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，包括膜重塑和蛋白质的正确时空靶向。虽然这些过程在真核生物中有相对较好的特征，但原核细胞器的生物发生和组装往往仍然知之甚少。在这个项目中，我的目标是分析趋磁细菌中独特的磁性细胞器--磁小体的蛋白质组装。我的初步结果表明，与Tic20相关的MamF样蛋白在磁小体的蛋白质组装过程中起着核心作用。Tic20是叶绿体内膜的蛋白质转运通道。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来鉴定在缺乏所有MAMF样基因的突变体中被错误定位的所有蛋白质。随后，我们将使用细菌双杂交分析和基于PAGE的方法来确定这些假定的底物是否直接与MamF样蛋白相互作用。在确定底物和MamF样蛋白中MM靶向决定因素后，我们将建立荧光显微磁小体靶向实验，以确定MamF介导的磁小体膜组装的机制和时机。随后，我们的目标是使用显微、生理或生化方法验证靶向机制。最后，我们对这些与Tic20相关的MamF样蛋白的功能鉴定不仅对于全面了解针对原核细胞器的蛋白质具有重要意义，而且有助于阐明Tic20样蛋白在非光合作用生物中的作用，并为真核生物中细胞器特异性蛋白转运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.