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. 2003 Sep;14(9):3650-63.
doi: 10.1091/mbc.e03-01-0057. Epub 2003 Jun 27.

DC3, the 21-kDa Subunit of the Outer Dynein Arm-Docking Complex (ODA-DC), Is a Novel EF-hand Protein Important for Assembly of Both the Outer Arm and the ODA-DC

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DC3, the 21-kDa Subunit of the Outer Dynein Arm-Docking Complex (ODA-DC), Is a Novel EF-hand Protein Important for Assembly of Both the Outer Arm and the ODA-DC

Diane M Casey et al. Mol Biol Cell. .
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Abstract

The outer dynein arm-docking complex (ODA-DC) is a microtubule-associated structure that targets the outer dynein arm to its binding site on the flagellar axoneme (Takada et al. 2002. Mol. Biol. Cell 13, 1015-1029). The ODA-DC of Chlamydomonas contains three proteins, referred to as DC1, DC2, and DC3. We here report the isolation and sequencing of genomic and full-length cDNA clones encoding DC3. The sequence predicts a 21,341 Da protein with four EF-hands that is a member of the CTER (calmodulin, troponin C, essential and regulatory myosin light chains) group and is most closely related to a predicted protein from Plasmodium. The DC3 gene, termed ODA14, is intronless. Chlamydomonas mutants that lack DC3 exhibit slow, jerky swimming because of loss of some but not all outer dynein arms. Some outer doublet microtubules without arms had a "partial" docking complex, indicating that DC1 and DC2 can assemble in the absence of DC3. In contrast, DC3 cannot assemble in the absence of DC1 or DC2. Transformation of a DC3-deletion strain with the wild-type DC3 gene rescued both the motility phenotype and the structural defect, whereas a mutated DC3 gene was incompetent to rescue. The results indicate that DC3 is important for both outer arm and ODA-DC assembly.

