Analysis of Thalassiosira pseudonana silicon transporters indicates distinct regulatory levels and transport activity through the cell cycle

Marine Biology Research Division, Scripps Institution of Oceanography, 9500 Gilman Drive, La Jolla, CA 92093-0202 USA

^* To whom correspondence should be addressed. Email: mhildebrand{at}ucsd.edu.

	Abstract

Analysis of expression and activity of silicon transporters (SITs) was done on synchronously growing cultures of the diatom, Thalassiosira pseudonana, to provide insight into the role these proteins play in cellular silicon metabolism during the cell cycle. The first SIT-specific polyclonal peptide antibody was generated and used in Western blot analysis of whole-cell protein lysates to monitor SIT protein levels during synchronized progression through the cell cycle. Peaks in SIT protein levels correlated with active periods of silica incorporation into cell wall substructures. Quantitative real-time PCR on each of the three distinct SIT genes (TpSIT1-3) showed mRNA levels of the most highly expressed SITs peaked during S phase of the cell cycle, a period prior to maximal silicon uptake and during which cell wall silicification does not occur. Variation in protein and mRNA levels did not correlate, suggesting a significant regulatory step of SITs is at the translational or post-translational level. Surge uptake rates also did not correlate with SIT protein levels suggesting SIT activity was internally controlled by the rate of silica incorporation. This is the first study to characterize SIT mRNA and protein expression and cellular uptake kinetics during the course of the cell cycle and cell wall synthesis, and provides novel insight into SIT regulation.