Assembly of a prokaryotic organelle by Tic20-like proteins

膜细胞器的生物发生和组装需要许多过程，包括膜重塑和正确的蛋白质时空靶向。虽然这些过程在真核生物中已经有了很好的描述，但对原核细胞器的生物发生和组装仍然知之甚少。在这个项目中，我的目标是分析磁小体的蛋白质组装，磁小体是来自趋磁细菌的独特磁性细胞器。我的初步结果表明，与叶绿体内膜的蛋白质转运通道Tic20相关的mamf样蛋白在磁小体的蛋白质组装过程中起核心作用。为了了解它们在磁小体组装过程中的作用，我们将首先通过蛋白质组学和免疫化学分析确定在缺乏所有mamf样基因的突变体中错误靶向的所有蛋白质。随后，我们将使用细菌双杂交试验和基于page的方法确定这些假定的底物是否直接与mamf样蛋白相互作用。在鉴定底物和mamf样蛋白中的MM靶向决定因素后，我们将建立荧光显微镜磁小体靶向分析，以确定mamf介导的磁小体膜组装的机制和时间。随后，我们的目标是通过显微镜，生理学或生化方法验证靶向机制。最后，我们对这些与tic20相关的mamf样蛋白的功能表征不仅对了解靶向原核细胞器的蛋白具有重要意义，而且有助于阐明tic20样蛋白在非光合生物中的作用，并为真核生物中细胞器特异性蛋白跨位子的进化提供新的线索。

The biogenesis and assembly of membranous cell organelles requires a multitude of processes, including membrane remodeling and correct spatio-temporal targeting of proteins. While these processes are relatively well characterized in eukaryotes, the biogenesis and assembly of prokaryotic organelles is often still poorly understood. In this project, I aim to analyze the protein assembly of magnetosomes, unique magnetic organelles from magnetotactic bacteria. My preliminary results indicate that MamF-like proteins which are related to Tic20, a protein translocating channel of the inner membrane of chloroplasts, play a central role during the protein assembly of magnetosomes. To understand their roles during magnetosome assembly we will first identify all proteins that are mistargeted in a mutant lacking all mamF-like genes by proteomic and immunochemical analyses. Subsequently, we will determine if these putative substrates directly interact with MamF-like proteins using bacterial two-hybrid assays and PAGE-based methods. After identification of MM targeting determinants within substrate and MamF-like proteins, we will establish a fluorescence microscopic magnetosome targeting assay to identify the mechanism and timing of the MamF-mediated magnetosome membrane assembly. Subsequently, we aim verify the targeting mechanism using microscopic, physiologic, or biochemical methods. Finally, our functional characterization of these Tic20-related MamF-like proteins will not only be important to generally understand protein targeting to a prokaryotic organelle but also help to elucidate the roles of Tic20-like proteins in non-photosynthetic organisms and shed new lights in the evolution of organelle-specific protein translocons in eukaryotes.