Abstract
Chloroplast membrane lipid synthesis relies on the import of glycerolipids from the ER. The TGD (TriGalactosylDiacylglycerol) proteins are required for this lipid transfer process. The TGD1, -2, and -3 proteins form a putative ABC (ATP-binding cassette) transporter transporting ER-derived lipids through the inner envelope membrane of the chloroplast, while TGD4 binds phosphatidic acid (PtdOH) and resides in the outer chloroplast envelope. We identified two sequences in TGD4, amino acids 1-80 and 110-145, which are necessary and sufficient for PtdOH binding. Deletion of both sequences abolished PtdOH binding activity. We also found that TGD4 from 18:3 plants bound specifically and with increased affinity PtdOH. TGD4 did not interact with other proteins and formed a homodimer both in vitro and in vivo. Our results suggest that TGD4 is an integral dimeric β-barrel lipid transfer protein that binds PtdOH with its N terminus and contains dimerization domains at its C terminus.
Highlights
The phosphatidic acid (PtdOH) binding site of TGD4 is not yet known
Plastid specific acyltransferases of the prokaryotic pathway are associated with the inner plastid envelope membrane and transfer the acyl groups, which are de novo synthesized in the chloroplast and attached to acyl carrier proteins (ACPs), to glycerol-3-phosphate producing PtdOH
The N termini of the respective truncations were fused to DsRED to enhance solubility, and the resulting recombinant proteins were produced in E. coli and purified to homogeneity using a C-terminal 6ϫ His tag
Summary
The phosphatidic acid (PtdOH) binding site of TGD4 is not yet known. Results: Amino acids 1– 80 and 110 –145 of TGD4 represent two PtdOH interacting sequences and TGD4 forms a homodimer in vitro and in vivo. Plastid specific acyltransferases of the prokaryotic pathway are associated with the inner plastid envelope membrane and transfer the acyl groups, which are de novo synthesized in the chloroplast and attached to acyl carrier proteins (ACPs), to glycerol-3-phosphate producing PtdOH. Glycolipids originating from the prokaryotic pathway have a 16-carbon acyl chain in the sn-2 position of the glyceryl backbone, while glycolipids derived from the eukaryotic pathway have an acyl chain of 18 carbons in the same position This difference has been attributed to the different substrate preferences of acyltransferases in the chloroplast and the ER [4, 5]. TGD4 Phosphatidic Acid Binding Site ing properties of TGD4 proteins in 18:3 plants, and the composition of the TGD4 complex
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