Figure 3. CaM-IP2 is associated with RSP3. Western blots of extracts iso- lated from wild-type (A), spoke headless (B; pf17), and spokeless (C; pf14) axonemes fractionated on sucrose gradients. Blots were probed with anti- bodies generated against CaM-IP2, -IP3, RSP2, RSP3, or CaM. In extracts isolated from both wild-type or pf17 axonemes, CaM-IP2 and -IP3 cosedi- ment with spoke stalk components. In extracts isolated from both pf17 and pf14 mutant axonemes, the sedimentation value for the CaM-IP2 complex is considerably lower than that in wild type. (D) Gel overlay of expressed RSP3. Bacterial extracts expressing fragments of CaM-IP2 (37 kD), -IP3 (32 kD), -IP4 (60 kD), and RSP3-GST (110 kD) were resolved on polyacryl- amide gels and either transferred to nitrocellulose (left) or stained with Coomassie blue (right). The membrane (left) was incubated with expressed RSP3 and probed with antibodies generated against RSP3. These results indicate that RSP3 binds to CaM-IP2. results is that CaM and CaM-IP2, -IP3, and -IP4 form a single complex that is associated with the radial spokes. CaM-IP2 binds to RSP3 To provide further evidence that the CaM-IP2, -IP3, and -IP4 complex is associated with the radial spokes, we investigated whether this complex cosediments with the radial spokes using sucrose density gradient centrifugation (Fig. 3). For axo- nemal extracts isolated from wild-type flagella, the polypep- tides comprising the radial spokes cosediment at 20S (Yang et al., 2001). Western blots of corresponding gradient fractions 518 JCB • VOLUME 179 • NUMBER 3 • 2007 reveal that CaM-IP2 and -IP3 cosediment with the radial spokes. For axonemal extracts isolated from flagella that lack the spoke heads (pf17), the polypeptides comprising the spoke stalks cosediment at 15S (Yang et al., 2001). Western blots of corresponding gradient fractions from pf17 extracts reveal that CaM-IP2 and -IP3 also cosediment with the radial spoke stalks (Fig. 3 B). For axonemal extracts isolated from flagella that completely lack the radial spokes (pf14), CaM-IP2 and -IP3 cosediment as a smaller complex at 11S (Fig. 3 C). Although we do not have antibodies that recognize CaM-IP4, immuno- precipitation experiments using sucrose gradient fractions con- firm that CaM-IP4 also cosediments with CaM-IP2, -IP3, and -IP4 (Fig. S3, available at http://www.jcb.org/cgi/content/full/ jcb.200703107/DC1). These results support the hypothesis that CaM, CaM-IP2, -IP3, and -IP4 form a single complex that is associated with the spoke stalk. As noted in our CaM-IP2 sequence analysis, CaM-IP2 is most similar to AAT-1, which is thought to form a quater- nary complex with an AKAP-binding protein. Based on previ- ous reports that RSP3 is an AKAP (Gaillard et al., 2001), that RSP3 is located at the base of the radial spoke stalk (Diener et al., 1993), and our results that CaM-IP2 cosediments with the spoke stalk (this study), we hypothesized that CaM-IP2 binds to RSP3. To test this hypothesis, we used a gel overlay assay. Bacterially expressed proteins (CaM-IP2, CaM-IP3, and RSP3) were resolved by SDS-PAGE and transferred to nitrocellulose membrane. The membrane was then incubated with bacterially expressed C. reinhardtii RSP3, and the overlain protein was de- tected by immunoblotting. As shown in Fig. 3 D, RSP3 binds only to CaM-IP2. These combined results indicate that CaM- IP2, -IP3, and -IP4 are associated with the radial spoke stalk via interactions between CaM-IP2 and RSP3. CaM binding is calcium sensitive The association of CaM with specific proteins is regulated by the calcium-binding state of CaM. All immunoprecipitation experiments were performed using low calcium conditions. Therefore, the eight polypeptides we precipitated do not re- quire calcium to form protein complexes with CaM. To inves- tigate whether CaM binding of these polypeptides is calcium sensitive, we first immunoprecipitated CaM from pf14 axo- nemal extracts under low calcium conditions; the protein A beads were then washed using high calcium buffer (see Materials and methods). CaM remained associated with the beads, whereas CaM-IP2, -IP3, and -IP4 were selectively extracted in the presence of high calcium (Fig. 4 A). These results demonstrate that the binding of CaM-IP2, -IP3, and -IP4 to CaM is calcium sensitive. We also performed immunoprecipitation experi- ments using the CaM-IP2 antibody under high calcium condi- tions. All three members of the complex are precipitated by the CaM-IP2 antibody in extracts from spokeless axonemes, indicating that the complex does not dissociate under high calcium conditions (Fig. 4 B). In addition, the CaM-IP2 anti- bodies precipitate the spokes from axonemal extracts isolated from either wild-type or central pairless axonemes, indicating that the complex does not dissociate from the spokes under high calcium conditions (Fig. 4 B).