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. 1998 Dec;9(12):3351-65.
doi: 10.1091/mbc.9.12.3351.

The Chlamydomonas IDA7 Locus Encodes a 140-kDa Dynein Intermediate Chain Required to Assemble the I1 Inner Arm Complex

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The Chlamydomonas IDA7 Locus Encodes a 140-kDa Dynein Intermediate Chain Required to Assemble the I1 Inner Arm Complex

C A Perrone et al. Mol Biol Cell. .
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Abstract

To identify new loci that are involved in the assembly and targeting of dynein complexes, we have screened a collection of motility mutants that were generated by insertional mutagenesis. One such mutant, 5B10, lacks the inner arm isoform known as the I1 complex. This isoform is located proximal to the first radial spoke in each 96-nm axoneme repeat and is an important target for the regulation of flagellar motility. Complementation tests reveal that 5B10 represents a new I1 locus, IDA7. Biochemical analyses confirm that ida7 axonemes lack at least five I1 complex subunits. Southern blots probed with a clone containing the gene encoding the 140-kDa intermediate chain (IC) indicate that the ida7 mutation is the result of plasmid insertion into the IC140 gene. Transformation with a wild-type copy of the IC140 gene completely rescues the mutant defects. Surprisingly, transformation with a construct of the IC140 gene lacking the first four exons of the coding sequence also rescues the mutant phenotype. These studies indicate that IC140 is essential for assembly of the I1 complex, but unlike other dynein ICs, the N-terminal region is not critical for its activity.

