Table I. Proteins precipitated with anti-CaM antibodies Name CaM-IP2 CaM-IP3 CaM-IP4 Predicted molecular mass ND 118 kD 97 kD Apparent molecular mass 183 kD 140 kD 100 kD pI ND 4.8 7.4 Similarities AAT-1, AKAP-binding protein pyridine-disulﬁde oxidoreductase domain WD repeats Flagellar proteome FAP91 FAP61 FAP251 Linkage group VII III III as NADH oxidases and peroxidases. The domain includes a small NADH-binding domain within a flavin adenine dinucleotide– binding domain and, thus, is thought to be involved in energy conversion. Searches of sequence databases reveal that the pre- dicted human protein C20orf26 is most similar to CaM-IP3 (E value of E = 6.8e-56), sharing 25% identity and 43% amino acid similarity along the entire length of the protein. The pre- dicted molecular mass of CaM-IP3 is 118 kD, although the protein has an apparent molecular mass of 140 kD as judged by SDS-PAGE. The acidic isoelectric point of CaM-IP3 (4.76) may partially account for this discrepancy. The identity of CaM-IP4 was determined from the amino acid sequence of five peptides. Based on database searches, CaM-IP4 is also located on linkage group III and corresponds to FAP251. CaM-IP4 contains seven WD-40 repeats that com- prise much of the N terminus of the protein. WD-40 repeats form propeller-like structures that serve as an interface for protein–protein interactions (Neer et al., 1994). In searches of sequence databases, a protein predicted from the Leishmania database (GenBank/EMBL/DDBJ accession no. XM811714; E value of E = 9e-53) is most similar to CaM-IP4 with 29% amino acid identity and 43% similarity along the entire length of this protein. CaM-IP4 is also 24% similar and 39% identical to a protein predicted from the human genome sequence (Gen- Bank/EMBL/DDBJ accession no. BC036233; E value of E = 6e-37). All of these polypeptides show similarity to micro- tubule-associated protein–like 5 from echinoderms. In addition to the WD-40 repeats, each of these proteins has a putative EF- hand domain at the C terminus. Although a motifs search does not predict an EF hand at the C terminus of CaM-IP4, the Inter- ProScan algorithm provided by the European Molecular Biol- ogy Laboratory predicts an EF hand–like domain. CaM-IP2, -IP3, and -IP4 form a single complex Based on CaM immunoprecipitation, it is possible that CaM- IP2, -IP3, and -IP4 bind CaM individually, in subsets, or together to form a single complex. To differentiate among these possi- bilities, we developed polyclonal antibodies in rabbits against either bacterially expressed protein fragments or synthetic pep- tides for each of CaM-IP2, -IP3, and -IP4 (see Materials and methods). CaM-IP4 proved to be nonantigenic. However, we were successful in obtaining antibodies that specifically recog- nize either CaM-IP2 or CaM-IP3 on Western blots (Fig. 2 A). We then used these antibodies in immunoprecipitation experiments. Antibodies generated against CaM-IP3 failed to precipitate CaM-IP3. On the other hand, the CaM-IP2 antibody specifically precipitated CaM-IP2 as well as -IP3 and -IP4 from extracts isolated from radial spokeless axonemes (Fig. 2 B, middle lane). For extracts isolated from wild-type and pf18 axonemes, additional polypeptides are precipitated by the CaM-IP2 antibodies that are not precipitated from extracts of the spokeless mutant pf14. Based on the molecular weights of these polypeptides (Yang et al., 2001) as well as their absence from precipitates of pf14 axonemal extracts, we suspected that these polypeptides were radial spoke components. Corresponding Western blots using antibodies generated against radial spoke protein 2 (RSP2) and RSP3 confirm that RSPs are precipitated by the CaM-IP2 antibodies. The simplest interpretation of these Figure 2. CaM-IP2, -IP3, and -IP4 form a single complex that is associ- ated with the radial spokes. (A) Western blots of axonemes (8 μg per lane) probed with antibodies generated against CaM-IP2 and -IP3. Both polypep- tides are present in all axonemes in approximately equal amounts. (B) Silver- stained gel (top) and corresponding Western blots (middle and bottom) of immunoprecipitation experiments using anti–CaM-IP2 antibodies and wild- type, radial spokeless (pf14), and central pairless (pf18) axonemal extracts. CaM-IP2 antibodies precipitate the CaM-IP2, -IP3, and -IP4 complex (asterisks) as well as RSPs (triangles). Blots were probed with antibodies generated against CaM-IP2, -IP3, RSP2, or RSP3. These results indicate that CaM-IP2, -IP3, and -IP4 form a single complex that is associated with the radial spokes. REGULATION OF DYNEIN ACTIVITY • DYMEK AND SMITH 517