Department of Microbiology, Ludwig Maximilian University of Munich, Germany.
Department of Microbiology, Ernst Moritz Arndt University of Greifswald, Germany.
J Proteomics. 2018 Feb 10;172:89-99. doi: 10.1016/j.jprot.2017.10.007. Epub 2017 Oct 17.
Magnetotactic bacteria produce chains of complex membrane-bound organelles that direct the biomineralization of magnetic nanoparticles and serve for magnetic field navigation. These magnetosome compartments have recently emerged as a model for studying the subcellular organization of prokaryotic organelles. Previous studies indicated the presence of specific proteins with various functions in magnetosome biosynthesis. However, the exact composition and stoichiometry of the magnetosome subproteome have remained unknown. In order to quantify and unambiguously identify all proteins specifically targeted to the magnetosome membrane of the Alphaproteobacterium Magnetospirillum gryphiswaldense, we analyzed the protein composition of several cellular fractions by semi-quantitative mass spectrometry. We found that nearly all genuine magnetosome membrane-integral proteins belong to a well-defined set of previously identified proteins encoded by gene clusters within a genomic island, indicating a highly controlled protein composition. Magnetosome proteins were present in different quantities with up to 120 copies per particle as estimated by correlating our results with available quantitative Western blot data. This high abundance suggests an unusually crowded protein composition of the membrane and a tight packing with transmembrane domains of integral proteins. Our findings will help to further define the structure of the organelle and contribute to the elucidation of magnetosome biogenesis.
Magnetosomes are one of the most complex bacterial organelles and consist of membrane-bounded crystals of magnetic minerals. The exact composition and stoichiometry of the associated membrane integral proteins are of major interest for a deeper understanding of prokaryotic organelle assembly; however, previous proteomic studies failed to reveal meaningful estimations due to the lack of precise and quantitative data, and the inherently high degree of accumulated protein contaminants in purified magnetosomes. Using a highly sensitive mass spectrometer, we acquired proteomic data from several cellular fractions of a magnetosome producing magnetotactic bacterium and developed a comparative algorithm to identify all genuine magnetosome membrane-integral proteins and to discriminate them from contaminants. Furthermore, by combining our data with previously published quantitative Western blot data, we were able to model the protein copy number and density within the magnetosome membrane. Our results suggest that the magnetosome membrane is specifically associated with a small subset of integral proteins that are tightly packed within the lipid layer. Our study provides by far the most comprehensive estimation of magnetosomal protein composition and stoichiometry and will help to elucidate the complex process of magnetosome biogenesis.
产磁细菌产生复杂的膜结合细胞器链，指导磁性纳米粒子的生物矿化，并用于磁场导航。这些磁小体隔室最近已成为研究原核细胞器亚细胞组织的模型。先前的研究表明，在磁小体生物合成中有各种功能的特定蛋白质的存在。然而，磁小体亚蛋白组的确切组成和化学计量仍然未知。为了定量和明确地鉴定 Alphaproteobacterium Magnetospirillum gryphiswaldense 的磁小体膜上专门靶向的所有蛋白质，我们通过半定量质谱分析了几个细胞级分的蛋白质组成。我们发现，几乎所有真正的磁小体膜整合蛋白都属于一组明确的先前通过基因组岛中的基因簇编码的蛋白质，这表明蛋白质组成具有高度的可控性。通过将我们的结果与可用的定量 Western blot 数据相关联，我们发现磁小体蛋白的数量不同，每个颗粒最多有 120 个拷贝。这种高丰度表明膜的蛋白质组成异常拥挤，并且与整合蛋白的跨膜结构域紧密包装。我们的发现将有助于进一步定义细胞器的结构，并有助于阐明磁小体的生物发生。
磁小体是最复杂的细菌细胞器之一，由膜结合的磁性矿物晶体组成。相关膜整合蛋白的精确组成和化学计量对于深入了解原核细胞器的组装具有重要意义；然而，由于缺乏精确和定量的数据以及在纯化的磁小体中固有地存在大量的蛋白质污染物，以前的蛋白质组学研究未能揭示有意义的估计值。使用高度灵敏的质谱仪，我们从产磁趋磁细菌的几个细胞级分中获取了蛋白质组学数据，并开发了一种比较算法来鉴定所有真正的磁小体膜整合蛋白，并将其与污染物区分开来。此外，通过将我们的数据与以前发表的定量 Western blot 数据相结合，我们能够模拟磁小体膜内的蛋白质拷贝数和密度。我们的结果表明，磁小体膜与紧密包装在脂质层内的一小部分整合蛋白特异性相关。我们的研究提供了迄今为止最全面的磁小体蛋白质组成和化学计量的估计值，并将有助于阐明磁小体生物发生的复杂过程。
