Mechanisms of Lipid Trafficking between the Endoplasmic Reticulum and the Chloroplast

智力优势：分隔成细胞器是所有真核细胞的一个特征，它依赖于含有甘油脂的生物膜。细胞器间脂质运输是必不可少的，因为不同的细胞器在特定亚细胞膜组装所需的脂质前体的生物合成中相互合作。一个突出的例子是植物叶绿体中光合作用类囊体膜的形成。半乳糖甘油脂是这些膜中最丰富的脂质成分，它们的生物合成可以作为研究脂质从内质网（ER）到叶绿体转移的范例。从长远来看，这一过程的遗传和生化分析有望为植物和其他生物的细胞器间脂质运输现象提供机制理解。一组25个拟南芥突变体被假设在er上质体脂质运输的不同方面受到影响。这些突变体的共同特点是具有复杂但强健的脂质表型，低聚半乳糖甘油脂的积累导致了三半乳糖二酰基甘油（tgd）突变体的命名。在目前鉴定的四个TGD位点中，TGD1、TGD2和TGD3编码渗透酶、底物结合蛋白和三磷酸腺苷酶亚基，这些亚基是叶绿体内包膜中假定的ABC转运蛋白复合物。一些间接证据表明，TGD123复合物介导磷脂酸从外包膜向内包膜的转移，在那里它被加工成二酰基甘油前体，用于半乳糖甘油脂的生物合成。最近发现的TGD4位点编码一种新的细胞质非内在膜蛋白，该蛋白似乎与内质网有关。据推测，TGD4蛋白直接参与脂质前体从内质网向叶绿体外膜的转移，可能是通过介导这两个膜之间直接接触区的形成。将追求三个具体目标：TGD4蛋白的生化和分子功能将被确定。2. 突变体收集中存在的未表征的tgd位点将主要由本科生绘制和表征。3. TGD123磷脂酸转运复合物的存在和功能的工作假设将被测试。更广泛的影响：除了上述科学影响外，将为研究生和本科生以及博士后研究人员提供适当的培训机会。在目标2下继续绘制tgd基因座需要本科生在高级研究员的指导下完成。拟南芥的遗传定位是基于成熟的技术和既定的程序。然而，它是智力上的挑战，并使训练新手在基本的实验室技能。这个过程可以根据学生的课程表分成小任务。学生参加每周一次的实验室会议，并在地区会议和校园活动中发表年度报告。本科生经常被列为出版物的共同作者。此外，该项目将参加最近在密歇根州立大学实施的植物基因组学暑期培训计划（www.plantgenomics.msu.edu）。

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).