Molecular characterization of lipid phosphatases in yeast

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Abstract

DGPP phosphatase is a membrane-associated 34-kDa enzyme from yeast that catalyzes the dephosphorylation of DGPP to yield phosphatidate (PA). Amino acid sequence information derived from DGPP phosphatase was used to identify and isolate the DPP1 gene encoding the enzyme. Multicopy plasmids containing the DPP1 gene directed a 10-fold overexpression of DGPP phosphatase activity in yeast. The heterologous expression of the yeast DPP1 gene in Sf-9 insect cells resulted in a 500-fold overexpression of DGPP phosphatase activity over that expressed in wild-type yeast. DGPP phosphatase possesses a type 2 PA phosphatase activity and its expression correlated with the overexpression of DGPP phosphatase activity in yeast and in insect cells. A second gene, LPP1, was isolated that shared 23% amino acid sequence identity with the DPP1 gene product. Both gene products contain a novel phosphatase sequence motif. Overexpression of the LPP1 gene in yeast and in Sf-9 insect cells resulted in an overexpression of PA phosphatase activity of 3-fold and 35-fold, respectively, over that found in wild-type yeast. Null allele dpp1 and lpp1 mutants were constructed. These mutants were viable and did not exhibit any obvious growth defects. Moreover, a double dpp1,lpp1 mutant was viable and showed no growth defects. Biochemical analyses of these mutants showed that the DPP1 gene product was responsible for all of the detectable DGPP phosphatase activity and half of the PA phosphatase activity in yeast. Together, the DPP1 and LPP1 gene products were responsible for 90% of the type 2 PA phosphatase activity in yeast. This work was supported by NIH grant GM-28140.

Original languageEnglish (US)
JournalFASEB Journal
Volume12
Issue number8
StatePublished - Dec 1 1998

All Science Journal Classification (ASJC) codes

  • Agricultural and Biological Sciences (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology

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