Biomedical subjects

Sew Peak-Chew

Publications and source records attributed to Sew Peak-Chew.

4 recordsLinked to original sources

PubMed2006

Control of phospholipid synthesis by phosphorylation of the yeast lipin Pah1p/Smp2p Mg2+-dependent phosphatidate phosphatase.

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.

Cell Membrane↗

PubMed2005

The yeast lipin Smp2 couples phospholipid biosynthesis to nuclear membrane growth.

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.

CDC28 Protein Kinase, S cerevisiae↗

PubMed2003

Interactions between centromere complexes in Saccharomyces cerevisiae.

We have purified two new complexes from Saccharomyces cerevisiae, one containing the centromere component Mtw1p together with Nnf1p, Nsl1p, and Dsn1p, which we call the Mtw1p complex, and the other containing Spc105p and Ydr532p, which we call the Spc105p complex. Further purifications using Dsn1p tagged with protein A show, in addition to the other components of the Mtw1p complex, the two components of the Spc105p complex and the four components of the previously described Ndc80p complex, suggesting that all three complexes are closely associated. Fluorescence microscopy and immunoelectron microscopy show that Nnf1p, Nsl1p, Dsn1p, Spc105p, and Ydr532p all localize to the nuclear side of the spindle pole body and along short spindles. Chromatin immunoprecipitation assays show that all five proteins are associated with centromere DNA. Homologues of Nsl1p and Spc105p in Schizosaccharomyces pombe also localize to the centromere. Temperature-sensitive mutations of Nsl1p, Dsn1p, and Spc105p all cause defects in chromosome segregation. Synthetic-lethal interactions are found between temperature-sensitive mutations in proteins from all three complexes, in agreement with their close physical association. These results show an increasingly complex structure for the S. cerevisiae centromere and a probable conservation of structure between parts of the centromeres of S. cerevisiae and S. pombe.

Amino Acid Sequence↗

PubMed2003

RPA is an initiation factor for human chromosomal DNA replication.

The initiation of chromosomal DNA replication in human cell nuclei is not well understood because of its complexity. To allow investigation of this process on a molecular level, we have recently established a cell-free system that initiates chromosomal DNA replication in an origin-specific manner under cell cycle control in isolated human cell nuclei. We have now used fractionation and reconstitution experiments to functionally identify cellular factors present in a human cell extract that trigger initiation of chromosomal DNA replication in this system. Initial fractionation of a cytosolic extract indicates the presence of at least two independent and non-redundant initiation factors. We have purified one of these factors to homogeneity and identified it as the single-stranded DNA binding protein RPA. The prokaryotic single-stranded DNA binding protein SSB cannot substitute for RPA in the initiation of human chromosomal DNA replication. Antibodies specific for human RPA inhibit the initiation step of human chromosomal DNA replication in vitro. RPA is recruited to DNA replication foci and becomes phosphorylated concomitant with the initiation step in vitro. These data establish a direct functional role for RPA as an essential factor for the initiation of human chromosomal DNA replication.

Cell Line↗