nuclear envelope by controlling phosphatidate levels. Overexpression of DGK1 causes the appearance of phosphatidate-enriched membranes around the nucleus and leads to its expansion, without proliferating the cortical endoplasmic reticulum membrane. Mutations that decrease phosphatidate levels decrease nuclear membrane growth in pah1⌬ cells. We propose that phosphatidate metabolism is a critical factor determining nuclear structure by regulating nuclear membrane biogenesis.Phospholipids are the major cellular components required for the assembly of biological membranes (1). The regulated production and distribution of phospholipids during the cell cycle or during development often underlie striking changes in membrane biogenesis, which, in turn, can impact on the size, shape, or number of organelles. For example, stimulation of phospholipid biosynthesis accompanies the expansion of the ER 3 in professional secretory cells (2) or neurite growth during neuronal differentiation (3). Despite these interesting observations, the molecular mechanisms responsible for coupling lipid production to organelle morphology remain largely unknown.An organelle that undergoes striking structural changes during the cell cycle is the nucleus. The nucleus is delimited by the nuclear envelope, which consists of a double lipid bilayer, the outer and inner nuclear membranes (4). The outer membrane is continuous with the ER, whereas the inner membrane faces the nucleoplasm and binds to chromatin. Nuclear membrane growth is essential for cell division. In yeast, nuclear membrane expansion allows anaphase to take place within a single nuclear compartment that partitions between mother and daughter cells (5). In metazoan cells, the nuclear membrane expands at the end of mitosis to accommodate chromatin decondensation and DNA replication (6). The source of the nuclear membrane and the mechanism by which it is added to the nuclear envelope remain unknown. Nuclear envelope remodeling is also important for cell types that undergo dramatic nuclear structure changes during their differentiation, such as mammalian blood cell types, where nuclei can be highly lobed and segmented, or spermatocytes and myocytes, where nuclei can take very elongated morphologies (7). The importance of proper nuclear envelope structure in cell physiology is underscored by the recent identification of several diseases that are associat...

Membrane traffic requires vesicles to fuse with a specific target, and SNARE proteins and Rab/Ypt GTPases contribute to this specificity. In the yeast Saccharomyces cerevisae, the Rab/Ypt GTPase Ypt6p is required for fusion of endosome‐derived vesicles with the late Golgi. We have shown previously that activation of Ypt6p depends on its exchange factor, Ric1p–Rgp1p, a peripheral membrane protein complex restricted to the Golgi. We show here that a conserved trimeric protein complex, VFT (Vps52/53/54), binds directly to Ypt6p:GTP. Localization of VFT to the Golgi requires Ypt6p, but is unaffected in gos1 and tlg1 mutants, in which late Golgi integral membrane proteins, including SNAREs, are mislocalized. The VFT complex also binds directly to the N‐terminal domain of the SNARE Tlg1p, both in vitro and in vivo, in a Ypt6p‐independent manner. We suggest that the VFT complex links vesicles containing Tlg1p to their target, which is defined by the local activation of Ypt6p.

Cells lacking the GTPase Ypt6p have defects in intracellular traffic and are temperature sensitive. Their growth is severely impaired by additional mutation of IMH1, which encodes a non‐essential Golgi‐associated coiled‐coil protein. A screen for mutants that, like ypt6, specifically impair the growth of imh1 cells led to the identification of RIC1. Ric1p forms a tight complex with a previously uncharacterized protein, Rgp1p. The Ric1p–Rgp1p complex binds Ypt6p in a nucleotide‐dependent manner, and purified Ric1p–Rgp1 stimulates guanine nucleotide exchange on Ypt6p in vitro. Deletion of RIC1 or RGP1, like that of YPT6, blocks the recycling of the exocytic SNARE Snc1p from early endosomes to the Golgi and causes temperature‐sensitive growth, but this defect can be relieved by overexpression of YPT6. Ric1p largely colocalizes with the late Golgi marker Sec7p. Ypt6p shows a similar distribution, but this is altered when RIC1 or RGP1 is mutated. We infer that the Ric1p–Rgp1p complex serves to activate Ypt6p on Golgi membranes by nucleotide exchange, and that this is required for efficient fusion of endosome‐derived vesicles with the Golgi.