Functional analysis of a protein involved in the endoplasmic reticulum-to-chloroplast lipid trafficking pathway in plants.

摘要

Photosynthesis is arguably the most important trait of plants for improving human conditions. Chloroplast is the dedicated cellular organelle where photosynthesis takes place. Chloroplast is made up of intricate membrane systems, where inner and outer envelopes enclose the photosynthetic thylakoid membranes. Chloroplast membranes mainly consist of non-phosphorous glycolipids that are synthesized through two pathways: the prokaryotic and the eukaryotic pathway.;In the prokaryotic pathway, fatty acids synthesized within the chloroplast are incorporated into glycerol-3-phosphate for glycolipids and phospholipids synthesis. All reactions take place inside the chloroplast. In the eukaryotic pathway, fatty acid residues are first exported from the chloroplast to the endoplasmic reticulum (ER) for the synthesis of extraplastidic membrane lipids. A certain portion of these lipids is transferred back from the ER to the chloroplast for chloroplast lipid synthesis.;Forward genetic screening has revealed a few Arabidopsis mutants impaired in the eukaryotic pathway. They accumulate an abnormal galactolipid trigalactosyldiacylglycerol (TGDG), and the mutants are, therefore, designated as tgd mutants. tgd mutants exhibit various phenotypes including dwarfed growth, pale-colored leaves, and partially aborted seeds. Four genes have been found responsible for the phenotypes: TGD1, 2, 3, 4. The TGD1, 2, 3 gene products, TGD1, 2, 3 proteins, form a putative ATP-binding cassette (ABC) transporter on the inner envelope of the chloroplast, however the molecular function of TGD4 protein cannot be inferred from previous knowledge.;In this work, TGD4 is found to be a transmembrane protein embedded in the chloroplast outer envelope. It is predicted to be a beta-barrel protein reminiscent of gram-negative bacteria outer membrane beta-barrel transporters. The recombinant TGD4 protein binds phosphatidic acid (PtdOH) specifically and two regions, amino acids 1-80 and 110-145 are responsible for PtdOH binding. tgd4 mutants accumulate PtdOH outside of the chloroplast suggesting TGD4 is a PtdOH transporter. TGD4 is indeed able to transfer PtdOH from one membrane bilayer to the other in vitro. TGD4 does not interact strongly with any other proteins but dose interact with itself and forms a homodimer in vivo. In conclusion, TGD4 is a putative chloroplast outer envelope PtdOH transporter that transfers PtdOH from the ER to the chloroplast intermembrane space.