axonemal central apparatus (Wargo et al., 2005). Here, we report the identities and localization of three polypeptides comprising the second CaM-containing complex and provide data support- ing the hypothesis that this complex plays an important role in modulating the activity of specific subsets of dynein arms. Results CaM immunoprecipitation and peptide identiﬁcation To identify CaM-containing complexes within the axoneme, we used anti-CaM antibodies in immunoprecipitation experi- ments (Wargo et al., 2005). Using extracts isolated from mutant axonemes lacking the radial spokes (pf14), our anti-CaM anti- bodies precipitated a total of eight polypeptides in addition to CaM (Fig. 1; see Fig. 4 a in Wargo et al., 2005). Five poly- peptides form a complex that includes PF6 and localizes to the C1a projection of the central apparatus (Wargo et al., 2005). However, the three additional polypeptides that are precipitated (designated CaM-IP2, -IP3, and -IP4; Fig. 1) are present in all central apparatus–defective mutants, including pf6. Therefore, these three polypeptides are not components of the radial spokes or central apparatus. These three polypeptides are also precipi- tated from extracts isolated from mutant axonemes lacking the outer dynein arms and inner arm I1 (pf28pf30; Fig. 1) as well as the inner arm–defective strains mia1 and mia2 (not depicted), Figure 1. Silver-stained gels of immunoprecipitation experiments using anti- CaM antibodies and extracts isolated from wild-type, radial spokeless (pf14), and central pairless (pf18) axonemes as well as axonemes lacking both the outer dynein arms and inner arm isoform I1 (pf28pf30). The precipitates were resolved on either a 7% (top) or 12% (bottom) acrylamide gel. Central apparatus proteins are indicated by dots; spoke proteins are indicated by triangles. CaM-IP2, -IP3, and -IP4 are indicated by asterisks. The darkly stained bands are the heavy (HC) and light (LC) chains from the antibodies. CaM-IP2, -IP3, and -IP4 are precipitated from all of the mutant extracts tested, indicating that these polypeptides are not components of the radial spokes, central apparatus, outer dynein arms, or inner dynein arm I1. 516 JCB • VOLUME 179 • NUMBER 3 • 2007 indicating that they do not localize to these dynein subforms. All three polypeptides are also precipitated from the move backward–only strains mbo1 and mbo2 (unpublished data), indicating that the assembly of these polypeptides is unaffected in these mutant strains. To determine the identities of CaM-IP2, -IP3, and -IP4, these polypeptides were excised from gels and analyzed by mass spectrometry (see Materials and methods; data summarized in Table I). The identity of CaM-IP2 was determined from four peptide sequences. Based on searches of the C. reinhardtii genome database (version 3.0; http://genome.jgi-psf.org/Chlre3/ Chlre3.home.html), the CaM-IP2 gene is located on the same contig as Zsp1; therefore, the CaM-IP2 gene is most likely on linkage group VII. In searches of the C. reinhardtii proteome database, CaM-IP2 is flagellar-associated protein 91 (FAP91; Pazour et al., 2005). BLAST searches reveal that CaM-IP2 (FAP91) is most similar to the human homologue of AAT-1 (C3orf15; GenBank/EMBL/DDBJ accession no. AAH35238; E value of E = 7e-49). AAT-1 was originally identified as a testis-specific protein in mice that forms a quaternary complex with AMY-1 (a c-myc–binding protein), an A-kinase anchoring protein (AKAP), and two regulatory subunits of PKA (cAMP- dependent protein kinase; Yukitake et al., 2002). More recently, a total of seven alternatively spliced isoforms of AAT-1 have been identified, and some of these isoforms are expressed in a va- riety of human tissues (Matsuda et al., 2005). The largest isoform of AAT-1, AAT-1L, is an 90-kD protein. The testis-specific isoform AAT-1α is reported to contain only the C-terminal 98 amino acids of AAT-1L. CaM-IP2 is 32% identical and 49% similar to AAT-1 over a stretch of 423 amino acids at the N terminus (Fig. S1, avail- able at http://www.jcb.org/cgi/content/full/jcb.200703107/DC1). Sequence similarity with AAT-1 may extend beyond the N ter- minus of the protein, but the C-terminal half of the CAM-IP2 sequence is not represented in the C. reinhardtii genome. We have tried to complete the CaM-IP2 coding sequence using a variety of approaches, including 5′ and 3′ rapid amplification of cDNA ends, obtaining small genomic fragments of CaM-IP2 for sequencing, and optimizing our sequencing reactions for GC-rich sequences. We have confirmed the predicted amino acid sequence for the N-terminal one third of the protein but were unsuccessful in obtaining the complete coding sequence. As judged by SDS-PAGE, the apparent molecular mass of CaM-IP2 is 183 kD, which is almost double that of AAT-1L. However, Northern blots of RNA isolated from wild-type cells indicate that the CaM-IP2 transcript is 4.4 kb (Fig. S2). Based on our experience comparing C. reinhardtii genomic and cod- ing sequences, the true molecular weight of this protein is most likely no greater than 120 kD. The identity of CaM-IP3 was determined from the amino acid sequence of five peptides. Based on database searches, CaM- IP3 is located on linkage group III and corresponds to FAP61. Using corresponding cDNA sequences in the C. reinhardtii expressed sequence tag (EST) database and RT-PCR, we de- termined that the CaM-IP3 gene encodes a 1,115–amino acid protein that contains a predicted NADH dehydrogenase domain. This domain is found in both class I and II oxidoreductases as well