Identification of proteins from a cell wall fraction of the diatom Thalassiosira pseudonana -: Insights into silica structure formation
Identification of proteins from a cell wall fraction of the diatom Thalassiosira pseudonana -: Insights into silica structure formation
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DOI：
10.1074/mcp.m500174-mcp200
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发表时间：
2006-01-01
影响因子：
7
通讯作者：
Hildebrand, M
中科院分区：
文献类型：
--
作者：
Frigeri, LG;Radabaugh, TR;Hildebrand, M
硅藻是单细胞真核藻类，细胞壁含有二氧化硅，在纳米尺度上结构复杂而华丽。通过膜结合的二氧化硅沉积囊泡的膨胀和成型形成整体二氧化硅结构。虽然二氧化硅聚合的分子细节正在澄清，我们有有限的洞察到二氧化硅沉积囊泡的分子组成部分，特别是膜相关的蛋白质，可能参与结构形成。为了鉴定这些蛋白质，我们改进了现有的程序，从第一个测序基因组的硅藻海链藻中分离出富集的细胞壁部分。我们应用串联质谱分析，这部分，确定31蛋白质作进一步评估。在细胞周期的同步进展过程中监测编码这些蛋白质的基因的mRNA水平，并与先前鉴定的两个具有不同mRNA模式的silaffin基因（参与二氧化硅聚合）进行比较，所述mRNA模式用作细胞壁形成的标志物。在鉴定的31种蛋白质中，10种具有与silaffins相关的mRNA模式，13种具有与silaffins无关的模式，7种具有相关但也显示出额外特征的模式。这些蛋白质在细胞壁合成的可能参与进行了讨论。特别是，谷氨酸乙酰转移酶被确定，促使多胺生物合成途径中的其他基因的mRNA模式的分析和细胞壁合成过程中诱导的那些识别。鸟氨酸脱羧酶的特定酶抑制剂的应用导致二氧化硅结构的显著改变，证实了多胺的参与，并表明操纵参与细胞壁合成的蛋白质可以改变结构。据我们所知，这是第一次对硅藻进行蛋白质组学分析，此外，我们确定了参与结构形成的新候选基因，并直接证明了一种酶（及其基因）参与结构形成过程。
Diatoms are unicellular eucaryotic algae with cell walls containing silica, intricately and ornately structured on the nanometer scale. Overall silica structure is formed by expansion and molding of the membrane-bound silica deposition vesicle. Although molecular details of silica polymerization are being clarified, we have limited insight into molecular components of the silica deposition vesicle, particularly of membrane-associated proteins that may be involved in structure formation. To identify such proteins, we refined existing procedures to isolate an enriched cell wall fraction from the diatom Thalassiosira pseudonana, the first diatom with a sequenced genome. We applied tandem mass spectrometric analysis to this fraction, identifying 31 proteins for further evaluation. mRNA levels for genes encoding these proteins were monitored during synchronized progression through the cell cycle and compared with two previously identified silaffin genes ( involved in silica polymerization) having distinct mRNA patterns that served as markers for cell wall formation. Of the 31 proteins identified, 10 had mRNA patterns that correlated with the silaffins, 13 had patterns that did not, and seven had patterns that correlated but also showed additional features. The possible involvements of these proteins in cell wall synthesis are discussed. In particular, glutamate acetyltransferase was identified, prompting an analysis of mRNA patterns for other genes in the polyamine biosynthesis pathway and identification of those induced during cell wall synthesis. Application of a specific enzymatic inhibitor for ornithine decarboxylase resulted in dramatic alteration of silica structure, confirming the involvement of polyamines and demonstrating that manipulation of proteins involved in cell wall synthesis can alter structure. To our knowledge, this is the first proteomic analysis of a diatom, and furthermore we identified new candidate genes involved in structure formation and directly demonstrated the involvement of one enzyme ( and its gene) in the structure formation process.