硅藻是单细胞藻类,通过次生内共生进化,红藻样真核生物被异养真核细胞吞噬的过程。这就产生了复杂结构和超微结构的质体,需要精心的蛋白质导入,贩运,信号和细胞内串扰途径。研究质体和线粒体及其在器官中的独特生理途径可能极大地有助于我们对光合作用的理解。线粒体呼吸和硅藻进化。如此复杂的细胞器的分离,然而,仍然要求,和现有的协议要么仅限于少数物种(对于质体),要么迄今尚未报道硅藻(对于线粒体)。在这项工作中,我们提出了第一个从硅藻模型中分离线粒体的方案。除此之外,我们扩展了协议,因此它也适用于高质量质体级分的纯化,并提供所得细胞器的详细结构和生理特征。分离的线粒体结构完整,显示了线粒体呼吸的明确证据,但部分仍含有残留的细胞碎片。相比之下,质体分离物几乎没有细胞污染物,具有结构保存的类囊体进行电子传输,但从Western印迹和质谱分析得出的结论是失去了大部分基质成分。线粒体和类囊体的液相色谱电喷雾电离质谱研究,此外,进行了详细的蛋白质组分析,从而为质体和线粒体提供了广泛的蛋白质组图,从而帮助我们扩大了对硅藻中细胞器代谢和功能的理解。
Diatoms are unicellular algae and evolved by secondary endosymbiosis, a process in which a red alga-like eukaryote was engulfed by a heterotrophic eukaryotic cell. This gave rise to plastids of remarkable complex architecture and ultrastructure that require elaborate protein importing, trafficking, signaling and intracellular cross-talk pathways. Studying both plastids and mitochondria and their distinctive physiological pathways in organello may greatly contribute to our understanding of photosynthesis, mitochondrial respiration and diatom evolution. The isolation of such complex organelles, however, is still demanding, and existing protocols are either limited to a few species (for plastids) or have not been reported for diatoms so far (for mitochondria). In this work, we present the first isolation protocol for mitochondria from the model diatom Thalassiosira pseudonana. Apart from that, we extended the protocol so that it is also applicable for the purification of a high-quality plastids fraction, and provide detailed structural and physiological characterizations of the resulting organelles. Isolated mitochondria were structurally intact, showed clear evidence of mitochondrial respiration, but the fractions still contained residual cell fragments. In contrast, plastid isolates were virtually free of cellular contaminants, featured structurally preserved thylakoids performing electron transport, but lost most of their stromal components as concluded from Western blots and mass spectrometry. Liquid chromatography electrospray-ionization mass spectrometry studies on mitochondria and thylakoids, moreover, allowed detailed proteome analyses which resulted in extensive proteome maps for both plastids and mitochondria thus helping us to broaden our understanding of organelle metabolism and functionality in diatoms.