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. 1998 Dec;9(12):3335-49.
doi: 10.1091/mbc.9.12.3335.

The Mr 140,000 Intermediate Chain of Chlamydomonas Flagellar Inner Arm Dynein Is a WD-repeat Protein Implicated in Dynein Arm Anchoring

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Free PMC article

The Mr 140,000 Intermediate Chain of Chlamydomonas Flagellar Inner Arm Dynein Is a WD-repeat Protein Implicated in Dynein Arm Anchoring

P Yang et al. Mol Biol Cell. .
Free PMC article

Abstract

Previous structural and biochemical studies have revealed that the inner arm dynein I1 is targeted and anchored to a unique site located proximal to the first radial spoke in each 96-nm axoneme repeat on flagellar doublet microtubules. To determine whether intermediate chains mediate the positioning and docking of dynein complexes, we cloned and characterized the 140-kDa intermediate chain (IC140) of the I1 complex. Sequence and secondary structural analysis, with particular emphasis on beta-sheet organization, predicted that IC140 contains seven WD repeats. Reexamination of other members of the dynein intermediate chain family of WD proteins indicated that these polypeptides also bear seven WD/beta-sheet repeats arranged in the same pattern along each intermediate chain protein. A polyclonal antibody was raised against a 53-kDa fusion protein derived from the C-terminal third of IC140. The antibody is highly specific for IC140 and does not bind to other dynein intermediate chains or proteins in Chlamydomonas flagella. Immunofluorescent microscopy of Chlamydomonas cells confirmed that IC140 is distributed along the length of both flagellar axonemes. In vitro reconstitution experiments demonstrated that the 53-kDa C-terminal fusion protein binds specifically to axonemes lacking the I1 complex. Chemical cross-linking indicated that IC140 is closely associated with a second intermediate chain in the I1 complex. These data suggest that IC140 contains domains responsible for the assembly and docking of the I1 complex to the doublet microtubule cargo.

