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. 1999 Aug 23;146(4):801-18.
doi: 10.1083/jcb.146.4.801.

Domains in the 1alpha Dynein Heavy Chain Required for Inner Arm Assembly and Flagellar Motility in Chlamydomonas

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Free PMC article

Domains in the 1alpha Dynein Heavy Chain Required for Inner Arm Assembly and Flagellar Motility in Chlamydomonas

S H Myster et al. J Cell Biol. .
Free PMC article

Abstract

Flagellar motility is generated by the activity of multiple dynein motors, but the specific role of each dynein heavy chain (Dhc) is largely unknown, and the mechanism by which the different Dhcs are targeted to their unique locations is also poorly understood. We report here the complete nucleotide sequence of the Chlamydomonas Dhc1 gene and the corresponding deduced amino acid sequence of the 1alpha Dhc of the I1 inner dynein arm. The 1alpha Dhc is similar to other axonemal Dhcs, but two additional phosphate binding motifs (P-loops) have been identified in the NH(2)- and COOH-terminal regions. Because mutations in Dhc1 result in motility defects and loss of the I1 inner arm, a series of Dhc1 transgenes were used to rescue the mutant phenotypes. Motile cotransformants that express either full-length or truncated 1alpha Dhcs were recovered. The truncated 1alpha Dhc fragments lacked the dynein motor domain, but still assembled with the 1beta Dhc and other I1 subunits into partially functional complexes at the correct axoneme location. Analysis of the transformants has identified the site of the 1alpha motor domain in the I1 structure and further revealed the role of the 1alpha Dhc in flagellar motility and phototactic behavior.

