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. 2004 Jun;15(6):2729-41.
doi: 10.1091/mbc.e03-11-0820. Epub 2004 Apr 2.

Oda5p, a Novel Axonemal Protein Required for Assembly of the Outer Dynein Arm and an Associated Adenylate Kinase

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Oda5p, a Novel Axonemal Protein Required for Assembly of the Outer Dynein Arm and an Associated Adenylate Kinase

Maureen Wirschell et al. Mol Biol Cell. .
Free PMC article

Abstract

Of the uncloned ODA genes required for outer dynein arm assembly in Chlamydomonas, ODA5 and ODA10 are of particular interest because they do not encode known subunits of the outer arm or the outer dynein arm-docking complex (ODA-DC), and because genetic studies suggest their products interact. Beginning with a tagged oda5 allele, we isolated genomic and cDNA clones of the wild-type gene. ODA5 predicts a novel, 66-kDa coiled-coil protein. Immunoblotting indicates Oda5p is an axonemal component that assembles onto the axoneme independently of the outer arm and ODA-DC and is uniquely missing in oda5 and oda10 axonemes. Oda5p is released from the axoneme by extraction with 0.6 M KCl, but the soluble Oda5p does not cosediment with the outer dynein arm/ODA-DC in sucrose gradients. Quantitative mass spectrometry by using isotope coded affinity tagging revealed that a previously unidentified adenylate kinase is reduced 35-50% in oda5 flagella. Direct enzymatic assays demonstrated a comparable reduction in adenylate kinase activity in oda5 flagella, and also in oda10 flagella, but not in flagella of other oda mutants. We propose that Oda5p is part of a novel axonemal complex that is required for outer arm assembly and anchors adenylate kinase in proximity to the arm.

