title: Lipins from plants are phosphatidate phosphatases that restore lipid synthesis in a pah1Δ mutant strain of Saccharomyces cerevisiae.
abstract: The identification of the yeast phosphatidate phosphohydrolase (PAH1) gene encoding an enzyme with phosphatidate phosphatase (PAP; 3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) activity led to the discovery of mammalian Lipins and subsequently to homologous genes from plants. In the present study, we describe the functional characterization of Arabidopsis and Brassica napus homologs of PAH1. Recombinant expression studies confirmed that homologous PAHs from plants can rescue different phenotypes exhibited by the yeast pah1Δ strain, such as temperature growth sensitivity and atypical neutral lipid composition. Using this expression system, we examined the role of the putative catalytic motif DXDXT and other conserved residues by mutational analysis. Mutants within the carboxy-terminal lipin domain displayed significantly decreased PAP activity, which was reflected by their limited ability to complement different phenotypes of pah1Δ. Subcellular localization studies using a green fluorescent protein fusion protein showed that Arabidopsis PAH1 is mostly present in the cytoplasm of yeast cells. However, upon oleic acid stimulation, green fluorescent protein fluorescence was predominantly found in the nucleus, suggesting that plant PAH1 might be involved in the transcriptional regulation of gene expression. In addition, we demonstrate that mutation of conserved residues that are essential for the PAP activity of the Arabidopsis PAH1 enzyme did not impair its nuclear localization in response to oleic acid. In conclusion, the present study provides evidence that Arabidopsis and B. napus PAHs restore lipid synthesis in yeast and that DXDXT is a functional enzymic motif within plant PAHs.
authors.affiliation: Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Canada.
authors.forename: Elzbieta
authors.initials: E
authors.lastname: Mietkiewska
authors.forename: Rodrigo M P
authors.initials: RM
authors.lastname: Siloto
authors.forename: Jay
authors.initials: J
authors.lastname: Dewald
authors.forename: Saleh
authors.initials: S
authors.lastname: Shah
authors.forename: David N
authors.initials: DN
authors.lastname: Brindley
authors.forename: Randall J
authors.initials: RJ
authors.lastname: Weselake
mesh_terms: D017360:Arabidopsis / Q000201:enzymology; D029688:Brassica napus / Q000201:enzymology; D002467:Cell Nucleus / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D010711:Phosphatidate Phosphatase / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*
chemical_list: D010940:Plant Proteins; D010711:Phosphatidate Phosphatase