The Saccharomyces cerevisiae PAH1-encoded phosphatidate phosphatase (PAP) catalyzes the penultimate step in the synthesis of triacylglycerol and plays a role in the transcriptional regulation of phospholipid synthesis genes. PAP is phosphorylated at multiple Ser and Thr residues and is dephosphorylated for in vivo function by the Nem1p-Spo7p protein phosphatase complex localized in the nuclear/endoplasmic reticulum membrane. In this work, we characterized seven previously identified phosphorylation sites of PAP that are within the Ser/Thr-Pro motif. When expressed on a low copy plasmid, wild type PAP could not complement the pah1⌬ mutant in the absence of the Nem1p-Spo7p complex. However, phosphorylation-deficient PAP (PAP-7A) containing alanine substitutions for the seven phosphorylation sites bypassed the requirement of the phosphatase complex and complemented the pah1⌬ nem1⌬ mutant phenotypes, such as temperature sensitivity, nuclear/endoplasmic reticulum membrane expansion, decreased triacylglycerol synthesis, and derepression of INO1 expression. Subcellular fractionation coupled with immunoblot analysis showed that PAP-7A was highly enriched in the membrane fraction. In fluorescence spectroscopy analysis, the PAP-7A showed tighter association with phospholipid vesicles than wild type PAP. Using site-directed mutagenesis of PAP, we identified Ser In the yeast Saccharomyces cerevisiae, the PAH1-encoded phosphatidate phosphatase (PAP) 2,3 catalyzes the dephosphorylation of PA, yielding DAG and P i (1, 2). This reaction is dependent on Mg 2ϩ ions and is based on a DXDX(T/V) catalytic motif within a haloacid dehalogenase-like domain in the enzyme (2-4). PAP is associated with the cytosolic and membrane fractions of the cell, and the association with the membrane is peripheral in nature (2). Chromatin immunoprecipitation analysis indicates that PAP is also localized in the nucleus (5). The DAG generated in the PAP reaction is used for the synthesis of TAG (2) and for the synthesis of phosphatidylethanolamine and phosphatidylcholine via the CDPethanolamine and CDP-choline branches, respectively, of the Kennedy pathway (4, 6). The enzyme also plays a major role in controlling the cellular concentration of its substrate PA (2), the precursor of phospholipids that are synthesized via the CDP-DAG pathway (6 -8). In addition, the substrate PA plays a signaling role in the transcriptional regulation of phospholipid synthesis genes (9). In fact, mutants defective in PAH1-encoded PAP activity exhibit a Ͼ90% reduction in TAG content, a derepression of phospholipid synthesis genes, and an expansion of the nuclear/ER membrane (3, 5). Thus, the regulation of PAP activity governs the synthesis of TAG, the pathways by which phospholipids are synthesized, PA signaling, and the growth of the nuclear/ER membrane (6).The importance of PAP in lipid metabolism and cell physiology is further emphasized by the fact that the overexpression of Lpin1-encoded PAP (also known as lipin 1) in mice leads to obesity and insulin sensitivity, w...

The influence of platelets on the cellular metabolism of atherogenic lipoproteins has not been characterized in detail. Therefore, we investigated the effect of platelet factor 4 (PF4), a cationic protein released in high concentration by activated platelets, on the uptake and degradation of low-density lipoprotein (

Saccharomyces cerevisiae cells lacking the amphiphysin-like orthologs, Rvs161 or Rvs167, are unable to thrive under many stress conditions. Here we show cells lacking Rvs161 require Cdc55, the B subunit of the yeast ceramide-activated protein phosphatase, for viability under heat stress. By using specific rvs mutant alleles, we linked this lethal genetic interaction to loss of Rvs161 endocytic domain function. Recessive mutations in the sphingolipid pathway, such as deletion of the very long-chain fatty acid elongase, Sur4, suppress the osmotic growth defect of rvs161 cells. We demonstrate that Cdc55 is required for sur4-dependent suppressor activity and that protein phosphatase activation, through overexpression of CDC55 alone, can also remediate this defect. Loss of SUR4 in rvs161 cells reinitiates Ste3 a-factor receptor endocytosis and requires Cdc55 function to do so. Moreover, overexpression of CDC55 reinitiates Ste3 endocytic-dependent degradation and restores fluid phase endocytosis in rvs161 cells. In contrast, loss of SUR4 or CDC55 overexpression does not remediate the actin polarization defects of osmotic stressed rvs161 cells. Importantly, remediation of rvs161 defects by protein phosphatase activation requires the ceramide-activated protein phosphatase catalytic subunit, Sit4, and the protein phosphatase 2A catalytic subunits, Pph21/Pph22. Finally, genetic analyses reveal a synthetic lethal interaction between loss of CDC55 and gene deletions lethal with rvs161, all of which function in endocytosis.