Figures

Figure 1.
Figure 1.
DC3 gene expression is induced by deflagellation. mRNA was isolated from wild-type cells that were either fully flagellated (non–deflagellated) or actively regrowing their flagella (30′ post deflagellation). mRNA was analyzed by northern hybridization using the DC3 cDNA as a probe (top panel). In non-deflagellated cells, a small amount of DC3 mRNA (∼1.4 kb) is visible; this message increases substantially upon deflagellation and growth of new flagella. With prolonged exposure, a second very minor message of ∼1.8 kb was also observed in the deflagellated samples. A probe (RBA1.1) to the fructose-bisphosphate aldolase gene, a gene involved in glycolysis, was used as a loading control; transcription of this gene is not induced by deflagellation (bottom panel).
Figure 2.
Figure 2.
DC3 sequence analysis. (A) Nucleic acid and deduced amino-acid sequence of DC3. The three in-frame STOP codons upstream of the start ATG are in bold. The peptides obtained by direct sequencing of DC3 tryptic fragments are underlined. An asterisk indicates the STOP codon at the end of the DC3 open reading frame. The consensus Chlamydomonas polyadenylation signal is double underlined. Nucleotide and amino-acid numbers are shown on the right (amino-acid numbers are in bold). These sequence data are available from GenBank/EMBL/DDBJ under accession number AY294291. (B) Comparison of the DC3 sequence and its putative homologue from Plasmodium falciparum. The alignment was generated by version 1.82 of ClustalW. Consensus symbols: “*” identical sequence; “:” conserved substitution; “.” semiconserved substitution. (C) Predicted secondary structure of the DC3 protein. The predicted secondary structure is typical of many EF-hand protein family members (reviewed in Kawasaki et al., 1998). The EF-hands are double underlined. L, loop; H, helix; E, strand; “.” no prediction.
Figure 3.
Figure 3.
Identification of insertional mutants with defects in the DC3 gene. Genomic DNA was isolated from insertional mutants having flagellar/cytoskeletal defects, cut with PstI, and analyzed by Southern blot using the DC3 cDNA as a probe. The probe hybridized to a single band (arrow) in wild type, but did not hybridize to any bands in strains V06 or F28. In strain V16, the probe hybridized to two bands, indicating the DC3 gene had been disrupted by an insertion.
Figure 4.
Figure 4.
Loss of DC3 results in partial loss of outer dynein arms. Top panel: Axonemal cross sections from a wild-type strain (left), the DC3-deletion strain (center), and the DC3-deletion strain rescued by transformation with the DC3 gene (right). DC3-null axonemes do not assemble a full complement of outer dynein arms. A gap was often seen between the A-tubule and the arm (arrowhead), suggesting that the arms are not as tightly associated with the outer doublet in the absence of DC3. Outer dynein arms (arrowhead) are completely restored in the null strain transformed with the cloned DC3 gene. Bottom panel: Longitudinal section showing the 24-nm periodicity of outer dynein arms (arrowheads) along a portion of a DC3-null outer doublet. The normal periodicity indicates that when outer arms are present, they assemble cooperatively. Bar, 50 nm.
Figure 5.
Figure 5.
The DC3-deletion strain assembles a “partial” docking complex. Axonemal cross sections from a strain lacking outer arms but retaining the ODA-DC (oda9), a strain lacking outer arms and the ODA-DC (oda3), and the DC3-deletion strain (DC3Δ). The ODA-DC is visible on oda9 axonemes as a small projection on the A-tubules at the sites where the outer dynein arms would normally attach (left, arrowheads). These projections are missing from the A-tubules of oda3 (center, arrowheads). DC3-null A-tubules that lacked the outer dynein arm frequently had a structure resembling the docking complex at sites where outer arms would normally attach (right, arrowheads). We refer to this structure as a “partial” docking complex. Bar, 25 nm.
Figure 6.
Figure 6.
(A) DC1 and DC2 assemble on the axoneme in the absence of DC3, but not vice versa. Top panel: Axonemes from wild type, the DC3-deletion strain (DC3Δ), oda1, and oda3 were isolated and analyzed by western blotting. oda1 is null for DC2; oda3 is null for DC1. The blot was probed with antibodies to DC1, DC2, and the inner arm IC, IC140 (used as a loading control). As expected, antibodies to DC1 detected protein in wild-type axonemes, but not in oda1 or oda3 axonemes, which lack the ODA-DC. Importantly, the antibody also detected protein in axonemes of the DC3-deletion strain. Essentially identical results were obtained with antibodies to DC2 (our unpublished results). These data indicate that DC1 and DC2 can assemble on the axoneme in the complete absence of DC3. Bottom panel: Axonemes from wild type, the DC3-deletion strain (DC3Δ), oda1, oda3, and oda9 were prepared as above (oda9 has a defect in IC1 and lacks outer dynein arms but retains the ODA-DC). The blot was probed with a polyclonal antibody to DC3. The antibody detects a single protein of Mr ∼25,000 in both oda9 and wild-type axonemes but detects no protein in DC3-null axonemes, indicating that it is specific for DC3. Axonemes from oda1 and oda3 do not contain DC3, indicating that DC3 assembly is dependent on the presence of DC1 and DC2. (B) Immunoprecipitation of the ODA-DC in the absence of Mg2+. The DC1 antibody was used to immunoprecipitate the ODA-DC from biotinylated 0.6 M KCl extracts of DC3-transformant (strain W215) and DC3-null axonemes. The immunoprecipitated proteins were then resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and detected using streptavidin-HRP. The anti-DC1 antibody immuno-precipitated three proteins of Mr ∼105,000, ∼70,000, and ∼25,000 (arrowheads) from DC3-transformant axonemal extracts (lane 3). These proteins, corresponding to DC1, DC2, and DC3, respectively, were not immunoprecipitated from DC3-transformant axonemal extracts using normal rabbit IgG (lane 4). In contrast, the anti-DC1 antibody immunoprecipitated only DC1 and DC2 (arrowheads) from the DC3-null axonemal extracts (lane 1). These proteins were not immunoprecipitated from DC3-null axonemal extracts using normal rabbit IgG (lane 2). These data confirm that a “partial” docking complex composed of DC1 and DC2 assembles on the axoneme when DC3 is missing and that transformation of the DC3-deletion strain with the DC3 gene restores DC3 to the ODA-DC. Numbers on left indicate molecular weight markers.
Figure 7.
Figure 7.
Top panel: The slow-swimming motility phenotype and the DC3 gene deletion cosegregate. The DC3-deletion strain was crossed to a wild-type strain, tetrads were dissected, and progeny from 16 different tetrads (lanes 1–16) were scored for motility by light microscopy and for the presence of the DC3 gene by Southern blotting. Whenever a cell line lacked the DC3 gene, it also had mutant motility (–). Lanes 17 and 19 contain DNA from the two DC3-deletion strains, V06 and F28, respectively. Lane 20 contains DNA from a wild-type strain. Bottom panel: Wild-type motility (+) segregates with the DC3 transgene. A DC3-deletion strain that had been rescued by transformation with the DC3 gene was crossed to a DC3-deletion strain of opposite mating type. Tetrads from the cross were dissected and the progeny scored for motility. DNA was isolated from the DC3-deletion strain (lane 1), the rescued strain (lane 2), and from randomly selected progeny representing 15 independent tetrads (lanes 3–17). Samples were analyzed by Southern blotting using the DC3 cDNA as a probe. Wild-type motility was observed only in progeny containing the inserted DC3 gene. +, wild-type motility; –, mutant motility; M, marker.
Figure 8.
Figure 8.
Phylogenetic tree showing the relatedness of DC3 to other CTER members. A cluster algorithm was used to build the tree. Branch lengths represent evolutionary relatedness. Numbers at branch points are bootstrap values. The 23-kDa T. thermophila calcium-binding protein (TCBP-23), which is an EF-hand protein but not a member of the CTER group, was used to root the tree. DC3 and its putative P. falciparum homologue (in bold) group independently of other CTER members, indicating they form a distinct subfamily within CTER. Protein names are followed by organism names; LC4 is the 18-kDa calcium-binding outer dynein arm light chain, O. cuniculus is rabbit. Accession numbers (in parentheses) are as follows: Calmodulin_O. cuniculus (1003191A); Calmodulin_P. falciparum (P24044); Calmodulin_C. reinhardtii (P04352); Troponin C_H. sapiens (P02590); Centrin_H. sapiens (NP_004335); Centrin_C. reinhardtii (P05434); Centrin_P. falciparum (NP_702332); LC4_C. reinhardtii (Q39584); DC3_C. reinhardtii (AY294291); Hypothetical protein_P. falciparum (NP_702309); and TCBP-23_T. thermophila (P20473).
Figure 9.
Figure 9.
Homology model of DC3. The atomic coordinates of DC3 were assigned by Swiss-Model using calmodulin (Taylor et al., 1991) as a template. The theoretical model was rendered in Protein Explorer; the amino terminus is at the top. The four EF-hands are labeled EF1–EF4. Short antiparallel β-sheets are predicted to link adjacent loop regions, perhaps stabilizing the EF-hands (Taylor et al., 1991). An asterisk marks the predicted nonhelical region that breaks what would be a long central helix into two shorter helices. Red: alpha-helices; blue and white: loops; yellow: beta strands.

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