A phosphatidic acid-binding protein of the chloroplast inner envelope membrane involved in lipid trafficking

Abstract

The biogenesis of the photosynthetic thylakoid membranes inside plant chloroplasts requires enzymes at the plastid envelope and the endoplasmic reticulum (ER). Extensive lipid trafficking is required for thylakoid lipid biosynthesis. Here the trigalactosyldiacylglycerol2 (tgd2) mutant of Arabidopsis is described. To the extent tested, tgd2 showed a complex lipid phenotype identical to the previously described tgd1 mutant. The aberrant accumulation of oligogalactolipids and triacylglycerols and the reduction of molecular species of galactolipids derived from the ER are consistent with a disruption of the import of ER-derived lipids into the plastid. The TGD1 protein is a permease-like component of an ABC transporter located in the chloroplast inner envelope membrane. The TGD2 gene encodes a phosphatidic acid-binding protein with a predicted mycobacterial cell entry domain. It is tethered to the inner chloroplast envelope membrane facing the outer envelope membrane. Presumed bacterial orthologs of TGD1 and TGD2 in Gram-negative bacteria are typically organized in transcriptional units, suggesting their involvement in a common biological process. Expression of the tgd2-1 mutant cDNA caused a dominantnegative effect replicating the tgd2 mutant phenotype. This result is interpreted as the interference of the mutant protein with its native protein complex. It is proposed that TGD2 represents the substrate-binding or regulatory component of a phosphatidic acid͞lipid transport complex in the chloroplast inner envelope membrane.Arabidopsis ͉ membrane transporter ͉ glycerolipids ͉ ABC transporter ͉ substrate-binding protein A s plant leaves expand the demand on the lipid biosynthetic machinery is high because leaf cells contain one of the most extensive membrane systems found in nature, the photosynthetic thylakoid membrane of chloroplasts. The most abundant thylakoid lipids include nonphosphorous galactolipids. Galactolipid biosynthesis involves the formation of phosphatidic acid (PA) in the plastid and at the endoplasmic reticulum (ER) in many plants (1, 2), including Arabidopsis. Fatty acids derived from de novo synthesis in the plastid are assembled into PA in the plastid or at the ER. In Arabidopsis, diacylglycerols derived from the plastid pathway or the ER pathway are present in galactolipids in approximately equal proportion (3). The Arabidopsis lipid galactosyltransferases MGD1 and DGD1, which successively galactosylate diacylglycerol, are associated with the inner and the outer chloroplast envelope membranes, respectively (4). The topology of the galactolipid biosynthetic machinery and the involvement of the ER pathway require extensive subcellular lipid trafficking, most of which is mechanistically not understood.To date, two mutants of Arabidopsis have been described that affect lipid trafficking from the ER to the plastid. The act1(ats1) mutant is deficient in the plastidic glycerol 3-phopshate acyltransferase, and most of the galactolipids in this mutant are derived from the ER pathway (5). In contrast, gala...

Arabidopsis Lipins, PDAT1 Acyltransferase, and SDP1 Triacylglycerol Lipase Synergistically Direct Fatty Acids toward β-Oxidation, Thereby Maintaining Membrane Lipid Homeostasis

“…SFR2 converts MGDG to DGDG and oligogalactolipids by transglycosylation with a concomitant production of DAG, which can be further metabolized to TAG (Moellering et al, 2010). The tgd1-1 mutant accumulates oligogalactolipids in leaves, indicating that SFR2 is activated in this mutant (Xu et al, 2003(Xu et al, , 2008Awai et al, 2006). To test the role of SFR2 in lipid metabolism in tgd1-1, we generated a double mutant between tgd1-1 and an SFR2 T-DNA knockout mutant, sfr2-3 (Moellering et al, 2010).…”

A 25-Amino Acid Sequence of the Arabidopsis TGD2 Protein Is Sufficient for Specific Binding of Phosphatidic Acid

“…Expression and Purification of DR-TGD2 Fusion ProteinsAll TGD2 truncated proteins used in this study were derived from the TGD2-dTMD-pQE31 plasmid (also known as TGD2C-pQE31) using a PCR-based strategy (5). Following digestion with NcoI and XhoI, the PCR fragment was ligated into the DsRed-plw01-His plasmid (provided by Dr. Michael Garavito, Michigan State University) to produce Discosoma sp.…”

“…The current hypothesis suggests that PtdCho returns to the outer plastid envelope, where it is converted to PtdOH by a phospolipase D (4). The TGD1, -2, and -3 proteins are postulated to be involved in the transfer of PtdOH from the outer envelope membrane to the inside of the inner envelope membrane (5)(6)(7)(8), where PtdOH is dephosphorylated by a PtdOH phosphatase to diacylglycerol, which is the precursor of galactoglycerolipid biosynthesis at the inner chloroplast envelope membrane. Alternatively, nascent fatty acids are assembled de novo at the chloroplast inner envelope into PtdOH, which then enters galactoglycerolipid biosynthesis, as described above.…”

“…The TGD1, -2, and -3 proteins resemble the permease, substrate binding, and ATPase subunits of a multipartite ABC transporter, which is localized in the inner chloroplast envelope (5)(6)(7)(8). Mutants with reduced function of these proteins have complex lipid phenotypes.…”

“…TGD2 appears anchored with its N-terminal domain into the inner envelope membrane such that the C terminus faces the intermembrane space (5). The C terminus of TGD2 was shown to specifically bind PtdOH.…”