title: Membrane topology of murine glycerol-3-phosphate acyltransferase 2.
abstract: Glycerol-3-phosphate acyltransferase (GPAT) is a rate-limiting enzyme in mammalian triacylglycerol biosynthesis. GPAT is a target for the treatment of metabolic disorders associated with high lipid accumulation. Although the molecular basis for GPAT1 activation has been investigated extensively, the activation of other isoforms, such as GPAT2, is less well understood. Here the membrane topology of the GPAT2 protein was examined using an epitope-tag-based method. Exogenously expressed GPAT2 protein was present in the membrane fraction of transformed HEK293 cells even in the presence of Na(2)CO(3) (100 mM), indicating that GPAT2 is a membrane-bound protein. Trypsin treatment of the membrane fraction degraded the N-terminal (FLAG) and C-terminal (myc-epitope) protein tags of the GPAT2 protein. Bioinformatic analysis of the GPAT2 protein sequence indicated four hydrophobic sequences as potential membrane-spanning regions (TM1-TM4). Immunoblotting of the myc-epitope tag, which was inserted between each TM region of the GPAT2 protein, showed that the amino acid sequence between TM3 and TM4 was protected from trypsin digestion. These results suggest that the GPAT2 protein has two transmembrane segments and that the N-terminal and C-terminal regions of this protein face the cytoplasm. These results also suggest that the enzymatically active motifs I-III of the GPAT2 protein face the cytosol, while motif IV is within the membrane. It is expected that the use of this topological model of GPAT2 will be essential in efforts to elucidate the molecular mechanisms of GPAT2 activity in mammalian cells.
authors.affiliation: Department of Nutrition and Metabolism, Institute of Health Biosciences, University of Tokushima Graduate School, 3-18-15, Kuramoto-cho, Tokushima City 770-8503, Japan.
authors.forename: Tadahiko
authors.initials: T
authors.lastname: Nakagawa
authors.forename: Nagakatsu
authors.initials: N
authors.lastname: Harada
authors.forename: Aiko
authors.initials: A
authors.lastname: Miyamoto
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Kawanishi
authors.forename: Masaki
authors.initials: M
authors.lastname: Yoshida
authors.forename: Masayuki
authors.initials: M
authors.lastname: Shono
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Mawatari
authors.forename: Akira
authors.initials: A
authors.lastname: Takahashi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Sakaue
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Nakaya
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology*; D003593:Cytoplasm / Q000201:enzymology; D005992:Glycerol-3-Phosphate O-Acyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary
chemical_list: D005992:Glycerol-3-Phosphate O-Acyltransferase; C571223:glycerol-3-phosphate acyltransferase 2, mouse