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...

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...

Lipins are the founding members of a novel family of Mg 2؉ -dependent phosphatidate phosphatases (PAP1 enzymes) that play key roles in fat metabolism and lipid biosynthesis. Despite their importance, there is still little information on how their activity is regulated. Here we demonstrate that the functions of lipin 1 and 2 are evolutionarily conserved from unicellular eukaryotes to mammals. The two lipins display distinct intracellular localization in HeLa M cells, with a pool of lipin 2 exhibiting a tight membrane association. Small interfering RNA-mediated silencing of lipin 1 leads to a dramatic decrease of the cellular PAP1 activity in HeLa M cells, whereas silencing of lipin 2 leads to an increase of lipin 1 levels and PAP1 activity. Consistent with their distinct functions in HeLa M cells, lipin 1 and 2 exhibit reciprocal patterns of protein expression in differentiating 3T3-L1 adipocytes. Lipin 2 levels increase in lipin 1-depleted 3T3-L1 cells without rescuing the adipogenic defects, whereas depletion of lipin 2 does not inhibit adipogenesis. Finally, we show that the PAP1 activity of both lipins is inhibited by phosphorylation during mitosis, leading to a decrease in the cellular PAP1 activity during cell division. We propose that distinct and non-redundant functions of lipin 1 and 2 regulate lipid production during the cell cycle and adipocyte differentiation.The phospholipid composition of biological membranes is crucial for many aspects of cell physiology, including growth, differentiation, and transport (1, 2). Phospholipids are also active participants of signaling cascades that control diverse cellular functions (3). Two key precursors of phospholipid biosynthesis are PA 3 and DAG, both of which have essential functions in signaling cascades, energy storage, and lipid biosynthetic pathways. Early biochemical studies identified a soluble Mg 2ϩ -dependent PA phosphatase (PAP1) activity that is key to catalyzing PA conversion to DAG (4 -6). DAG has multiple functions. First, DAG is used for the synthesis of the most abundant phospholipids found in biological membranes, phosphatidylcholine, and phosphatidylethanolamine (6). Second, DAG is also used for the synthesis of the neutral lipid triacylglycerol, an essential storage form of energy and fatty acids, which accumulates as lipid droplets in adipocytes (7,8). Despite the key role of the PAP1 reaction, the identity of the enzyme(s) responsible for this activity remained unknown until recently. Han et al. (9) have demonstrated that Pah1p/Smp2p is the PAP1 enzyme controlling biosynthetic production of phospholipids and triacylglycerol in yeast. A separate study found that Pah1p regulates transcription of many phospholipid biosynthetic enzymes, nuclear/ER membrane biogenesis, and nuclear structure (10).Mammals express three Pah1p-related proteins called lipins 1, 2, and 3 (11). Two closely related lipin 1 isoforms have been described, the ␣ form and the ␤ form, which contains a short insertion close to its N-terminal end (12). All members of the Pah1...

Fetal growth plays a role in programming of adult cardiometabolic disorders, which in men, are associated with lowered testosterone levels. Fetal growth and fetal androgen exposure can also predetermine testosterone levels in men, although how is unknown, because the adult Leydig cells (ALCs) that produce testosterone do not differentiate until puberty. To explain this conundrum, we hypothesized that stem cells for ALCs must be present in the fetal testis and might be susceptible to programming by fetal androgen exposure during masculinization. To address this hypothesis, we used ALC ablation/regeneration to identify that, in rats, ALCs derive from stem/progenitor cells that express chicken ovalbumin upstream promoter transcription factor II. These stem cells are abundant in the fetal testis of humans and rodents, and lineage tracing in mice shows that they develop into ALCs. The stem cells also express androgen receptors (ARs). Reduction in fetal androgen action through AR KO in mice or dibutyl phthalate (DBP) -induced reduction in intratesticular testosterone in rats reduced ALC stem cell number by ∼40% at birth to adulthood and induced compensated ALC failure (low/normal testosterone and elevated luteinizing hormone). In DBP-exposed males, this failure was probably explained by reduced testicular steroidogenic acute regulatory protein expression, which is associated with increased histone methylation (H3K27me3) in the proximal promoter. Accordingly, ALCs and ALC stem cells immunoexpressed increased H3K27me3, a change that was also evident in ALC stem cells in fetal testes. These studies highlight how a key component of male reproductive development can fundamentally reprogram adult hormone production (through an epigenetic change), which might affect lifetime disease risk.adult Leydig stem/progenitor cells | compensated Leydig cell failure | GATA4 | ethane dimethane sulfonate

A fully functional central and peripheral circadian clock is not essential for reproduction and development to term, but has critical roles peri-natally and post-partum.

