Characterization of the fenestrin, a cytoskeletal protein involved in Tetrahymena cell polarity and of its coding gene

Fenestrin is a cytoskeletal protein, which participates in conjugation and cell cycle regulation of Tetrahymena by formation of specific structures during these processes. In cell cycle it appears at the anterior poles of future proter and opisth cells. By 2-D electrophoresis, we found that in starved T. thermophila cells fenestrin appears in four isoforms that have the pH in the range of 5.2–5.5. The differences of pH of about 0.1–0.3 between isoforms could suggest that they differ by the phosphorylation state. By mass spectrometry, we identified some of the peptides derived form the enzyme digestion of fenestrin. These peptides were used for searching through T. thermophila genome, available at the site http://www.tigr.org. A nucleotide sequence coding one of the peptides was initially found. Then, by extension, the sequence of all fenestrin gene appeared, containing the other peptides identified by spectrometry. By RT-PCR we characterized one intron in this gene. The analysis of the aminoacid sequence of the exons revealed that fenestrin is composed from at least two domains: N-terminal leucine-rich domain and C-terminal glutamic acid-rich domain. The BLAST search on general databases showed the existence of human and mouse proteins homologous to fenestrin in sequence, molecular mass and domain organization. Thus, fenestrin appears to be a protein correlated to cell polarity with an evolutionarily conserved structure. Its function should be cleared by mutational analysis and gene knock out.