Abstract
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 pah1delta cells.
Highlights
Over the years there has been significant progress in understanding the mechanisms by which proteins are targeted and assembled into the various intracellular compartments
Identification of Phosphorylation Sites on Pah1p—To identify the amino acid residues phosphorylated in Pah1p, a Pah1p-PtA fusion expressed under the control of the endogenous PAH1 promoter from a centromeric plasmid was purified from extracts of pah1⌬ nem1⌬ spo7⌬ cells by IgG-Sepharose chromatography
We identified four phosphorylation sites matching the minimal Ser/Thr-Pro motif phosphorylated by cyclindependent kinases (Cdk): Ser-168, Ser-602, Ser-744, Ser-748 (Fig. 1A and Table 3)
Summary
Over the years there has been significant progress in understanding the mechanisms by which proteins are targeted and assembled into the various intracellular compartments. Little is still known about how eukaryotic cells regulate the growth of membrane-bound organelles. Cell-free assays suggest that nuclear membrane expansion and nuclear growth takes place via homotypic fusion of vesicles with the outer nuclear membrane [3, 5,6,7]. It is not clear 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. RS453 nem1::HIS3 spo7::HIS3 RS453 pah1::TRP1 ϩ YCplac111-LEU2-PAH1-PtA RS453 nem1::HIS3 spo7::HIS3 pah1::TRP1 ϩ YCplac111-LEU2-SMP2-PtA RS453 pah1::TRP1 ϩ pRS313-HIS3-PAH1-PtA ϩ YCplac111-LEU2-GAL1/10-NEM1 ϩ
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