Proteins and Membrane Compartments involved in Silica Biogenesis

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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.