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. 2000 Jul;11(7):2297-313.
doi: 10.1091/mbc.11.7.2297.

Insights Into the Structural Organization of the I1 Inner Arm Dynein From a Domain Analysis of the 1beta Dynein Heavy Chain

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Insights Into the Structural Organization of the I1 Inner Arm Dynein From a Domain Analysis of the 1beta Dynein Heavy Chain

C A Perrone et al. Mol Biol Cell. .
Free PMC article

Abstract

To identify domains in the dynein heavy chain (Dhc) required for the assembly of an inner arm dynein, we characterized a new motility mutant (ida2-6) obtained by insertional mutagenesis. ida2-6 axonemes lack the polypeptides associated with the I1 inner arm complex. Recovery of genomic DNA flanking the mutation indicates that the defects are caused by plasmid insertion into the Dhc10 transcription unit, which encodes the 1beta Dhc of the I1 complex. Transformation with Dhc10 constructs encoding <20% of the Dhc can partially rescue the motility defects by reassembly of an I1 complex containing an N-terminal 1beta Dhc fragment and a full-length 1alpha Dhc. Electron microscopic analysis reveals the location of the missing 1beta Dhc motor domain within the axoneme structure. These observations, together with recent studies on the 1alpha Dhc, identify a Dhc domain required for complex assembly and further demonstrate that the intermediate and light chains are associated with the stem regions of the Dhcs in a distinct structural location. The positioning of these subunits within the I1 structure has significant implications for the pathways that target the assembly of the I1 complex into the axoneme and modify the activity of the I1 dynein during flagellar motility.

