title: Biochemical and topological properties of type A MGDG synthase, a spinach chloroplast envelope enzyme catalyzing the synthesis of both prokaryotic and eukaryotic MGDG.
abstract: MGDG synthase, the enzyme that catalyzes the synthesis of the major chloroplast membrane lipid monogalactosyldiacylglycerol (MGDG), is encoded by a multigenic family. We have analyzed the biochemical properties, subcellular localization and membrane topology of a spinach chloroplast MGDG synthase, a representative member of the type A family from Spinacia oleracea (soMGD A), using a recombinant protein that was functionally overexpressed in Escherichia coli and specific polyclonal antibodies. We demonstrated that soMGD A could catalyze the synthesis of both 'prokaryotic' and 'eukaryotic' MGDG molecular species in vitro, with a selectivity for diacylglycerol similar to that of purified chloroplast envelope MGDG synthase activity. Furthermore, soMGD A was shown to be sensitive to chemical reagents (dithiothreitol, N-ethylmaleimide and o-phenanthroline) known to affect MGDG synthesis by the partially purified enzyme, as well as in isolated chloroplast envelope membranes. In spinach chloroplasts, soMGD A was localized by Western blot analysis in the inner envelope membrane. Topological studies demonstrated that soMGD A is a monotopic enzyme, embedded within one leaflet of the inner envelope membrane from spinach chloroplasts, a structure which may involve amphipathic alpha helices. We further demonstrated that in vitro, soMGD A precursor is imported and processed to its correct mature form in intact chloroplasts. These results show that soMGD A corresponds to a mature polypeptide of approximately 45 kDa. In addition, inactivation kinetics after gamma-ray irradiation strongly suggest that both native chloroplast envelope MGDG synthase and recombinant soMGD A have a functional molecular mass of 95-100 kDa, indicating that they are probably active as homodimers made of two 45-kDa subunits. This study suggests that, in spite of the growing evidence that MGDG synthesis is catalyzed by a multigenic family of enzymes, in spinach leaves both prokaryotic and eukaryotic MGDG syntheses could be attributable to a unique dimeric enzyme, provided that diacylglycerol is transported from the outer membrane to the inner membrane of the chloroplast envelope.
authors.affiliation: Department de Biologie Moléculaire et Structurale, Laboratoire de Physiologie Cellulaire Végétale, Commissariat à lEnergie Atomique-Grenoble, URA CNRS 576, France.
authors.forename: C
authors.initials: C
authors.lastname: Miège
authors.forename: E
authors.initials: E
authors.lastname: Maréchal
authors.forename: M
authors.initials: M
authors.lastname: Shimojima
authors.forename: K
authors.initials: K
authors.lastname: Awai
authors.forename: M A
authors.initials: MA
authors.lastname: Block
authors.forename: H
authors.initials: H
authors.lastname: Ohta
authors.forename: K
authors.initials: K
authors.lastname: Takamiya
authors.forename: R
authors.initials: R
authors.lastname: Douce
authors.forename: J
authors.initials: J
authors.lastname: Joyard
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002736:Chloroplasts / Q000201:enzymology; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D004075:Diglycerides / Q000096:biosynthesis*; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D038983:Galactolipids; D005700:Galactosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006017:Glycolipids / Q000096:biosynthesis*; D007425:Intracellular Membranes / Q000201:enzymology; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D018724:Spinacia oleracea / Q000201:enzymology* / Q000235:genetics
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D018744:DNA, Plant; D004075:Diglycerides; D038983:Galactolipids; D006017:Glycolipids; D011994:Recombinant Proteins; C009909:monogalactosyldiacylglycerol; D005700:Galactosyltransferases; C109990:1,2-diacylglycerol 3-beta-galactosyltransferase