IMMUNOFLUORESCENCE LOCALIZATION OF A 25-KDA TETRAHYMENA EF-HAND CA2-BINDING PROTEIN, TCBP-25, IN THE CELL CORTEX AND POSSIBLE INVOLVEMENT IN CONJUGATION()

The Tetrahymena Ca2+-binding protein of 25 kDa (TCBP-25) is a member o f the calmodulin family containing four EF-hand Ca2+-binding loops, bu t its biological role has not yet been investigated. In this study, TC BP-25 was expressed in Escherichia coli as a glutathione S-transferase fusion protein and then purified. Purified TCBP-25 showed a typical C a2+-dependent shift in electrophoretic mobility, consistent with confo rmational change caused by Ca2+-binding. Localization of TCBP-25 was e xamined by indirect immunofluorescence using an antiserum specific for TCBP-25. Strong immunofluorescence was observed all over the cell cor tex except in and around basal bodies, From the results of immunofluor escence using detergent extracted cells, TCBP-25 is suggested to exist as an insoluble form in the cell cortex. TCBP-25 appears to be locali zed in the cortical alveoli or the epiplasm and exists around both the migratory and the stationary gametic pronuclei at the pronuclear exch ange stage during conjugation. Therefore, we speculate that TCBP-25 ma y play crucial roles in Ca2+-mediated signaling processes in the cell cortex and in a Ca2+-dependent pronuclear exchange process during conj ugation. (C) 1995 Academic Press, Inc.