Figures

Figure 1
Figure 1
Dendrogram of Dhc sequences and mapping of the Dhc10 gene. (A) The deduced amino acid sequences surrounding the conserved ATP hydrolytic site (P-loop 1) were aligned with the GCG program Pileup. The subset of Dhc sequences most closely related to Dhc1 is shown here. The Chlamydomonas reinhardtii (Cr) Dhc1 (U61364, AF243806) and Dhc10 (AJ132799) sequences are marked by asterisks. The GenBank accession numbers for the other sequences are as follows: Tripneustes gratilla (Tg), U03976, U03973; Rattus norvegicus (Rn), D26502, D26493; Mus musculus (Mm), Z83812, Z83813; Paramecium tetraurelia (Pt) L18803; Drosophila melanogaster (Dm) L23200. (B) Genetic map location of the Dhc10 gene. The Dhc10 gene was mapped to linkage group XV based on linkage to the genetic marker ida2 (<2.6 cM, no recombinants in 18 tetrads) and the molecular marker Dhc7 (∼7.4 cM).
Figure 2
Figure 2
Inner arm structure in wild-type and mutant strains. Longitudinal images of the 96-nm axoneme repeat from wild type, ida2-6, and the rescued ida2-6 strain, D11, are shown here. The averages for wild type (A), ida2-6 (C), and the rescued ida2-6 strain (E) are based on 8, 10, and 9 individual axonemes and 74, 87, and 85 axoneme repeats, respectively. (B) Model of the 96-nm axoneme repeat with the major lobes of density in the inner arm region labeled 1–10. The outer arms (OA) are on top, the inner arm (IA) region is below, and the proximal and distal radial spokes are labeled S1 and S2, respectively. (D) Difference plot between wild type and ida2-6. (F) Difference plot between wild type and the rescued ida2-6 strain. The difference plots are derived from a pixel-by-pixel analysis of variance between averages, where statistically significant differences between the averages for two strains are indicated by the gray areas. Differences not significant at the 0.005 confidence level have been set to zero.
Figure 3
Figure 3
Dynein polypeptide defects in ida2-6 axonemes. (A) The high-molecular-weight region of a 3–5% polyacrylamide, 3–8 M urea gradient gel that was loaded with whole axonemes from wild type, pf9-2, and ida2-6 is shown here. The 1α and 1β Dhcs of the I1 complex, which migrate between the outer arm β and γ Dhcs, are indicated by the asterisks on the right. Both pf9-2 and ida2-6 lack the 1α and 1β Dhcs. (B) Sucrose density gradient centrifugation of dynein extracts from pf28, wild type, ida2-6, and pf9-3. All sucrose gradient fractions were loaded on 5–15% polyacrylamide gels. The subunits of the I1 complex sediment in the 18–19S region, along with contaminating outer arm components (Piperno et al., 1990; Porter et al., 1992). Only the 18–19S peaks are shown here. IC140, IC138, and IC110 are visible in pf28 (which lacks the outer arm components) and wild type. (C) Western blot of wild-type and mutant axonemes probed with the antibody to the Tctex1 LC.
Figure 4
Figure 4
Recovery of genomic DNA flanking the site of the ida2-6 mutation. (A) Cosegregation of plasmid sequences with the ida2-6 motility defect. Shown here is an autoradiogram of a genomic Southern blot of SphI-digested DNA from a wild-type strain, ida2-6, and the slow-swimming progeny from four successive generations (F1, F2, F3, and F4) of ida2-6 × nit1 crosses. The blot was hybridized with the vector sequence from the NIT1 plasmid. Three copies of the plasmid are present in ida2-6 and all of the slow-swimming progeny. (B) Diagram of the plasmid insertion in ida2-6 and the strategy used to clone genomic DNA flanking the site of insertion. Genomic DNA from ida2-6 was digested with ClaI, which cuts once per plasmid, and KpnI, which does not cut within the plasmid sequences. Southern blots probed with the NIT1 plasmid identified a unique 3.4-kb KpnI–ClaI fragment in ida2-6 containing genomic DNA adjacent to the inserted plasmids. This fragment was cloned from a size-fractionated mini library to yield the plasmid p27B3 (see MATERIALS AND METHODS). p27B3 was then digested with KpnI–Sau3A to release a 350-bp fragment containing only genomic DNA. (C) Genomic Southern blot of wild-type and ida2-6 DNA probed with the 350-bp KpnI–Sau3A fragment. A RFLP is clearly visible between the two strains, confirming that the genomic DNA is close to the site of plasmid insertion in ida2-6. (D) Northern blot of wild-type and ida2-6 RNA hybridized with a 3.2-kb SacI fragment from the end of phage clone C. A single large (>13 kb) transcript is visible in wild type, and its expression is increased after deflagellation (compare 0 and 45 min). The Dhc10 transcript is significantly smaller in ida2-6 (∼4 kb), but it is still expressed at a high level after deflagellation. This transcript also hybridizes with a NIT8 probe (see text). To ensure that equal quantities of RNA were loaded in all lanes, Northern blots were also hybridized with the CRY1 gene encoding the ribosomal S14 subunit.
Figure 5
Figure 5
Recovery of the IDA2/Dhc10 transcription unit. (A) Shown on the top is a partial restriction map of the IDA2/Dhc10 genomic region in wild type. Shown below is a diagram of the genomic region in two different insertional mutants, ida2-6 and ida2-7. Vertical marks indicate SacI sites; the approximate sizes of the restriction fragments are also indicated. The shaded rectangle corresponds to the 350-bp KpnI–Sau3A probe recovered as the genomic fragment flanking the site of plasmid insertion in ida2-6; the small open rectangle corresponds to the 150-bp Dhc10 PCR product; and the rectangle with hash marks indicates the 3.2-kb SacI fragment isolated from the end of phage clone C. The approximate location of the Dhc10 transcription unit is illustrated by the arrow. The ida2-6 mutation was generated by plasmid insertion into the Dhc10 coding region. The plasmid insertion event in ida2-7 was associated with a large (>20 kb) deletion of the Dhc10 transcription unit. (B) Wild-type phage clones recovered