Figures

Figure 1
Figure 1
Analysis of longitudinal images of axonemes isolated from wild-type, ida7, and a rescued ida7 strain (AH1). Grand averages from wild-type (A), ida7 (C), and the rescued ida7 (E) are based on 8, 8, and 9 individual axonemes and 74, 65, and 84 axoneme repeats, respectively. (B) Model of axoneme structures within the 96-nm axoneme repeat, with the major lobes of density in the inner arm region labeled 1–10. The proximal and distal radial spokes are labeled S1 and S2, respectively; the outer arms (OA) are on top, and the inner arm (IA) region is below. (D) Difference plot between wild-type and ida7, and (F) difference plot between wild-type and the rescued ida7 strain (AH1). Differences not significant at 0.005 confidence level have been set to zero.
Figure 2
Figure 2
SDS-PAGE analysis of dynein polypeptides in wild-type and mutant axonemes. (A) Shown here is the high molecular weight region of a 3–5% polyacrylamide gradient loaded with whole axonemes from wild-type, pf9–2, and ida7. The outer arm DHCs (α, β, and γ) are indicated on the left in wild type. The 1α and 1β DHCs of the I1 complex migrate between the β and γ DHCs of the outer arm, as indicated by asterisks on the right. Note that both pf9–2 and ida7 axonemes lack the 1α and 1β DHCs. (B) Sucrose density gradient centrifugation of dynein extracts from pf28, wild-type, ida7, and pf9–3. Shown here is a 5–15% polyacrylamide gradient gel loaded with fractions from the 19S region. This region typically contains the peak of the I1 complex and the trailing edge of the outer arm components that peak at 21S. The three ICs associated with the I1 complex are indicated on the left in the pf28 sample, which lacks the outer arms (Mitchell and Rosenbaum, 1985). The I1 ICs are also present in the wild-type sample, but are missing in both ida7 and pf9–3.
Figure 3
Figure 3
Genomic Southern analyses of ida7 probed with plasmid sequences and the IC140 gene. (A) Cosegregation of the ida7 motility defects and the NIT1 plasmid. Shown here are Southern blots of PstI-digested genomic DNA isolated from wild-type, the original ida7 strain, and the slow swimming progeny from two successive generations (F1, F2) of ida7 x nit1 crosses. The blot was hybridized to a probe containing vector sequence from the pMN24 (NIT1) plasmid. No vector bands are detectable in the wild-type sample, but three bands are visible in ida7 and in all of its slow swimming progeny. (B) Disruption of the IC140 gene by plasmid insertion. A Southern blot of NotI-digested genomic DNA from wild-type and ida7 was hybridized with an 11.5-kb XbaI genomic fragment containing the IC140 gene. One of the restriction fragments (indicated by the asterisk to the right) is shifted in the ida7 lane. (C) Shown here is a NotI restriction map of the genomic region surrounding the IC140 gene in wild-type (top), and a diagram of the pMN24 plasmid insertions in ida7 (bottom).
Figure 4
Figure 4
Genetic map location of the IC140 gene/IDA7 locus. The IC140 gene maps to a new linkage group, approximately 18.8 cM from the DHC gene Dhc8 and 22.3 cM from the centromere. The ratio of parental ditype:nonparental ditype:tetratype tetrads (PD:NPD:TT) was 15:0:9 for IC140 vs. Dhc8 and 6:11:14 for IC140 vs. the centromere-linked marker ac17.
Figure 5
Figure 5
Rescue of the ida7 motility defects by transformation with constructs of the IC140 genomic region. (A) Diagram of the 11.5-kb wild-type genomic fragment containing the IC140 gene. Areas of interest based on the sequence analysis of Yang and Sale (1998) are shown here. The open rectangles indicate the exons of the IC140 gene, and the solid lines indicate the introns. The predicted translation start and stop sites are indicated, as well as the probable polyadenylation site. The regions containing TATA boxes and “tub” boxes are denoted by brackets. (B) Diagram of the IC140 gene constructs used to transform the ida7 mutant. The constructs are drawn with respect to the diagram of the IC140 gene above. The name of each plasmid construct is listed on the left, and the number of rescued transformants relative to the total number of transformants screened is presented as a ratio on the right.
Figure 6
Figure 6
Expression of IC140 transcripts in wild-type, ida7, and rescued strains. Shown here are Northern blots of total RNA isolated before (0) or after (45) deflagellation. RNA (20 μg per lane) was separated on a 1% formaldehyde agarose gel, transferred to a Magnagraph membrane, and hybridized to a 1.6-kb cDNA fragment of the IC140 gene. A single 3.7-kb transcript is present in both the wild-type and AH1, and the expression of this transcript is up-regulated by deflagellation. The 3.7-kb transcript is completely missing in ida7. A smaller transcript is expressed at high levels both before and after deflagellation in the 5A strain. Hybridizing the blot with a control probe for the CRY1 gene, which encodes the S14 ribosomal protein subunit, confirmed that equal amounts of RNA were loaded in each lane (Perrone and Porter, unpublished results).
Figure 7
Figure 7
Assembly of I1 complex polypeptides in wild-type and mutant axonemes. Whole axonemes were isolated from wild-type, the ida7 mutant, and two of the rescued transformants, strain AH1 and strain 5A, and then loaded in duplicate onto a 7% polyacrylamide gel (25 μg/lane), and blotted to two membranes. (A) The first blot was stained with colloidal gold to confirm that equal amounts of total protein were loaded in all the lanes. (B) The high molecular weight region of the second blot was incubated with an antibody specific for the 1α DHC of the I1 complex. A single, high molecular weight band was seen in the wild-type, AH1, and 5A lanes, but not in the ida7 lane. (C) The bottom of the blot was incubated with an antibody specific for the C-terminal half of the IC140 (Yang and Sale, 1998). No band was detected in the ida7 lane, whereas both wild-type and AH1 contained a single immunoreactive band at approximately 140 kDa. Strain 5A, which was recovered after transformation with a fragment of the IC140 gene, has an immunoreactive band at ∼108 kDa.
Figure 8
Figure 8
Functional domains in dynein ICs. The IC140 is shown diagramatically in comparison to other dynein ICs. The shaded boxes indicate WD repeats; the double loops indicate predicted coiled-coil regions, and the asterisks denote WD repeats that have been implicated in subunit interactions (redrawn following Yang and Sale, 1998). IC78 is the outer arm IC that has been implicated in binding to the axoneme (see MT regions indicated, King et al., 1995). IC69 is another IC required for outer arm assembly (Mitchell and Kang, 1991). Mutations in the N-terminal region can alter flagellar beat frequency (see BF, Mitchell and Kang, 1993). IC2C (IC74) is a cytoplasmic dynein IC that appears to mediate interaction with the p150glued subunit of the dynactin complex (see p150, Vaughan and Vallee, 1995; Steffen et al., 1997).

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