Immunofluorescence localization of a 25-kDa Tetrahymena EF-hand Ca(2+)-binding protein, TCBP-25, in the cell cortex and possible involvement in conjugation

The Tetrahymena Ca(2+)-binding protein of 25 kDa (TCBP-25) is a member of the calmodulin family containing four EF-hand Ca(2+)-binding loops, but its biological role has not yet been investigated. In this study, TCBP-25 was expressed in Escherichia coli as a glutathione S-transferase fusion protein and then purified. Purified TCBP-25 showed a typical Ca(2+)-dependent shift in electrophoretic mobility, consistent with conformational change caused by Ca(2+)-binding. Localization of TCBP-25 was examined by indirect immunofluorescence using an antiserum specific for TCBP-25. Strong immunofluorescence was observed all over the cell cortex except in and around basal bodies. From the results of immunofluorescence using detergent-extracted cells, TCBP-25 is suggested to exist as an insoluble form in the cell cortex. TCBP-25 appears to be localized in the cortical alveoli or the epiplasm and exists around both the migratory and the stationary gametic pronuclei at the pronuclear exchange stage during conjugation. Therefore, we speculate that TCBP-25 may play crucial roles in Ca(2+)-mediated signaling processes in the cell cortex and in a Ca(2+)-dependent pronuclear exchange process during conjugation.