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. 2005 Dec;16(12):5661-74.
doi: 10.1091/mbc.e05-08-0732. Epub 2005 Sep 29.

Differential Light Chain Assembly Influences Outer Arm Dynein Motor Function

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Free PMC article

Differential Light Chain Assembly Influences Outer Arm Dynein Motor Function

Linda M DiBella et al. Mol Biol Cell. .
Free PMC article

Abstract

Tctex1 and Tctex2 were originally described as potential distorters/sterility factors in the non-Mendelian transmission of t-haplotypes in mice. These proteins have since been identified as subunits of cytoplasmic and/or axonemal dyneins. Within the Chlamydomonas flagellum, Tctex1 is a subunit of inner arm I1. We have now identified a second Tctex1-related protein (here termed LC9) in Chlamydomonas. LC9 copurifies with outer arm dynein in sucrose density gradients and is missing only in those strains completely lacking this motor. Zero-length cross-linking of purified outer arm dynein indicates that LC9 interacts directly with both the IC1 and IC2 intermediate chains. Immunoblot analysis revealed that LC2, LC6, and LC9 are missing in an IC2 mutant strain (oda6-r88) that can assemble outer arms but exhibits significantly reduced flagellar beat frequency. This defect is unlikely to be due to lack of LC6, because an LC6 null mutant (oda13) exhibits only a minor swimming abnormality. Using an LC2 null mutant (oda12-1), we find that although some outer arm dynein components assemble in the absence of LC2, they are nonfunctional. In contrast, dyneins from oda6-r88, which also lack LC2, retain some activity. Furthermore, we observed a synthetic assembly defect in an oda6-r88 oda12-1 double mutant. These data suggest that LC2, LC6, and LC9 have different roles in outer arm assembly and are required for wild-type motor function in the Chlamydomonas flagellum.

