Mechanisms of Lipid Import into Chloroplasts

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INTELLECTUAL MERIT The chloroplast is the defining organelle of plant cells. It contains the photosynthetic membrane, which converts sunlight into chemical energy, a process essential to most forms of life on earth. The photosynthetic membrane is not able to make its building blocks, the polar lipid constituents, but relies on lipid precursors assembled at the inner and outer chloroplast envelope membranes and the endoplasmic reticulum (ER), a membrane system outside the chloroplast. Therefore, lipid transfer into the chloroplast and through the envelope membranes to the photosynthetic membranes is essential. Gaining a mechanistic understanding of lipid transfer processes involved in the assembly of the photosynthetic membrane is the long-term goal of this project.The isolation of genetic mutants of the model plant Arabidopsis disrupted in ER-to-chloroplast lipid transfer has enabled first mechanistic studies of the underlying phenomena. The tgd mutants of Arabidopsis are disrupted in chloroplast lipid import and are named for their characteristic phenotype, the accumulation of the oligogalactolipid trigalactosyldiacylglycerol. Availability of these mutants led to the identification of four key proteins (TGD1,2,3,4) involved in the process. TGD1, 2 and 3 resemble components of ATP Binding Cassette (ABC) transporters and are hypothesized to assemble into a complex that facilitates transfer of lipids between the chloroplast envelope membranes and through the inner envelope membrane. The TGD4 protein on the other hand is integrated in the chloroplast outer envelope membrane and is hypothesized to transfer lipids from the ER to the TGD1,2,3 complex. This project will investigate how TGD4 and TGD1,2,3 complexes assemble and form a lipid conduit from the ER through the chloroplast envelope membranes, an essential feature of chloroplast membrane biogenesis.BROADER IMPACTS Mechanisms of interorganelle lipid transfer in any eukaryotic cell are poorly understood but essential to organelle formation. Thus, it is expected that the analysis of chloroplast lipid import will provide mechanistic insights into general lipid transfer phenomena relevant beyond plant cells. The project also provides excellent training opportunities for a postdoctoral researcher, a graduate student, and undergraduate student, who collaborate effectively in a team. Elements of the project are specifically designed for the participation of undergraduate students, and will provide training in basic laboratory techniques in molecular biology and biochemistry and require critical thinking towards proper experimental controls and data analysis. All participants including the undergraduate students present their results in weekly lab meetings, present posters or talks at scientific meetings, and participate in the publication of the results as coauthors. All coauthors actively participate in manuscript writing. Co-mentoring of the graduate and undergraduate students by the postdoctoral researcher provides experience in the management of people and projects. In addition, undergraduate students working on the project participate in a summer training program in Plant Genomics at Michigan State University. This program has successfully recruited a diverse group of students and faculty in the past five years.