Cloning and functional characterization of a novel mitochondrial N-ethylmaleimide-sensitive glycerol-3-phosphate acyltransferase (GPAT2)

Shuli Wang, Douglas P. Lee, Nan Gong, Nicole M.J. Schwerbrock, Douglas G. Mashek, Maria R. Gonzalez-Baró, Cliona Stapleton, Lei O. Li, Tal M. Lewin, Rosalind A. Coleman

Research output: Contribution to journalArticlepeer-review

60 Scopus citations

Abstract

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial and rate-limiting step in glycerolipid synthesis. Several mammalian GPAT activities have been recognized, including N-ethylmaleimide (NEM)-sensitive isoforms in microsomes and mitochondria and an NEM-resistant form in mitochondrial outer membrane (GPAT1). We have now cloned a second mitochondrial isoform, GPAT2 from mouse testis. The open-reading frame encodes a protein of 798 amino acids with a calculated mass of 88.8 kDa and 27% amino acid identity to GPAT1. Testis mRNA expression was 50-fold higher than in liver or brown adipose tissue, but the specific activity of NEM-sensitive GPAT in testis mitochondria was similar to that in liver. When Cos-7 cells were transiently transfected with GPAT2, NEM-sensitive GPAT activity increased 30%. Confocal microscopy confirmed a mitochondrial location. Incubation of GPAT2-transfected Cos-7 cells with trace (3 μM; 0.25 μCi) [1-14C]oleate for 6 h increased incorporation of [14C]oleate into TAG 84%. In contrast, incorporation into phospholipid species was lower than in control cells. Although a polyclonal antibody raised against full-length GPAT1 detected an ∼89-kDa band in liver and testis from GPAT1 null mice and both 89- and 80-kDa bands in BAT from the knockout animals, the GPAT2 protein expressed in Cos-7 cells was only 80 kDa. In vitro translation showed a single product of 89 kDa. Unlike GPAT1, GPAT2 mRNA abundance in liver was not altered by fasting or refeeding. GPAT2 is likely to have a specialized function in testis.

Original languageEnglish (US)
Pages (from-to)347-358
Number of pages12
JournalArchives of Biochemistry and Biophysics
Volume465
Issue number2
DOIs
StatePublished - Sep 15 2007

Keywords

  • Glycerolipid
  • Gpam
  • Steatosis
  • Testis
  • Triacylglycerol

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