Control of Phospholipid Synthesis by Phosphorylation of the Yeast Lipin Pah1p/Smp2p Mg2+-dependent Phosphatidate Phosphatase

Abstract

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...

Insulin Controls Subcellular Localization and Multisite Phosphorylation of the Phosphatidic Acid Phosphatase, Lipin 1

“…None of the highly conserved sites were found to be phosphorylated in a recent analysis of phosphorylation sites of lipin (Pah1p) in S. cerevisiae (34). In this study, the PAP activity of Pah1p harboring Ala mutations in seven (Ser/Thr)-Pro phosphorylation sites exhibited enhanced PAP activity (34).…”

“…In this study, the PAP activity of Pah1p harboring Ala mutations in seven (Ser/Thr)-Pro phosphorylation sites exhibited enhanced PAP activity (34). Curiously, the large majority of phosphorylation sites in yeast Pah1p were found in a region of the protein lacking homology with lipins from other species (Fig.…”

“…In view of the findings that both yeast (34) and mammalian lipins (Fig. 4) are phosphorylated in several (Ser/Thr)-Pro phosphorylation sites, it is interesting to speculate that one or more of the protein kinases and/or phosphatases acting on lipin proteins in yeasts and mammals have been conserved.…”

Colorimetric determination of pure Mg2+-dependent phosphatidate phosphatase activity

“…However, this limitation is also a major benefit because the screen for inhibitors (or activators) should be carried out under well-defined conditions that are free from other reactions that might generate orthophosphate and interfere with the interpretation of results. Obtaining large quantities of pure PAP1 enzyme is facilitated by the overexpression and purification of yeast [13] and human proteins [6]. As advertised by commercial vendors of the malachite green-molybdate reagent, the PAP1 colorimetric assay was applicability to a 96-well format (data not shown), which should facilitate a large-scale screen of PAP1 inhibitors (or activators).…”

“…Saccharomyces cerevisiae PAH1-encoded PAP1 [6] was expressed and purified to homogeneity as described by O'Hara et al [13]. The yeast enzyme was used a model PAP1 to develop the colorimetric assay.…”