Proteins and Membrane Compartments involved in Silica Biogenesis

硅藻利用物种特有的蛋白质来产生它们特有的生物硅结构。阻碍我们理解生物二氧化硅形态发生的两个主要瓶颈是：(a)分离生物二氧化硅形成的细胞内隔室（二氧化硅沉积囊泡= sdv），以及(b)缺乏与系统发育相关但形态不同的硅藻物种（假海硅藻，T. oceanica，隐环硅藻）的生物二氧化硅相关蛋白质组学信息。我们已经鉴定了超过26种新的生物二氧化硅相关蛋白和13种不同的翻译后修饰赖氨酸，它们对生物二氧化硅的形成很重要。在下一个资助期内，我们将使用全离子碎片LC-MS/MS将翻译后修饰映射到三种生物硅体蛋白质中的特定赖氨酸残基。我们进一步鉴定了一个新的保守的硅藻膜蛋白，硅蛋白-1 (Sin1)，并证明它是假单胞菌SDV膜的一个完整蛋白。它的n端结构域负责通过与sdv内的有机基质结合将Sin1结合到生物二氧化硅中。体外实验表明，Sin1的重组n端结构域通过ph触发组装成大簇，并通过与长链多胺的协同作用促进二氧化硅的形成。Sin1是SDV膜控制生物二氧化硅形成有机基质的分子链接。Sin1的发现为sdv及其相关膜室的分离和生化表征提供了新的机会，这将在下一个资助期进行。膜相关Sin1的结构和功能将在体外和体内进行表征。体内实验将包括敲除或敲除Sin1，并分析由此产生的突变体是否存在生物矿化缺陷。与Baldus （SP-4）和Steinem （SP-7）团队合作，将确定Sin1的三维结构，并在体外研究脂质双分子层结合Sin1的自组装性质和矿物形成活性。

Diatoms employ species-specific sets of proteins to generate their characteristic biosilica structures. The two major bottlenecks that hamper our understanding of biosilica morphogenes are (a) isolating the intracellular compartments for biosilica formation (silica deposition vesicles = SDVs), and (b) the lack of information on biosilica-associated proteomes from phylogenetically related, yet morphologically distinct diatom species (Thalassiosira pseudonana, T. oceanica, Cyclotella cryptica). We have identified more than 26 new biosilica-associated proteins and 13 different post-traslationally modified lysines that are important for biosilica formation. In the next funding period we will map the post-translational modifications to specific lysine residues in proteins of the three biosilicomes using all-ions fragmentation LC-MS/MS.We have further identified a novel conserved diatom membrane protein, silicanin-1 (Sin1), and demonstrated that it is an integral protein of SDV membranes in T. pseudonana. Its N-terminal domain is responsible for incorporating Sin1 into the biosilica via association with the organic matrix inside the SDVs. In vitro experiments showed that the recombinant N-terminal domain of Sin1 undergoes pH-triggered assembly into large clusters, and promotes silica formation by synergistic interaction with long-chain polyamines. Sin1 is a molecular link by which SDV membranes exert control on the assembly of biosilica forming organic matrices. The discovery of Sin1 has provided new opportunities for isolation and biochemical characterization of SDVs and related membrane compartments, which will be pursued in the next funding period. Structure and function of membrane-associated Sin1 will be characterized in vitro and also in vivo. The in vivo experiments will include knocking-down or knocking-out of Sin1 and analyzing the resulting mutants for deficiencies in biomineralization. In collaboration with the Baldus (SP-4) and Steinem (SP-7) groups the 3D structure of Sin1 will be determined, and the self-assembly properties and mineral-forming activity of lipid bilayer bound Sin1 will be investigated in vitro.