Proteins and Membrane Compartments involved in Silica Biogenesis

硅藻利用物种特异性的蛋白质组来产生其特有的生物硅结构。阻碍我们理解生物硅形态基因的两个主要瓶颈是（a）分离用于生物硅形成的细胞内区室（硅沉积囊泡= SDV），和（B）缺乏关于来自生物硅遗传学相关但形态学上不同的硅藻物种的生物硅相关蛋白质组的信息（Thalassiosira damana，T. oceanica、小环藻（Cyclotella cryptica）。我们已经确定了超过26种新的生物硅相关蛋白和13种不同的翻译后修饰的赖氨酸，这对生物硅的形成很重要。在下一个资助期内，我们将使用全离子裂解LC-MS/MS来定位三种生物硅体蛋白中特定赖氨酸残基的翻译后修饰。我们进一步鉴定了一种新的保守硅藻膜蛋白silicanin-1（Sin 1），并证明它是T.是的其N-末端结构域负责通过与SDV内的有机基质结合将Sin 1并入生物二氧化硅中。体外实验表明，重组的Sin 1的N-末端结构域进行pH触发组装成大的集群，并促进二氧化硅形成的协同作用与长链多胺。Sin 1是SDV膜对生物二氧化硅形成有机基质的组装施加控制的分子连接。Sin 1的发现为SDV和相关膜隔室的分离和生物化学表征提供了新的机会，这将在下一个资助期内继续进行。膜相关Sin 1的结构和功能将在体外和体内进行表征。体内实验将包括敲低或敲除Sin 1，并分析所得突变体的生物矿化缺陷。与Baldus（SP-4）和Steinem（SP-7）团队合作，将确定Sin 1的三维结构，并在体外研究脂质双层结合Sin 1的自组装特性和矿物形成活性。

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.