using the 350-bp KpnI–Sau3A probe are shown with respect to the genomic region above. Clones C, B, and H were tested for their ability to rescue the motility defect of ida2-6 by cotransformation. Clone C encodes up to residue 989 of the 1β Dhc sequence, and clone H encodes up to residue 1695. The resulting N-terminal fragments are predicted to have molecular masses of ∼113 and 195 kDa, respectively, and completely lack the dynein motor domain. (C) Phage clones recovered using the 150-bp Dhc10 PCR fragment. (D) A BAC clone that was recovered using an RT-PCR product from the 3′ end of the Dhc10 gene. (E) Diagram of smaller subclones used in cotransformation experiments to rescue ida2-6 and ida2-7. These subclones are drawn to scale with respect to parts A–D. pCAP1 encodes up to residue 989 of the 1β Dhc; pCAP2 encodes up to residue 508; and pCAP3 encodes up to residue 811. (F) Diagram of the intron/exon structure of the Dhc10 gene as determined by RT-PCR. The exons are indicated by the open rectangles, and the introns are indicated by solid lines. A TATA box sequence located ∼140 bp upstream of the proposed translation start site is also shown. The identification of the translation start site was based on the recovery of an RT-PCR product using a forward primer downstream of the TATA box sequence and a reverse primer from exon 3. This RT-PCR product contained stop codons in all three frames preceding the proposed start codon. The region upstream of the TATA box sequence also contains six tub box sequences thought to enhance the expression of flagellar genes (Davies and Grossman, 1994). The 3′ end of the gene is contained within the 7.2-kb SacI subclone (see A). Two putative polyadenylation signals (TGTAA) were identified 116 and 445 bp downstream of the proposed stop site. Also shown are the sites that correspond to the junction between Dhc10 and the plasmid insertions in ida2-6, and the 3′ end of the Dhc10 sequence in phage clone C and pCAP1. The scale of this diagram is enlarged relative to parts A–E.
Figure 6
Figure 6
Predicted amino acid sequence of the 1β Dhc. The deduced amino acid sequence (residues 1–4513) of the Dhc10 gene product is shown here. The four regions corresponding to P-loops 1–4 are underlined. The regions containing the degenerate P-loops identified by comparison with the cytoplasmic Dhc from Dictyostelium (Neuwald et al., 1999) are indicated by the dashed boxes. The peptide sequence (VALQTDKQRRDMED) used to generate a specific antibody (residues 945–959) and the C-terminal amino acid residues encoded by the different Dhc10 constructs used for cotransformation are also shown.
Figure 7
Figure 7
Diagram depicting regions of high coiled-coil probability for the Dhc10 gene product. The plot was generated using the COILS program (Lupus et al., 1991; Lupus 1996) with a window size of 28 and the MTIDK matrix. The approximate positions of the four central P-loops are indicated by the arrowheads.
Figure 8
Figure 8
The Dhc10 antibody identifies the 1β Dhc as the gene product of the IDA2/Dhc10 locus. (A) Specificity of I1 Dhc antibodies. The I1 complex was purified by sucrose density gradient centrifugation and analyzed on 5% polyacrylamide gels to resolve the 1α and 1β Dhcs (left lane). Duplicate immunoblots were probed with an affinity-purified Dhc1 antibody (middle lane) and an affinity-purified Dhc10 antibody (right lane). (B) Reassembly of I1 complex subunits in Dhc10 transformants. Axonemes were isolated from wild-type and mutant strains, run on 5% polyacrylamide gels, and blotted to either nitrocellulose or polyvinylidene difluoride. One strip containing only the Dhc region was probed with the Dhc1 antibody to detect the 1α Dhc (top blot). A second strip containing the region above 85 kDa was probed with the Dhc10 antibody to detect the 1β Dhc and related N-terminal fragments (middle blot). A third strip containing the 140-kDa region was probed with the IC140 antibody (bottom blot). ida2-6 and ida2-7 are two different insertional mutants (see Figure 5A). Both strains were cotransformed with Dhc10 constructs encoding N-terminal 1β Dhc fragments of varying lengths (see Figure 5, B and E). Note that the 1β Dhc fragment encoded by the pCAP3 subclone does not contain the peptide epitope used to generate the Dhc10 antibody, so the ∼92-kDa fragment cannot be detected by Western blot analysis of axoneme polypeptides.
Figure 9
Figure 9
Formation of an I1 complex in the rescued ida2 strain. Dynein extracts from wild type and the rescued ida2-6 strain, D11, were subjected to sucrose density gradient centrifugation. The resulting fractions were run on duplicate 5–15% polyacrylamide gradient gels, and the gels were either silver stained or transferred to Western blots. (A) Gel of wild-type fractions sedimenting in the 19S region. The 110-, 138-, and 140-kDa ICs of the I1 complex (indicated by the solid circles) cosediment in fractions 6 and 7. (B) Gel of the rescued ida2-6 strain, D11. The I1 ICs cosediment at 16S in fractions 9 and 10. A novel polypeptide migrating at ∼113 kDa in fractions 9 and 10 is indicated by a diamond. This polypeptide is not present in the wild-type gradient. (C) Blots of the wild-type fractions shown in A were probed with antibodies against the 1α Dhc (top panel), IC140 (second panel), 1β Dhc (third panel), and Tctex1 LC (bottom panel). (D) Blots of the rescued ida2-6 fractions shown in B were probed with I1 complex antibodies as described in C. Note the presence of the 1β Dhc fragment at ∼113 kDa.
Figure 10
Figure 10
(A) Scheme of 1β Dhc and N-terminal fragments analyzed in this study. Also indicated are the locations of the P-loops (P1, P2, P3, and P4) and the regions predicted to form α-helical coiled coils (curlicues). (B) Scheme illustrating the location of I1 complex components within the structure of the 96-nm axoneme repeat.

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