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. 2002 Mar;13(3):1015-29.
doi: 10.1091/mbc.01-04-0201.

The Outer Dynein Arm-Docking Complex: Composition and Characterization of a Subunit (oda1) Necessary for Outer Arm Assembly

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The Outer Dynein Arm-Docking Complex: Composition and Characterization of a Subunit (oda1) Necessary for Outer Arm Assembly

Saeko Takada et al. Mol Biol Cell. .
Free PMC article

Abstract

To learn more about how dyneins are targeted to specific sites in the flagellum, we have investigated a factor necessary for binding of outer arm dynein to the axonemal microtubules of Chlamydomonas. This factor, termed the outer dynein arm-docking complex (ODA-DC), previously was shown to be missing from axonemes of the outer dynein armless mutants oda1 and oda3. We have now partially purified the ODA-DC, determined that it contains equimolar amounts of M(r) approximately 105,000 and approximately 70,000 proteins plus a third protein of M(r) approximately 25,000, and found that it is associated with the isolated outer arm in a 1:1 molar ratio. We have cloned a full-length cDNA encoding the M(r) approximately 70,000 protein; the sequence predicts a 62.5-kDa protein with potential homologs in higher ciliated organisms, including humans. Sequencing of corresponding cDNA from strain oda1 revealed it has a mutation resulting in a stop codon just downstream of the initiator ATG; thus, it is unable to make the full-length M(r) approximately 70,000 protein. These results demonstrate that the ODA1 gene encodes the M(r) approximately 70,000 protein, and that the protein is essential for assembly of the ODA-DC and the outer dynein arm onto the doublet microtubule.

