Mechanisms of Lipid Trafficking between the Endoplasmic Reticulum and the Chloroplast

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Intellectual Merit: Compartmentation into organelles is a signature trait of all eukaryotic cells and depends on biological membranes containing glycerolipids. Interorganelle lipid trafficking is essential, because different organelles cooperate in the biosynthesis of lipid precursors required for the assembly of specific subcellular membranes. A prominent example is the formation of the photosynthetic thylakoid membranes in plant chloroplasts. Galactoglycerolipids are the most abundant lipid components of these membranes and their biosynthesis serves as a paradigm for the study of lipid transfer from the endoplasmic reticulum (ER) to the chloroplast. Long-term, the genetic and biochemical analysis of this process is expected to provide a mechanistic understanding of interorganelle lipid trafficking phenomena in plants and other organisms. A collection of 25 Arabidopsis mutants is hypothesized to be affected in different aspects of ER-toplastid lipid trafficking. Common to these mutants is a complex, but robust lipid phenotype, the accumulation of oligogalactoglycerolipids giving rise to the trigalactosyldiacylglycerol (tgd) mutant designation. Of the four TGD loci identified at this time, TGD1, TGD2, and TGD3 encode the permease, the substrate-binding protein, and the ATPase subunits of a putative ABC transporter complex in the inner chloroplast envelope membrane. Several lines of indirect evidence suggest that the TGD123 complex mediates the transfer of phosphatidic acid from the outer envelope to the inside of the inner envelope membrane, where it is processed into the diacylglycerol precursor for galactoglycerolipid biosynthesis. The recently identified TGD4 locus encodes a novel cytosolic non-intrinsic membrane protein, which appears to be associated with the ER. It is hypothesized that the TGD4 protein is directly involved in the transfer of lipid precursors from the ER to the outer chloroplast envelope, possibly by mediating the formation of direct contact zones between these two membranes. Three specific aims will be pursued: 1.The biochemical and molecular function of the TGD4 protein will be determined. 2. Uncharacterized tgd loci present in the mutant collection will be mapped and characterized largely by undergraduate students. 3. The working hypothesis for the existence and function of the proposed TGD123 phosphatidic acid transport complex will be tested. Broader Impacts: Aside from the scientific impacts mentioned, appropriate training opportunities will be provided for graduate and undergraduate students, as well as postdoctoral researchers. The continuing mapping of tgd loci under aim 2 involves undergraduate students supervised by senior researchers. Genetic mapping in Arabidopsis is based on mature technology and established procedures. However, it is intellectually challenging and enables the training of novices in fundamental lab skills. The procedures can be divided into small tasks fitting with a student's class schedule. Students participate in weekly lab meetings and give annual presentations on their work at regional meetings and during on-campus events. Undergraduate students are regularly included as coauthors on publications. In addition, the project will participate in a summer training program in Plant Genomics that was recently implemented at MSU (www.plantgenomics.msu.edu).