The cilia of Paramecium tetraurelia contain both Ca2+-dependent and Ca2+-inhibitable calmodulin-binding proteins.
To identify protein targets for calmodulin (CaM) in the cilia of Paramecium tetraurelia, we employed a 125I-CaM blot assay after resolution of ciliary proteins on SDS/polyacrylamide gels. Two distinct types of CaM-binding proteins were detected. One group bound 125I-CaM at free Ca2+ concentrations above 0.5-1 microM and included a major binding activity of 63 kDa (C63) and activities of 126 kDa (C126), 96 kDa (C96), and 36 kDa (C36). CaM bound these proteins with high (nanomolar) affinity and specificity relative to related Ca2+ receptors. The second type of protein bound 125I-CaM only when the free Ca2+ concentration was below 1-2 microM and included polypeptides of 95 kDa (E95) and 105 kDa (E105). E105 may also contain Ca2+-dependent binding sites for CaM. Both E95 and E105 exhibited strong specificity for Paramecium CaM over bovine CaM. Ciliary subfractionation experiments suggested that C63, C126, C96, E95, and E105 are bound to the axoneme, whereas C36 is a soluble and/or membrane-associated protein. Additional Ca2+-dependent CaM-binding proteins of 63, 70, and 120 kDa were found associated with ciliary membrane vesicles. In support of these results, filtration binding assays also indicated high-affinity binding sites for CaM on isolated intact axonemes and suggested the presence of both Ca2+-dependent and Ca2+-inhibitable targets. Like E95 and E105, the Ca2+-inhibitable CaM-binding sites showed strong preference for Paramecium CaM over vertebrate CaM and troponin C. Together, these results suggest that CaM has multiple targets in the cilium and hence may regulate ciliary motility in a complex and pleiotropic fashion.
Evans
T C
TC
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison 53706.
Nelson
D L
DL
D008841:Actin Cytoskeleton / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002923:Cilia / Q000378:metabolism* / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D010247:Paramecium / Q000378:metabolism*
D002148:Calmodulin-Binding Proteins; D002118:Calcium