Figures

Figure 1
Figure 1
ODA-DC composition and association with dynein. (A and B) SDS-PAGE analyses of fractions from sucrose density gradient centrifugations of high-salt extracts from axonemes of mutant strains oda6 (A) and oda3 (B). Oda6 axonemes have ODA-DCs but lack outer arms; oda3 axonemes lack both ODA-DCs and outer arms. Three proteins (Mr ∼105,000, ∼70,000, and ∼25,000, black arrowheads in A) cosediment at 7S in the sucrose gradient fractionation of the oda6 extract, but are specifically missing in comparable fractions from the mutant oda3 (B, white arrowheads). The left lane in each gel was loaded with molecular weight standards (97.4, 66.2, 45.0, 31.0, 21.5, and 14.4 kDa). Gradients shown in A and B were centrifuged under standard conditions in the absence of Mg2+ (Piperno and Luck, 1979; Pfister et al., 1982). (C) Immunoprecipitation of the ODA-DC in the absence of Mg2+. The anti-DC105 antibody was used to immunoprecipitate the complex from biotinylated 0.6 M KCl extracts of wild-type (wt) and oda1 (oda1) axonemes. The immunoprecipitated proteins were then separated by SDS-PAGE, transferred to nitrocellulose, and probed with streptavidin-HRP (lanes a–d). The anti-DC105 antibody immunoprecipitated three proteins (arrowheads, Mr ∼105,000, ∼70,000, ∼25,000) from wild-type axonemal extracts (lane b). None of these three proteins were immunoprecipitated from wild-type extracts by using rabbit normal IgG (lane a), or from oda1 axonemal extracts by using the anti-DC105 antibody (lane d) or rabbit normal IgG (lanes c). The wild-type biotinylated 0.6 M KCl extract also was analyzed in Western blots probed with antibodies specific for the Mr ∼105,000 and ∼70,000 proteins to confirm the relative mobilities of the biotinylated proteins (lanes e and f). During the two overnight dialyses necessary to prepare the 0.6 M KCl extracts for immunoprecipitation, some proteolysis of the Mr ∼105,000 and ∼70,000 proteins occurred, resulting in minor immunoreactive fragments (*, lane b) running just below the intact proteins. The samples used for the Western blots were stored at 4°C for several days longer to accentuate the proteolytic fragments (*, lanes e and f). Numbers on left indicate molecular weight markers. (D) SDS-PAGE analyses of extract of wild-type axonemes centrifuged in a sucrose density gradient at low hydrostatic pressure in the presence of Mg2+; under these conditions the three ODA-DC proteins (arrowheads) cosediment with the three-headed outer arm dynein at 23S. Outer arm dynein intermediate chains (IC69 and IC78) and light chains are indicated by arrows and a bracket, respectively. A, B, and D are 5–20% acrylamide gradient gels (5–20%); Coomassie blue stain. (E) Dynein coimmunoprecipitates with the ODA-DC in the presence of Mg2+. Wild-type axonemes were extracted with 0.6 M KCl in HMDEK, the ODA-DC immunoprecipitated by using the rabbit polyclonal anti-DC105 antibody, and the immunoprecipitate analyzed by Western blotting. Antibodies specific for the Mr ∼105,000 (α-DC105, lane c) and Mr ∼70,000 (α-DC70, lane d) ODA-DC polypeptides and the outer dynein arm intermediate chains IC69 (α-IC69, lane a) and IC78 (α-IC78, lane b) each recognized a protein of the appropriate size in the immunoprecipitate. None of these proteins were immunoprecipitated by the normal rabbit IgG (lanes e–h). The anti-rabbit IgG secondary antibody used to probe lanes c, d, g, and h also detected the rabbit IgG used for the immunoprecipitation (dark bands at Mr ∼50,000 in lanes c, d, g, and h); the anti-mouse IgG secondary antibodies used to probe lanes a, b, e, and f did not detect the rabbit IgG. Numbers on right indicate molecular weight markers.
Figure 2
Figure 2
Sequence and predicted coiled-coil structure of the Mr ∼70,000 ODA-DC protein. (A) Nucleotide sequence of a cDNA clone encoding the protein, and its deduced amino acid sequence. An in-frame stop codon just upstream of the predicted translation initiator ATG is indicated by an underline and an asterisk; the stop codon at the end of the long open reading frame is marked by an asterisk. Lines under the deduced amino acids indicate sequence that exactly matches that obtained by direct microsequencing of three tryptic peptides from the Mr ∼70,000 protein. Two nucleotide sequences that were used for PCR primers to amplify the complete protein coding region from oda1-1 and wild-type first-strand cDNA are indicated by double underlines in the 5′- and 3′-untranslated regions. A complete Chlamydomonas polyadenylation signal sequence (TGTAA) is marked by a row of asterisks. The C at nucleotide position 283, which is changed to a T in oda1-1, is shown in bold. These sequence data are available from GenBank/European Molecular Biology Laboratory/DNA Data Bank of Japan under accession no. AY039618. (B) The deduced amino acid sequence of the Mr ∼70,000 protein was analyzed using the program COILS (MTIDK matrix, with a 2.5 weighting of hydrophobic positions a and d), which estimates the probability that a region of polypeptide will form a coiled-coil structure (Lupas, 1996a). Regions A, B, and D (amino acids 27–60, 120–215, and 323–370, respectively) have a high probability (>99%) of forming coiled-coils. Region C (amino acids 234–262) is predicted to be α-helical, but when the NEWCOILS program is run with weighting of hydrophobic positions a and d, this region is not predicted to form a coiled coil.
Figure 3
Figure 3
Comparison of the Chlamydomonas Mr ∼70,000 ODA-DC sequence (Cr DC2) with potential homologs from L. major (Lm DC2) (accession no. CAB55364.1), D. melanogaster (Dm DC2-1) (accession no. AAF55345), and Homo sapiens (Hs DC2-1 and Hs DC2-2) (accession nos. AK057357 and AK057488, respectively). Predicted amino acid sequences from GenBank were aligned with the CLUSTAL W program. Residues identical to those in the Chlamydomonas sequence are shaded in dark gray, whereas amino acids substituted to similar residues are shaded in light gray; both types are surrounded by boxes.
Figure 4
Figure 4
Amplification, in vitro transcription, and in vitro translation of cDNAs encoding the Mr ∼70,000 ODA-DC protein. (A) Amplified cDNA made from first strand cDNA by using a pair of primers (Figure 2A) designed to amplify the full-length open reading frame encoding the Mr ∼70,000 protein. Template RNAs were from wild-type cells (lane 1), oda1-1 mutant cells that had been deflagellated and were in the process of regenerating new flagella (lane 2), and oda1-1 cells that were not deflagellated (lane 3). The left lane was loaded with a 1-kb DNA ladder (Invitrogen). (B) Formaldehyde-agarose gel (1.2%) of RNA transcribed from cDNA clones derived from the PCR products shown in A. The products were cloned in pBluescript II KS (−) and transcribed in vitro by using T7 RNA polymerase. Two wild-type (wt) and four oda1-1 (oda1) clones were examined; all produced RNA of the expected size. The left lane shows an RNA ladder (0.24–9.5 kb) (Invitrogen). (C) SDS-PAGE analysis of products obtained by in vitro translation of RNAs shown in B. The wild-type RNAs (wt) produced a protein of Mr ∼70,000 (arrow), whereas the oda1-1 RNAs (oda1) failed to produce a protein of this size. The right lane (L) is a product from luciferase mRNA (61 kDa). Bars at the left indicate molecular weight markers (76,000 and 52,000).
Figure 5
Figure 5
Cross-linking of the ODA-DC proteins by EDC. A 0.6 M KCl extract of wild-type axonemes was incubated with increasing concentrations of EDC (0, 0.2, 0.5, 1, 2, 4, 6, 10, and 20 mM). The proteins were then separated on a 6% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. The right panel shows the blot probed with the anti-DC70 antibody; the left panel shows the same blot stripped and reprobed with the anti-DC105 antibody. Multiple cross-linked products migrating between Mr ∼175,000 and ∼210,000 appear simultaneously, are recognized by both antibodies, and become progressively more prominent as the EDC concentration is increased. Numbers on left indicate molecular weight markers.
Figure 6
Figure 6
Models for ODA-DC structure. (A) Diagram illustrating how the Mr ∼70,000 and ∼105,000 ODA-DC proteins might interact. In this model, the two proteins (Oda1 and Oda3, respectively) interact via their coiled-coil domains to form a rod-shaped heterodimer; noncoiled-coil regions loop out from the rod. (B) Diagram illustrating how the ODA-DCs might link up end-to-end to form a filament running the length of the A-tubule. The Mr ∼25,000 ODA-DC protein (blue subunit marked 25) is shown attached to one end of the rod-shaped ODA-DC; this placement is purely speculative. In this model, the ODA-DCs repeat with the same 24-nm periodicity as the outer dynein arms, two of which are shown attached to the ODA-DC and A-tubule. The α, β, and γ heavy chains of the outer arms are labeled α, β, and γ, respectively; other labels identify the dynein intermediate and light chains by apparent molecular weight (King and Witman, 1989).

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