Figures

Figure 1
Figure 1
Schematic picture illustrates the strategy and steps used to clone Chlamydomonas IC140 gene and message. The 11.5-kb XbaI genomic subclone contains the complete IC140 gene consisting of 14 exons (solid bars) and 13 introns. Open boxes indicate 5′ and 3′ untranslated regions. Arrows indicate the position and the direction of primers for RT-PCR (see MATERIALS AND METHODS). The pC1 clone was recovered from screening a cDNA library. PC2 and pC3 resulted from RT-PCR using the gene-specific primer RT1 for reverse transcription and primer pairs FUS/Cr5 and T3/FUSrev, respectively (see RESULTS for details).
Figure 2
Figure 2
(A) Southern blot analysis of 5 μg genomic DNA digested with EcoRI, XhoI, SalI, and SacI (lanes a–d, respectively). (B) Northern blot analysis of 3 μg poly(A) RNA prepared from Chlamydomonas regenerating half-length of flagella (lane a) and from control cells (no deflagellation) (lane b). Both membranes were probed with the EcoRI–NotI fragment of pC1. The Northern blot was reprobed with the CRY1 gene to confirm the equal loading.
Figure 3
Figure 3
The IC140 genomic and protein sequence. The amino acid sequence predicted from cDNA sequence aligned with the exons of the genomic sequence. Bold italic letters, aa sequences from peptide microsequencing; box, predicted tub boxes; asterisk, stop codons preceding the predicted first ATG and for terminating IC140 translation; arrow, primer for RT-PCR; arrowhead, 5′ end of the first partial cDNA clone from cDNA library; letter in an open triangle, transcription initiation site; letter in an open square, polyadenylation site. The bold TGTAA is probably the polyadenylation signal.
Figure 4
Figure 4
Dot plot (A) and dendrogram (B) reveal the homology among the ICs: IC140 of the Chlamydomonas inner arm dynein I1, IC69 and IC78 of Chlamydomonas outer arm dynein, and IC2C of rat cytoplasmic dynein. The dot plots were generated by the program Compare with a stringency 18 to find 18 similarity matching values in a comparison window size of 30 (Maizel and Lenk, 1981). The dendrogram was compiled by the PileUp program using default parameters.
Figure 5
Figure 5
(A) Sequence alignment of the seven predicted WD/β-sheet repeats in dynein ICs. A–E WD repeats were based on the WD consensus structure discussed by Wilkerson et al. (1995). The C′ and E′ repeats were assigned based on β-sheet location discussed in RESULTS and DISCUSSION. The asterisks indicate the consensus amino acid residues for WD repeats (Neer et al., 1994). The underlined asterisk indicates that two of the three residues were required to match the consensus sequence. The residues in the ICs matching the consensus sequence are shown in bold characters. Underlined regions (a–c) correspond to the first three β-strands as seen in the structure of Gβb. The fourth strand (d) is not illustrated, because it is located in the variable region between WD repeats. (B) Schematic depicting the predicted Chou-Fasman β-sheets (sharp sawtooth wave), WD repeats (open oval), and the coiled coils (box) of the IC proteins listed in Figure 4A.
Figure 6
Figure 6
Polyclonal antibody raised against the 53-kDa fusion protein of IC140 specifically recognized IC140. Western blots for Figure 6, A and B, were probed with the anti-IC140 serum followed by HRP-conjugated secondary antibody and colorimetric development. (A) Lane a, wild-type high-salt extract; lane b, pf28 axonemes; lane c, pf28pf30 axonemes; lane d, I1 fraction from sucrose gradient of pf17 dynein extract; lane e, 53-kDa fusion protein. Inset, immunofluorecent microscopy illustrating that IC140 is located along both flagellar axonemes in wild-type Chlamydomonas cells. (B) Lane a, Amido Black staining revealing the proteins of the I1 fraction frompf28 axonemes including 3 ICs: 140, 138, and 97 kDa (dot); lane b, corresponding Western blot of the same 21S fraction. Molecular weight markers (M.) between lanes a and b were used for alignment. (C) Western blots and protein stains of overloaded gels revealed that IC140 is present in minor but detectable quantity in axonemes from pf28pf30 and pf9-3. For comparison, axonemes from control strains oda1, pf17, and mbo2 bear wild-type amounts of IC140. Axonemes (50 μg/lane) were separated in SDS-PAGE (4.5% for α HC, 7% for IC140 and Tubulin). Top panel, Western blot probed with affinity-purified anti-α HC (Myster, et al., 1997). Middle panel, Western blot probed with anti-IC140 serum. Bottom panel, tubulin (25 μg/lane) was revealed by Amido Black staining, showing equal loading of samples.
Figure 7
Figure 7
Selective binding of the C1 fusion protein of IC140 to axonemes lacking the I1 complex. Increasing amounts of purified fusion protein were mixed and incubated with isolated axonemes from either pf28pf30, lacking the I1 complex (lanes a–d), or pf28 (lanes e–h) (see MATERIALS AND METHODS). (A) Western blots using anti-IC140 serum, which reveals the 53-kDa fusion protein (double arrowhead) and IC140 (arrowhead) and using HRP secondary antibodies and developed with chemiluminescent reagents. (B) Duplicate gel stained with Commasie brilliant blue, confirming equal loads for each sample.
Figure 8
Figure 8
Western blot analysis, using anti-IC140 antibodies, of EDC cross-linked axonemal proteins. (A) Axonemes (right) or dynein-containing high-salt extracts (left), derived from pf28, were treated with 0, 1.5, 3.5, or 8.5 mM EDC (A, lanes a–d). Notably a novel 240-kDa band (arrow) in addition to IC140 (arrow) appears after exposure to EDC. (B) The same 240-kDa immunoreactive band appears in the 19S-I1 fraction (left, EDC gradient fractions 2, 4, 6, and 8 shown). In contrast, the same gradient fractions of the control, nontreated axonemes do not reveal the 240-kDa band (right, Control, fractions 2, 4, 6, and 8). In each case samples were separated by 5% SDS-PAGE followed by Western blot using anti-IC140 serum and the enhanced chemiluminescent reagents.

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