Androgens such as testosterone are steroid hormones essential for normal male reproductive development and function. Mutations of androgen receptors (AR) are often found in patients with disorders of male reproductive development, and milder mutations may be responsible for some cases of male infertility. Androgens exert their action through AR and its signalling in the testis is essential for spermatogenesis. AR is not expressed in the developing germ cell lineage so is thought to exert its effects through testicular Sertoli and peri-tubular myoid (PTM) cells. AR signalling in spermatogenesis has been investigated in rodent models where testosterone levels are chemically supressed or models with transgenic disruption of AR. These models have pinpointed the steps of spermatogenesis that require AR signalling, specifically maintenance of spermatogonial numbers, blood-testis barrier integrity, completion of meiosis, adhesion of spermatids and spermiation, together these studies detail the essential nature of androgens in the promotion of male fertility.

Sertoli cells (SCs) regulate testicular fate in the differentiating gonad and are the main regulators of spermatogenesis in the adult testis; however, their role during the intervening period of testis development, in particular during adult Leydig cell (ALC) differentiation and function, remains largely unknown. To examine SC function during fetal and prepubertal development we generated two transgenic mouse models that permit controlled, cell-specific ablation of SCs in pre-and postnatal life. Results show that SCs are required: (1) to maintain the differentiated phenotype of peritubular myoid cells (PTMCs) in prepubertal life; (2) to maintain the ALC progenitor population in the postnatal testis; and (3) for development of normal ALC numbers. Furthermore, our data show that fetal LCs function independently from SC, germ cell or PTMC support in the prepubertal testis. Together, these findings reveal that SCs remain essential regulators of testis development long after the period of sex determination. These findings have significant implications for our understanding of male reproductive disorders and wider androgen-related conditions affecting male health.

The Saccharomyces cerevisiae DGK1 gene encodes a diacylglycerol kinase enzyme that catalyzes the formation of phosphatidate from diacylglycerol. Unlike the diacylglycerol kinases from bacteria, plants, and animals, the yeast enzyme utilizes CTP, instead of ATP, as the phosphate donor in the reaction. Dgk1p contains a CTP transferase domain that is present in the SEC59-encoded dolichol kinase and CDS1-encoded CDP-diacylglycerol synthase enzymes. Deletion analysis showed that the CTP transferase domain was sufficient for diacylglycerol kinase activity. Point mutations (R76A, K77A, D177A, and G184A) of conserved residues within the CTP transferase domain caused a loss of diacylglycerol kinase activity. Analysis of DGK1 alleles showed that the in vivo functions of Dgk1p were specifically due to its diacylglycerol kinase activity. The DGK1-encoded enzyme had a pH optimum at 7.0 -7.5, required Ca 2؉ or Mg 2؉ ions for activity, was potently inhibited by N-ethylmaleimide, and was labile at temperatures above 40°C. The enzyme exhibited positive cooperative (Hill number ‫؍‬ 2.5) kinetics with respect to diacylglycerol (apparent K m ‫؍‬ 6.5 mol %) and saturation kinetics with respect to CTP (apparent K m ‫؍‬ 0.3 mM). dCTP was both a substrate (apparent K m ‫؍‬ 0.4 mM) and competitive inhibitor (apparent K i ‫؍‬ 0.4 mM) of the enzyme. Diacylglycerol kinase activity was stimulated by major membrane phospholipids and was inhibited by CDP-diacylglycerol and sphingoid bases.In the yeast Saccharomyces cerevisiae, PA 2 is an important phospholipid intermediate in the synthesis of membrane phospholipids and the neutral lipid triacylglycerol (see Fig. 1) (1-5). The major phospholipids phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine are derived from PA via the liponucleotide intermediate CDP-DAG (1-3). The mitochondrial phospholipids phosphatidylglycerol and cardiolipin are similarly derived from PA via CDP-DAG (not shown in Fig. 1) (6). Alternatively, phosphatidylethanolamine and phosphatidylcholine may be derived from PA via DAG, which is also utilized for the synthesis of the storage lipid triacylglycerol (1-3, 5). In the de novo biosynthetic pathway, PA is made from lyso-PA that is derived from either glycerol 3-phosphate or dihydroxyacetone phosphate (1-4). Besides the de novo pathway, PA is produced from the phospholipase D-mediated turnover of phosphatidylethanolamine and phosphatidylcholine (1-4). In bacteria, plants, and animals, PA may be produced from DAG via an ATP-dependent DAG kinase reaction (7-10). However, an enzyme catalyzing this reaction has not been identified from S. cerevisiae, and there are no yeast genes that encode a homologous protein in the superfamily of DAG kinase enzymes from bacteria, plants, and animals.In addition to its role as an intermediate of lipid metabolism, PA plays a central role in the transcriptional regulation of phospholipid synthesis in S. cerevisiae (1). PA, along with the Scs2p protein at the nuclear/ER membrane, binds and inactivates the t...