Figures

Figure 1
Figure 1
Dhc1 gene structure. (a) Partial restriction map of the Dhc1 gene. The black box labeled P1 represents the 227 bp fragment of the Dhc1 gene that was recovered in the first PCR screen for Dhc genes in Chlamydomonas (Porter et al. 1996). This sequence was used to screen a large insert genomic library and recover >35 kb of genomic DNA surrounding the region encoding the hydrolytic ATP binding site (P1) (Porter et al. 1996; Myster et al. 1997). Indicated below are the approximate positions of selected SacI subclones (A–G) used as probes and other subclones (pSM8 and p14SE) that were used to construct a truncated Dhc1 gene. (b) Intron/exon structure of the Dhc1 transcription unit. The approximate size and position of the 29 exons (open boxes) and 28 introns (intervening lines) predicted from the analysis of the Dhc1 nucleotide sequence are shown. Also indicated are other features such as the TATA box, the regions encoding predicted P-loops, and a predicted polyadenylation signal at the 3′ end of the gene. Bracketed regions numbered 1–5 indicate the position of the primers used for RT-PCR.
Figure 5
Figure 5
Constructs of the Dhc1 gene used in the cotransformation experiments. (A) Partial restriction map of the Dhc1 gene. The arrow above identifies the position of the Dhc1 transcription unit within this region (Myster et al. 1997). SacI subclones (black and gray boxes) were used to screen two cosmid libraries to identify clones that contain the complete Dhc1 transcription unit. (B) Partial restriction map of the cosmid cA1. The approximate locations of the regions encoding the primary ATP hydrolytic site (P1) and the peptide (peptide) recognized by the 1α Dhc specific antibody (Myster et al. 1997) are shown. The cA1 clone is incomplete on the 3′ end of the Dhc1 gene; it encodes up to residue 4,105 of the 1α Dhc sequence followed by the addition of 87 novel amino acids. Before transformation, the cA1 cosmid was linearized with BspE1, leaving >8 kb of flanking genomic DNA upstream of the predicted translation start site. The ARG7 sequence used as a selectable marker is located downstream of the Dhc1 sequence. (C) Partial restriction map of the cosmid cW1 containing a full-length Dhc1 gene. The regions encoding P1 and the peptide epitope are indicated, as are the predicted translation stop site (stop), and the consensus polyadenylation signal TGTAA (poly A signal). Before transformation, the cW1 cosmid was digested with PvuI, which releases the Dhc1 transcription unit as a 26-kb fragment flanked by ∼1.6 kb of genomic DNA on the 5′ end and ∼4 kb of genomic and vector DNA on the 3′ end. (D) Construction of a truncated version of the Dhc1 gene. An 11-kb SalI-AscI fragment from pSM8 was subcloned into the SalI-AscI digested p14SE plasmid. The resulting pD1SA construct contains ∼1.7 kb of genomic DNA 5′ of the predicted translation start site, the region encoding the first 1,956 amino acids, including the epitope recognized by the 1α Dhc antibody, and an ∼1-kb fragment containing the 3′ end of the gene. The shift in the open reading frame after the AscI site results in the addition of nine novel amino acids (QCHGCGPGV) followed by a stop. This construct was linearized with SalI before transformation. (e) Recovery of BAC clones containing the Dhc1 gene. Two different, large insert BAC clones were used in cotransformation experiments. The insert in the N24-1 clone contains ∼18 kb upstream and ∼52 kb downstream of the Dhc1 gene, whereas the insert in J1-5 contains ∼52 kb of genomic DNA both upstream and downstream of the Dhc1 gene. These constructs were not linearized before transformation.
Figure 2
Figure 2
Predicted amino acid sequence of the 1α Dhc. Sequences identified as P-loop motifs are indicated by a single underline. The amino acid sequence of the peptide used for antibody production by Myster et al. 1997 is indicated by asterisks. The arginine (R) residue at the COOH-terminal end of the truncated 1α Dhc in the G3 transformant is indicated by bold and an underline. The complete nucleotide sequence is available from GenBank/EMBL/DDBJ under accession number AJ243806.
Figure 3
Figure 3
Comparisons between Dhc sequences. The 1α Dhc was compared with the Chlamydomonas outer arm Dhc sequences (α, β, and γ) and the Dictyostelium cytoplasmic Dhc using the GCG program COMPARE with a window of 50 residues and a stringency of 22. The accession numbers for these sequences are as follows: L26049, U02963, U15303, and Z15124.
Figure 4
Figure 4
Secondary structure of the 1α Dhc. Shown here is a graphical representation of the regions of the 1α Dhc predicted to form α-helical coiled coils, as determined by the program COILS version 2.2 (Lupus et al. 1991; Lupus 1996). The six regions of the sequence predicted to encode P-loops are identified by arrows. The position of the peptide sequence (amino acids 1,059-1,073) used to generate an isoform specific antibody (Myster et al. 1997) is also indicated.
Figure 6
Figure 6
Western blots of isolated axonemes from Dhc1 transformants. Isolated axonemes were prepared from the Dhc1 transformants, split into triplicate for separation on 5% polyacrylamide gels, and then either stained with Coomassie blue or transferred to membranes and incubated with the affinity-purified 1α Dhc antisera or the IC140 antisera. (A, left panel) A 5% polyacrylamide gel loaded with 20-μg whole axonemes from pf28, pf9 pf28, and the 11 motile Dhc1 transformants generated by transformation with the cW1 cosmid. The additional bands in the pf9 pf28 sample are most likely contaminating flagellar membrane proteins. (Middle panel) A 5% gel loaded with whole axonemes from pf28, pf9-3, and the four rescued strains generated by transformation with the cA1 cosmid. (Right panel) A 5% gel loaded with whole axonemes from pf28 and the five rescued strains obtained with the BAC clones. (B) Duplicate immunoblots probed with the affinity-purified 1α Dhc antibody. (C) Duplicate immunoblots probed with the IC140 antisera. Control blots probed with tubulin antibodies confirmed that roughly equivalent amounts of flagellar protein were loaded in each lane (data not shown).
