Figure 7. Western blots of isolated axonemes (10 g/lane) probed with antibodies gener- ated against the I1 intermediate chain IC138. Intermediate chain IC138, left. As indicated, some axoneme samples were treated with ei- ther anti–CaM-IP2 or anti–C1a-32 antibodies (C1a; Wargo et al., 2005) in the presence or absence of calf intestinal alkaline phosphatase (CIP). In the absence of calf intestinal alkaline phosphatase treatment, IC138 appears hyper- phosphorylated in both central pairless (pf18) and spokeless (pf14) axonemes compared with wild type. No observable difference in phos- phorylation is detected after the addition of CaM-IP2 antibodies. The 120–150-kD molecular mass range of Coomassie blue–stained gels of the same samples are shown as a loading con- trol (right). possesses a single CaM-IP2 transcript that increases in abun- dance after deflagellation. Therefore, in C. reinhardtii, CaM-IP2 is most likely present in a single isoform that primarily local- izes to the flagellum. The presence of multiple splice variants of axonemal proteins in mammals, which are not observed for their C. reinhardtii homologues, has been previously reported (Zhang et al., 2002). The fact that CaM-IP2 is associated with both CaM and an AKAP (RSP3) raises the intriguing possibility that this com- plex may play a role in integrating the calcium- and cAMP- mediated signaling pathways to produce particular changes in motility. Yukitake et al. (2002) found that mammalian AAT-1α weakly stimulated the activity of PKA and that AAT-1α itself was phosphorylated by PKA in vivo and in vitro. Although bona fide PKA homologues are not represented in the C. reinhardtii flagellar proteome, several AKAPS as well as proteins with domains similar to the PKA RII subunit have been identified (Gaillard et al., 2001; Yang et al., 2006). In addition, pharma- cological and functional studies have implicated a role for PKA in modulating motility (Howard et al., 1994; Habermacher and Sale, 1995, 1997; Gaillard et al., 2006). We are currently in- vestigating whether CaM-IP2 is posttranslationally modified in vivo in C. reinhardtii. The presence of an NADH dehydrogenase domain in CaM-IP3 (FAP61) is suggestive of a role for this protein in energy conversion. This domain is found in oxidoreductases as well as in NADH oxidases and peroxidases. Wakabayashi and King (2006) have recently shown that in C. reinhardtii, motility changes that occur in response to light are altered by oxidative/reductive stress and that this effect is mediated by the redox poise of the outer dynein arms (Wakabayashi and King, 2006). Given that motility changes in response to light are generally preceded by changes in intraflagellar calcium, that CaM-IP3 is part of a complex that contains CaM, and that CaM-IP3 possesses an NADH dehydro- genase domain, one possibility is that CaM-IP3 plays a role in mediating calcium-induced changes in the flagellar redox state that affect the activity of the outer dynein arms. CaM-IP2, -IP3, and -IP4 form a CaM- and spoke-associated complex The conclusion that the CaM-IP2, -IP3, and -IP4 complex is associated with and localizes to the base of the radial spokes is 522 JCB • VOLUME 179 • NUMBER 3 • 2007 based on the following observations. First, antibodies raised against CaM-IP2 precipitate all members of this complex as well as the radial spokes. Second, the complex cosediments with the radial spokes and spoke stalks on sucrose gradients of axonemal extracts. Finally, gel overlay assays using recom- binant RSP3 reveal that RSP3 binds specifically to CaM-IP2. Previous studies have shown that mutations in RSP3 (pf14) re- sult in the failure of the entire radial spoke to assemble (Huang et al., 1981) and that recombinant RSP3 binds to pf14 axonemes (Diener et al., 1993). Therefore, RSP3 is predicted to localize to the base of the spoke and serve as a point of stalk attachment to the axonemal microtubule. Based on the clear association of the CaM-IP2, -IP3, and -IP4 complex with both CaM and the radial spokes, we propose to use the acronym CSC (CaM- and spoke-associated complex) to refer to this complex. Given that the CSC is associated with the radial spokes in wild-type axonemal extracts, it may seem surprising that this complex was not identified in analyses of the radial spoke pro- teome (Yang et al., 2006). However, two important factors indi- cate that this complex is not part of the spoke structures. First, the pf14 mutation results in the complete failure of the spokes to assemble; therefore, comparisons of polypeptides lacking in this mutant with those present in wild-type axonemes have his- torically defined radial spoke components (Huang et al., 1981; Piperno et al., 1981). Because we initially identified this com- plex in immunoprecipitation experiments using axonemal ex- tracts isolated from pf14 axonemes, its presence in this mutant would eliminate the possibility that it is part of the radial spoke structure. Second, although candidate spots possibly correspond- ing to these proteins are visible on the two-dimensional gels of isolated spokes by Yang et al. (2001), they are clearly present in substoichiometric amounts relative to other spoke components. Therefore, if these spots are in fact CSC components, it is pos- sible that not every spoke is associated with a CSC. Given their presence in spokeless mutants and the fact that they may be pres- ent in substoichiometric amounts relative to spoke proteins, the CSC polypeptides do not appear to be components of the spoke structures. Experiments are currently underway to define the pre- cise stoichiometry of this complex to radial spokes as well as their more precise localization within the axoneme. Based on the apparent localization of the CSC to the base of the spokes, the mutant phenotype for members of this