Figures

Figure 1.
Figure 1.
The ODA5 gene rescues the Oda5- motility phenotype and the transforming DNA is recovered in the oda5-2 rescued strains. (A) The upper line illustrates the intron-exon structure of the ODA5 gene. Rectangles indicate exons and solid lines indicate introns. The initial exon is marked by an arrow indicating the direction of transcription. The second line is the restriction map for the relevant portion of the rescuing BAC (N, Nco1; B, BamH1; E, EcoR1; S, Sal1). Subclones from the rescuing BAC were tested for their ability to rescue the Oda5- motility phenotype. The left column indicates the number of rescued transformants/total number of cotransformants screened. The right column indicates the construct name and the size of the genomic fragment. The smallest rescuing fragment was a 6.1-kb Sal1-BamH1 fragment (50.1). (B) Motility assays were performed on wild type, oda5-2, and one of the rescued strains (oda5-2 rescued). Both swimming speed and flagellar beat frequency were rescued to near wild-type levels in the rescued strain (n = 30 for each strain). (C) Southern blots of SacI/BamH1-digested DNA from wild type, oda5-2, and two strains rescued by transformation of oda5-2 were probed with the transforming DNA. Wild-type DNA contains hybridizing sequences from the ODA5 region. Oda5-2 does not contain hybridizing sequences as these regions are deleted. The two rescued strains contain hybridizing sequences, demonstrating that these sequences have been recovered.
Figure 2.
Figure 2.
The oda5-2 insertional mutant lacks outer dynein arms and the rescued strains have restored outer dynein arms. Electron micrographs of axonemal cross sections from (A) wild type, (B) an oda5-2 insertional mutant rescued with the 6.1-kb fragment containing the ODA5 gene (oda5–2 rescued), and (C and D) the oda5-2 insertional mutant. Arrows indicate the outer dynein arms in the wild-type and rescued flagella, but the absence of outer arms in the oda5-2 flagella. In occasional micrographs, some outer arms, or partial arms, were observed in oda5-2 (arrowhead in D). Bar, 100 nm.
Figure 3.
Figure 3.
Sequence and predicted structure of the ODA5 gene and its product. (A) ODA5 cDNA sequence and its predicted amino acid sequence. In bold and underlined are a Sal1 restriction enzyme site (567–572), which denotes the 5′ end of the 50.1 rescuing fragment; an in-frame stop codon (704–706) that is upstream of the predicted start codon; a GTA codon (1046–1048), encoding valine 108, that is converted to a TAA stop codon in the oda5-1 mutant strain; a polyadenylation signal sequence (2828–2832); and a BamH1 restriction site (2854–2859), which denotes the 3′ end of the 50.1 rescuing genomic fragment. The sequence from the polyadenylation site to the BamH1 site is derived from the ODA5 genomic sequence. Additional 5′ untranslated region (UTR) sequences were identified by PCR amplification of cDNA and EST database searches. The additional 5′ UTR sequence matches genomic sequence 5′ to the Sal1 site. This indicates that the 50.1 rescuing fragment does not contain the entire 5′ UTR of ODA5, but it does contain the entire coding region and 3′ polyadenylation signal. These sequences have been deposited in GenBank/EMBL/DDBJ with accession no. AY452532. (B) Graphical representation of the predicted coiled-coil regions in the Oda5 protein as determined using the COILS program (MTIDK matrix, with a 2.5 weighting of hydrophobic positions a and d) (Lupas et al., 1991). The x-axis is amino acid number, and the y-axis is the probability that the sequence will form coiled-coil secondary structure.
Figure 4.
Figure 4.
ODA5 gene expression is up-regulated by deflagellation. PolyA+ mRNA was isolated from wild-type non-deflagellated (nd) cells and cells that were deflagellated and actively regenerating their flagella (r). (A) An ODA5 cDNA probe was hybridized to Northern blots of the isolated mRNA. The probe identifies an induced message at ∼2.7 kb, in good agreement with the 2.8-kb size of the ODA5 cDNA. (B) A cDNA probe to fructose-biphosphate aldolase recognizes an ∼2.0-kb mRNA that serves as a loading control; transcription of this gene is not up-regulated by deflagellation.
Figure 5.
Figure 5.
Western blot analysis indicates Oda5p is a salt-extractable, Mr 76,000 axonemal protein that sediments at ∼5S in sucrose density gradients. (A) The Oda5 antibody recognizes an Mr 76,000 band in wild-type whole cells that is absent from the oda5-2 whole cells, confirming the antibody recognizes the correct protein. This band is not detected in cell bodies lacking flagella (middle), yet it is readily detected in whole flagella (right). (B) Oda5p remains associated with the axoneme after Nonidet P-40 detergent extraction (demembranated axonemes) and is not detected in the Nonidet P-40 detergent-soluble membrane + matrix fraction (membrane + matrix). Extraction of demembranated axonemes with 0.6 M KCl releases Oda5p into the KCl extract; none remains in the KCl-extracted axonemes. (C) Sucrose gradient fractions were probed with antibodies to outer dynein arm components IC2 and γ-DHC, and with the Oda5p-antibody. The outer dynein arm/ODA-DC complex sediments at ∼23S as expected; however, Oda5p sediments at ∼5S.
Figure 6.
Figure 6.
Oda5p assembles onto the axoneme independently of the outer dynein arm and the ODA-DC. (A) Western blot of axonemes isolated from wild type (wt) and oda5-2, oda9, oda1, oda3, oda8, and oda10 and probed with the Oda5p-antibody. Oda5p is detected on axonemes from wild type and all the oda mutants except oda5-2 and oda10. (B) Western blot of axonemes from the same strains was probed with an antibody to the DC2 component of the ODA-DC. DC2 assembles onto axonemes in all strains except oda1 and oda3, which are defective in DC2 and DC1, respectively. (C) A Western blot of axonemes from the same strains was probed with an antibody to the IC140 component of the inner dynein arm I1. As expected, IC140 assembles onto axonemes in all of the strains and serves as a loading control for these Western blots.
Figure 7.
Figure 7.
Adenylate kinase activity is reduced in oda5 and oda10 mutant flagella. (A) Electron micrographs of flagella extracted with Tergitol and Nonidet P-40 detergents. Tergitol-extracted axonemes contain cosedimenting flagellar membranes (left, arrows), whereas Nonidet-extracted axonemes are virtually free of flagellar membranes (right). Bar, 300 nm. (B) Adenylate kinase activity was measured in Tergitol-treated axonemes from wild type, oda1, oda3, oda5, oda8, oda9, oda10, and an HA-tagged ODA5 rescued strain (r). The histogram shows AK activity as a percentage relative to that of wild type. Adenylate kinase activity is specifically reduced in oda5 and oda10 mutant axonemes, but it is restored to the wild-type level in the strain transformed with an HA-tagged ODA5 gene construct. (C) When flagella are treated with Tergitol, the Oda5p-associated AK activity remains in the pellet. However, when flagella are treated with Nonidet P-40, the Oda5p-associated AK activity is released into the supernatant. For each fraction, the AK activity of oda5 is shown as a percentage relative to that of wild type. Error bars in B and C represent the standard deviation calculated from three independent measurements.
Figure 8.
Figure 8.
The flagellar AK gene structure and predicted cDNA and protein sequences. (A) Intron-exon structure of the AK gene. Black boxes indicate the exons; solid lines indicate the noncoding regions; arrow indicates the direction of transcription. (B) AK cDNA sequence and deduced protein sequence. Underlined and in bold are an upstream, in-frame stop codon, TAG; a consensus polyadenylation signal sequence, TGTAA, located 521 bp downstream of the stop codon (*); and the peptides identified by ICAT. The conserved AK domain is present in three nearly identical repeats; the shaded regions denote the AK P-loop motif and the AK signature motif in each of the three domains. Nucleotides 1–1075 and 1328–2619 have been confirmed by PCR amplification of cDNA; nucleotides 1076-1327 are derived from the JGI version 2.0 gene model for this gene; nucleotides 2620–2703 are derived from EST sequences. These sequences have been reported in GenBank/EMBL/DDBJ with accession no. AY452531. (C) Comparison of the C. reinhardtii flagellar AK and H. sapiens AK1 and AK5. The highly conserved AK domains, containing the P-loop motif and the AK signature motif, are indicated by black boxes, whereas unique sequences are represented by a line. The human AK5 enzyme occurs as a small isoform (AK5) and two longer isoforms called variant 1 and 2 (AK5–1 and AK5–2). The greatest percent identity/percent similarity between one of the three Chlamydomonas AK domains and one of the AK domains of each human AK isoform are indicated.
Figure 9.
Figure 9.
Model for assembly of the Oda5p/Oda8p/Oda10p complex in the wild-type axoneme. The outer dynein arm attaches to the A-tubule via the ODA-DC and the Oda5p/Oda8p/Oda10p complex; the flagellar AK is held near the outer arm by its association with the latter. α, β, and γ indicate the α, β, and γ dynein heavy chains; numbers indicate dynein LC subunits.

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