Identification of Proteins from a Cell Wall Fraction of the Diatom Thalassiosira pseudonana Insights into Silica Structure Formation

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. Molecular & Cellular Proteomics 5:182–193, 2006. The silicified cell walls of the unicellular algae known as diatoms are fascinating products of biomineralization processes. Each diatom species constructs a uniquely structured cell wall called a frustule, consisting of valves and girdle bands (1, 2) composed of organic material and silica, which is often ornately and intricately shaped with fine features on the nanometer scale. Silica structures are made in the specialized membrane-bound intracellular compartment known as the silica deposition vesicle, or SDV (3–6). The SDV is a dynamic organelle that is expanded and molded during frustule formation, and these movements are essential to produce structures characteristic of the valve and girdle bands (1, 7–10). Upon completion, the entire silica structure is exocytosed to form an integral part of the cell wall (1). A first step to understand structure formation at the molecular level would be to isolate a purified SDV fraction and analyze its protein component. Unfortunately in over 3 decades of attempts, the SDV has not been purified. At best, researchers have isolated cell wall fractions enriched in components of the SDV (11, 12) but very likely also containing other constituents. Despite their incomplete purity, examination of cell wall fractions has yielded valuable insights into components involved in cell wall synthesis. Initial attempts relied on complete removal of surrounding cell wall organic material followed by dissolution of the silica with hydrofluoric acid (HF), suggesting that proteins were specifically associated with, and likely occluded within, the silica (12). More refined approaches were developed by Kroger et al. (11, 13), who initially identified calcium-binding cell wall coat proteins called frustulins extracted from the cell wall by EDTA treatment. They then examined proteins tightly associated with the silica by pretreatment with EDTA and SDS followed by dissolution of the silica with HF (and later ammonium fluoride) combined with chromatographic purification (14–17). These studies identified the pleuralins, proteins associated with the region of overlap between the two halves of the cell of the pennate diatom Cylindrotheca fusiformis (17, 18), the silaffins, one of the major components involved in silica polymerization (14, 19), and long chain polyamines, another major silica polymerization component (15). Continuing work highlights the importance of silaffins and polyamines From the ‡Marine Biology Research Division, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, California 92093-0202 and the §Department of Biochemistry, University of Arizona, Tucson, Arizona 85721 Received, June 7, 2005, and in revised form, October 3, 2005 Published, MCP Papers in Press, October 5, 2005, DOI 10.1074/ mcp.M500174-MCP200 1 The abbreviations used are: SDV, silica deposition vesicle; ASWT, artificial seawater tryptone; DAPDH, 1,3-diaminoproprane dihydrochloride; HF, hydrofluoric acid; bis-Tris, 2-[bis(2-hydroxyethyl)amino]2-(hydroxymethyl)propane-1,3-diol; SEM, scanning electron microscopy. Research