To determine the size of the p44 transcript and also to detect its up-regulation upon deflagellation, total RNA was isolated from wild-type cells 45 min after deflagellation. results suggest that inner-arm dynein d and VEGFA its subunit organization are widely conserved. Ciliary and flagellar beating is driven by axonemal dyneins that are contained in the outer and inner dynein arms. Several lines of evidence have indicated that the outer-arm and inner-arm dyneins are functionally distinct and differ totally in their subunit organization and arrangement in the axoneme (for a review, see reference 8). In mutant, which has a mutation in the p28 gene and lacks dyneins a, c, and d (7), or from the mutant, which has a mutation in the gene for conventional actin and lacks dyneins a, c, d, and e (12, 13). It seemed likely that the 44- and 38-kDa proteins were subunits of dynein d. We have recently cloned the cDNA of the 38-kDa protein and shown that it is actually associated with isolated dynein d. This protein has been registered in the flagellar proteome database (17) as FAP146 and has been described as a zinc finger-like flagellum-associated protein (24). In the present study, we cloned and sequenced the cDNA of the 44-kDa protein (p44). Our results suggest that it functions together with p38 in the docking of dynein d to the outer doublet microtubules. Homologues of this protein, as well as those of p38, are found in a wide variety of organisms with motile cilia and flagella, indicating that dyneins structurally related to dynein d are widely conserved and serve some unique functions in cilia and flagellar motility. MATERIALS AND METHODS Strains and culture. The following strains were used: 137c (wild type) and the (lacking outer-arm dynein) (10), (lacking inner-arm dynein f) (9), (lacking inner-arm dyneins a, c, and d) (9), (lacking inner-arm dyneins a, c, d, and e) (12), and (lacking inner-arm dynein e) (12) mutants. Cells were grown in liquid Tris-acetic acid-phosphate (TAP) medium with aeration on a cycle of 12 h of light and 12 h of darkness. Isolation of axonemes. Flagella were isolated by the dibucaine method of Witman (21) and were demembranated to yield axonemes by extraction with 0.2% Nonidet P-40 in HMDEK solution (30 mM HEPES, 5 mM MgSO4, 1 mM dithiothreitol, 1 mM EGTA, and 50 mM potassium acetate [pH 7.4]). Crude dynein extract and isolation of dynein. Crude extracts containing various dyneins were obtained by high-salt extraction of wild-type or axonemes as described IRAK-1-4 Inhibitor I elsewhere (23) and were fractionated into individual dynein species by chromatography on a Uno Q ion-exchange column (Bio-Rad). We used a Uno Q column instead of a Mono Q column to achieve better separation of dynein d. Sucrose density gradient centrifugation. A crude dynein extract from wild-type or axonemes was layered on top of a 4.9-ml linear 5 to 20% sucrose density gradient that was prepared in HMDEK solution containing 0.2 mM protease inhibitor (Pefabloc). The gradients were centrifuged in a Hitachi RPS55T-2 swing rotor at 180,000 for 5.5 h at 4C. Catalase (11.4S), aldolase (7S), bovine IRAK-1-4 Inhibitor I serum albumin (BSA) (4.4S), and RNase A (2S) were also centrifuged as sedimentation coefficient markers on separate sucrose gradients prepared in HMDEK solution. Thirteen fractions (400 l each) were collected. Protein identification. The p44 protein band of dynein d was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and was subsequently excised from the gels and digested with trypsin. The peptide mixture was then eluted and analyzed using an oMALDI-Qq-TOF MS/MS IRAK-1-4 Inhibitor I QSTAR IRAK-1-4 Inhibitor I Pulsar (Applied Biosystems). The data were then used to search the genome database (JGI, version 2.0; http://genome.jgi-psf.org/chlre2/chlre2.home.html) using the Mascot search algorithm (http://www.matrixscience.com/) to identify the genomic sequences that encode each peptide. Determination of the cDNA sequence of p44. The sequences at the 5 and 3 ends of the p44 cDNA were obtained following RACE (rapid amplification of cDNA ends) PCR. The primers used were as follows: for 5 RACE, forward primer AUAP (5-GGCCACGCGTCGACTAGTAC-3) and reverse primer p44-R1 (5-GCAGCAGACCCAGAGCCT-3); for nested PCR, forward primer AUAP and reverse primer p44-R9 (5-GGTTGTGTGTGAGGCTGAAA-3); for 3 RACE, forward primer p44-F1 (5-GAAGCTGCACAACCTCATTGC-3) and reverse primer AUAP; and for nested PCR, forward primer p44-F2 (5-GGCTATCGCAGGACACACAG-3) and reverse primer AUAP. The resulting sequence of the 3 RACE was confirmed using primers p44-F5 (5-GAACCTCACCACAGTGTACCTGAGAC-3), p44-F9 (5-GCGGTGTCGTACTTTGAGAAG-3), and p44-R6 (5-CTTCCGCTCTGGTCTACATTAGTTCC-3). The cDNA used had been.