Figure 7
Figure 7
Cosedimentation of the truncated 1α Dhc with other subunits of the I1 complex. Whole axonemes were isolated from pf28 and the Dhc1 transformant G4 and extracted with high salt to release the dynein arms. The crude dynein extracts were loaded onto a 5–20% sucrose gradient and centrifuged for 15 h at 33,500 g. Duplicate samples from the 19S region of the gradients were separated on 5% polyacrylamide gels and either stained with Coomassie blue or transferred to nitrocellulose and incubated with the affinity-purified 1α Dhc antibody. (A) Gel and corresponding immunoblot from the pf28 extract. (B) Gel and corresponding immunoblot from the G4 extract. The asterisk identifies the truncated 1α Dhc that cosediments at ∼16S with the other subunits of the I1 complex.
Figure 8
Figure 8
Structural defects in Dhc1 transformants. Analysis of longitudinal images of axonemes from (a) wild-type, (c) pf9-3, (e) G4+OA, and (g) E2+OA. Grand averages for wild-type, pf9-3, G4+OA, and E2+OA based on 9, 6, 10, and 9 individual axonemes and 62, 44, 89, and 77 repeating units, respectively. (b) Outline of the individual densities in the 96-nm axonemal repeat. OA indicates positions of four outer dynein arm complexes per 96-nm repeat. S1 and S2 indicate the proximal and distal radial spokes, respectively. IA indicates the structures (numbered 1–10) seen in the inner arm region. (d) Difference plot between wild-type and pf9-3, with differences not significant at the 0.005 confidence level set to zero. (f) Difference plot between wild-type and G4+OA. (h) Difference plot between wild-type and E2+OA.
Figure 9
Figure 9
Northern blot analysis of Dhc1 transcripts in rescued strains. (A) Partial restriction map of the Dhc1 gene and subclones used as probes in the Northern analysis. (B) Northern blots of total RNA from wild-type, G4+OA, and E2+OA, isolated before (0) and 45 min (45) after deflagellation. Parallel samples of 20 μg total RNA were separated on 0.75% formaldehyde-agarose gels, transferred to Zetabind, and hybridized with selected Dhc1 subclones. Each probe hybridized to a single, large (>14 kb) transcript in wild-type (wt). Probe A3′ (left panel) also hybridized to truncated Dhc1 transcripts that are upregulated in response to deflagellation in G4+OA and E2+OA. Probes B and C gave similar results. Probe D (middle panel) hybridized to the truncated Dhc1 transcript in E2+OA, but not in G4+OA. (Probe D, which corresponds to the most highly conserved region of the Dhc1 gene, also cross-hybridized weakly with other Dhc transcripts in these samples.) Probe G (right panel) did not detect any transcripts in either G4+OA or E2+OA, and similar results were seen with probes E and F. (C) Northern blots of total RNA from wild-type, G3, A2, and G9 isolated 45 min (45) after deflagellation. Each probe hybridized to a single transcript in wild-type (wt) as well as the endogenous Dhc1 transcript present in the pf9-2 background of the transformants (Myster et al. 1997). Probe C (left panel) hybridized to the truncated Dhc1 transcripts in G3, A2, and G9, but probes D–F (middle panel) did not.
Figure 10
Figure 10
Modification of the Dhc1 transgenes in the transformants. (A) Southern blot analysis of the Dhc1 transgenes after integration into the pf9-2 pf28 host strain. DNA was isolated from pf9-3, pf28, pf9-2, the H10 strain obtained by transformation with pD1SA, and the 11 rescued strains generated by transformation with cW1. 4 μg of genomic DNA was digested with SacI, separated on an 0.8% agarose gel, transferred to Magnagraph, and hybridized with probe C (Fig. 1). Probe C identifies the endogenous 4.2-kb SacI fragment in every sample except pf9-3, which contains a 13-kb deletion of the Dhc1 gene (Myster et al. 1997). Probe C also hybridizes to novel restriction fragments in a subset of the transformants, which indicates that this region was a frequent site of rearrangement of the Dhc1 transgenes. Rearrangements in the region corresponding to probe D were identified in the remaining samples (data not shown). (B) Determination of the predicted amino acid sequence of the 1α Dhc fragment in the G3 transformant. The truncated Dhc1 transgene was recovered by screening a mini-library constructed from G3 genomic DNA (see Materials and Methods). The 3′ end of the transgene was sequenced with Dhc1 specific primers. The nucleotide sequence revealed that the Dhc1 transgene was fused to an unidentified DNA sequence. The resulting hybrid gene encodes up to residue 1,249 of the 1α Dhc, and then adds 17 novel amino acids before encountering a stop. (C) Schematic representation of the truncated 1α Dhc polypeptide present in transformant G3. The top line is the wild-type 1α Dhc showing the approximate positions of the P-loops, the predicted coiled-coil domains, and the peptide epitope recognized by the 1α Dhc antibody. Below is a similar diagram of the 1α Dhc fragment in G3. The sequence ends at amino acid 1,249, shortly after the epitope recognized by the 1α Dhc antibody.

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