Nunn Brook L, Aker Jocelyn R, Shaffer Scott A, Tsai Shannon, Strzepek Robert F, Boyd Philip W, Freeman Theodore Larson, Brittnacher Mitchell, Malmström Lars, Goodlett David R
Medicinal Chemistry Department, University of Washington, Box 335351, Seattle, Washington 98195, USA.
Aquat Microb Ecol. 2009 Jun 3;55(3):241-253. doi: 10.3354/ame01284.
Diatoms play a critical role in the oceans' carbon and silicon cycles; however, a mechanistic understanding of the biochemical processes that contribute to their ecological success remains elusive. Completion of the Thalassiosira pseudonana genome provided 'blueprints' for the potential biochemical machinery of diatoms, but offers only a limited insight into their biology under various environmental conditions. Using high-throughput shotgun proteomics, we identified a total of 1928 proteins expressed by T. pseudonana cultured under optimal growth conditions, enabling us to analyze this diatom's primary metabolic and biosynthetic pathways. Of the proteins identified, 70% are involved in cellular metabolism, while 11% are involved in the transport of molecules. We identified all of the enzymes involved in the urea cycle, thereby describing the complete pathway to convert ammonia to urea, along with urea transporters, and the urea-degrading enzyme urease. Although metabolic exchange between these pathways remains ambiguous, their constitutive presence suggests complex intracellular nitrogen recycling. In addition, all C(4) related enzymes for carbon fixation have been identified to be in abundance, with high protein sequence coverage. Quantification of mass spectra acquisitions demonstrated that the 20 most abundant proteins included an unexpectedly high expression of clathrin, which is the primary structural protein involved in endocytic transport. This result highlights a previously overlooked mechanism for the inter- and intra-cellular transport of nutrients and macromolecules in diatoms, potentially providing a missing link to organelle communication and metabolite exchange. Our results demonstrate the power of proteomics, and lay the groundwork for future comparative proteomic studies and directed analyses of specifically expressed proteins and biochemical pathways of oceanic diatoms.
硅藻在海洋碳和硅循环中起着关键作用；然而，对于促成其生态成功的生化过程的机制性理解仍然难以捉摸。拟南芥基因组的完成提供了硅藻潜在生化机制的“蓝图”，但对于它们在各种环境条件下的生物学特性仅提供了有限的见解。利用高通量鸟枪法蛋白质组学，我们鉴定出了在最佳生长条件下培养的拟南芥总共表达的1928种蛋白质，这使我们能够分析这种硅藻的主要代谢和生物合成途径。在鉴定出的蛋白质中，70%参与细胞代谢，而11%参与分子运输。我们鉴定出了尿素循环中涉及的所有酶，从而描述了将氨转化为尿素的完整途径，以及尿素转运蛋白和尿素降解酶脲酶。尽管这些途径之间的代谢交换仍不明确，但它们的组成性存在表明存在复杂的细胞内氮循环。此外，所有与碳固定相关的C(4)酶都被鉴定为大量存在，蛋白质序列覆盖率高。质谱采集的定量分析表明，20种最丰富的蛋白质中包括衣被蛋白的意外高表达，衣被蛋白是参与内吞运输的主要结构蛋白。这一结果突出了硅藻中营养物质和大分子细胞间和细胞内运输这一先前被忽视的机制，可能为细胞器通讯和代谢物交换提供了一个缺失的环节。我们的结果证明了蛋白质组学的强大作用，并为未来海洋硅藻的比较蛋白质组学研究以及对特异性表达蛋白质和生化途径的定向分析奠定了基础。
