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 a 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 < 45 kDa. In addition, inactivation kinetics after g-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.

Galactolipid deficiency and abnormal chloroplast development in the Arabidopsis MGD synthase 1 mutant

“…Previous studies led to the identification of cDNAs encoding MGD synthases from cucumber (11) and spinach (12). In addition to these cDNAs, and to the Arabidopsis MGD1 gene described in this report, two MGD synthase-related Arabidopsis sequences can be found in the GenBank database (12).…”

“…CA A04005) and MGD3 (GenBank accession no. AAD28678) have previously been referred to as atMGD type B and atMGD type C, respectively (12). MGD1 shares 71.4% amino acid sequence identity with csMGD1, but only 53.2% identity with the MGD2 and 55.4% identity with the MGD3.…”

“…In addition to these cDNAs, and to the Arabidopsis MGD1 gene described in this report, two MGD synthase-related Arabidopsis sequences can be found in the GenBank database (12). MGD1 and the cucumber and spinach MGD synthases (referred to here as csMGD1 and soMGD1) are very closely related, each sharing at least 70% amino acid sequence identity.…”

“…This enzyme transfers D-galactose from UDP-galactose to sn-1,2-diacylglycerol (DAG) (10). Cucumber and spinach MGD synthases have been described in detail (11,12), and similar Arabidopsis sequences are present in the GenBank database (12). The DAG used in MGD synthesis is thought to be derived, in Arabidopsis, from two different biosynthetic pathways (13,14).…”

Analysis of the Plastidic phosphate translocator Gene Family in Arabidopsis and Identification of New phosphate translocator-Homologous Transporters, Classified by Their Putative Substrate-Binding Site

“…They are possibly involved in the biosynthesis of galactolipids (monogalactosyldiacylglycerol [MGDG] and digalactosydiacylglycerol) in plastids, which are the major constituents of plastid membranes (Joyard et al, 1996). MGDG synthase transfers Gal from the donor (UDP-Gal) to a hydrophobic acceptor molecule, diacylglycerol, to synthesize MGDG and UDP, a reaction that occurs within the envelope membranes (Miège et al, 1999). Another lipid, sulfoquinovosyldiacylglycerol, is found in the photosynthetic membranes of plants and bacteria (Essigmann et al, 1998).…”