Figures

Figure 1.
Figure 1.
Molecular analysis of LC9. (a) Nucleotide and predicted protein sequence of LC9 originally derived from the overlapping ESTs BG849022, BG847628, BI721765, BI530285, BG847627, and BG849020 and confirmed by analysis of a full-length cDNA. The 5′-UTR contains two in-frame stop codons, and a perfect copy of the Chlamydomonas polyadenylation signal (underline) is present in the 3′-UTR. This sequence is available under GenBank accession no. DQ114947. (b) RNA samples from nondeflagellated cells (NDF) and from cells that had been deflagellated by pH shock and allowed to regenerate flagella for 30 min (30′postDF) were probed with the LC9 coding region. A single message of ∼1.3 kb is greatly up-regulated after flagellar excision. (c) Rooted neighbor-joining phylogenetic tree illustrating the relationship between LC9, Chlamydomonas inner arm Tctex1 (AF039437), sea urchin outer arm LC3 (JC6573), murine Tctex1 (A32995), and human rp3 (U02556). Also included are three members of the Tctex2 light chain subclass, which form a distinct grouping: murine Tctex2 (U21673), Chlamydomonas outer arm LC2 (U89649) and Chlamydomonas inner arm Tctex2b (BK004867). (d) Sequence alignment of the Tctex1-related proteins shown in c was performed using ClustalW. The secondary structure of Chlamydomonas Tctex1 determined by NMR spectroscopy (Wu et al., 2001, 2005) is shown above the alignment. The strand-switched dimer interface involves the β3 strand (white), which hydrogen bonds to the β2 strand from the other monomer. Residues in mammalian Tctex1 that interact with the cytoplasmic dynein IC (Mok et al., 2001) are indicated by *.
Figure 2.
Figure 2.
Oligomeric state of LC9 and specificity of the CT231 antibody. (a) The oligomeric state of fusion proteins containing LC9 and Chlamydomonas inner arm Tctex1 attached to MBP was determined by multi-angle laser light scattering. The absorbance at 214 nm of the gel filtration column elution profile for MBP-Tctex1 (solid line) and MBP-LC9 (dotted line) is shown. Individual molecular weight determinations are indicated by • (MBP-Tctex1) and □ (MBP-LC9), respectively. MBP-Tctex1 is dimeric, whereas MBP-LC9 is mostly monomeric under the same solution conditions; a small peak of MBP-LC9 dimer is evident coeluting with MBP-Tctex1. (b) The Tctex1 and LC9 fusion proteins were digested with factor Xa, electrophoresed in a 15% acrylamide gel and either stained with Coomassie blue (top) or blotted to nitrocellulose and probed with CT231 (bottom). The antibody recognizes LC9 but not Tctex1 with which it shares 40% sequence identity. (c) Recombinant outer arm dynein LCs were probed with CT231 raised against LC9. No other outer arm LC component is recognized by this antibody. (d) Approximately 120 μgof wild-type axonemes were electrophoresed in a 5–15% acrylamide gradient gel and stained with Coomassie blue (left) or probed with CT231 (right). Only a single axonemal protein of the appropriate Mr is recognized.
Figure 3.
Figure 3.
LC9 is a component of the outer dynein arm. (a) Approximately 150 μg of flagella were treated with detergent to yield the microtubular axoneme and solubilize the membrane and flagellar matrix components. Subsequently, the axonemes were extracted with 0.6 M NaCl to remove proteins associated via ionic interactions. Equivalent amounts of each sample were separated in a 5–15% acrylamide gradient gel and stained with Coomassie blue (top) or blotted and probed with CT231 (bottom). LC9 is specifically associated with the axonemes and most is solubilized by high salt treatment. The Mr markers are indicated at left. (b) The 0.6 M NaCl extract was sedimented through a 5–20% sucrose density gradient in the presence of Mg2+, and equivalent volumes of each fraction were electrophoresed in a 5–15% acrylamide gradient gel (top, Coomassie blue; bottom, CT231 blot); the outer dynein arm sediments at ∼23 S in fractions 5–7, as does LC9. The Mr markers are indicated at left. (c) Axonemes from wild-type Chlamydomonas (strain cc124) and from mutants lacking inner arm I1 (ida1), a subset of inner arms I2/3 (ida4), the outer arm (oda9), the outer arm and docking complex (oda3), both outer arms and inner arm I1 (pf28pf30), radial spokes (pf14), and central pair microtubule complex (pf18) were electrophoresed and probed with CT231. Also included is the strain oda15, which lacks LC7a and only assembles ∼30% of outer arms (DiBella et al., 2004a). The LC9 protein is specifically missing only in those strains that do not assemble outer dynein arms.
Figure 4.
Figure 4.
LC9 interacts with both outer arm dynein intermediate chains. Purified outer arm dynein from the ida1 strain that lacks inner arm I1 was treated with 0 or 20 mM EDC, electrophoresed, and probed with the CT231 antibody (left). In the absence of EDC, only the LC9 band was observed. However, after EDC treatment two additional prominent bands of Mr ∼80,000–90,000 were obtained. In the center and right panels, single lanes of 20 mM EDC-treated dynein were cut lengthwise, and half were probed for LC9 and the other half for either IC2 (monoclonal antibody [mAb] 1869A) or IC1 (mAb 1878A). Reassembly of the blots revealed that the two LC9 cross-linked products also contain IC2 (Mr ∼80,000 band) and IC1 (Mr ∼90,000 band).
Figure 5.
Figure 5.
An oda6 pseudorevertant lacks LC2, LC6, and LC9. (a) Diagram of the IC2 protein indicating the location of WD-repeats, the C-terminal coiled coil domain, a putative LC9 interaction site, and the original oda6-95 frame-shift that results in a premature stop codon is shown at top. Maps of IC2 from oda6-95 and the two pseudorevertants (oda6-r75 and oda6-r88) are shown below the wild type (data are from Mitchell and Kang, 1993). In these diagrams, residues that are altered from the original and their location are indicated. Whether the strain is capable of outer arm dynein assembly and flagellar beat frequency analysis is indicated at right. (b) Axonemes from wild-type, oda6, oda6-r75, and oda6-r88 strains were electrophoresed in a 5–15% acrylamide gradient gel and stained with Coomassie blue. The Mr markers are at left. (c) Identical samples to those shown in b were blotted and probed with antibodies R5932, R5391, R4930, R4928, and CT231 to detect LC1, LC2, LC3, LC6, and LC9, respectively. All these outer arm proteins are missing in oda6 but are restored in oda6-r75. In contrast, LC2, LC6, and LC9 are not present in axonemes from the oda6-r88 pseudorevertant, which assembles outer arms but does not rescue the beat frequency defect.
Figure 6.
Figure 6.
LC9 assembly is not dependent on LC6. (a) Flagellar axonemes were isolated from wild-type and the oda13 strain, and ∼100 μg of each was electrophoresed in a 5–15% acrylamide gradient gel and stained with Coomassie blue. The Mr markers are at left. (b) Similar samples were blotted to nitrocellulose and probed with antibodies R5391, R4930, R4928, and CT231 to detect LC2, LC3, LC6, and LC9, respectively (right). Only LC6 is missing in the oda13 mutant axonemes, indicating that this protein is not required for the assembly of either LC2 or LC9. (c) FFT analysis of the oda13 mutant and the parental wild-type strain (g1) reveals only a subtle reduction in the peak flagellar beat frequency for the mutant.
Figure 7.
Figure 7.
The oda6-r88 allele is not an extragenic suppressor of oda12-1. (a) PCR analysis of genomic DNA from the progeny of an oda6-r88× oda12-1 cross and both wild-type and parental mutant strains. Top, a 585-base pair segment of the LC2 gene that is completely missing in the oda12-1 null allele. Bottom, a BamHI digest of a 495-base pair region of the IC2 gene that includes the novel restriction site introduced by the oda6-r88 reversion. In this tetratype tetrad, progeny “b” represents the double mutant. (b) Flagellar beat frequency analysis of the four tetrad products identified in a. The double mutant exhibits a beat frequency similar to oda12-1 and is clearly reduced from that observed for oda6-r88. (c) Axoneme samples from each tetrad product were electrophoresed in a 5–15% acrylamide gradient gel and stained with Coomassie blue. The Mr markers are indicated at left. (d) Samples identical to those shown in c were blotted to nitrocellulose and probed to detect various components of the outer dynein arm. The Tctex1 protein from inner arm I1 was used as a loading control. (e) Cross-section electron micrographs of wild-type and oda6-r88 oda12-1 mutant axonemes. No outer arms are detectable in the double mutant.
Figure 8.
Figure 8.
Model for IC/LC interactions within the outer dynein arm. A model for protein-protein associations within the IC/LC complex of outer arm dynein is shown. Both ICs consist of an N-terminal region (in IC1 this interacts directly with α-tubulin), seven WD-repeats that form a β-propeller, and a C-terminal segment (in IC2 this is predicted to form a coiled-coil). LC2, LC6, and LC9 likely interact with IC2 via the region defective in oda6-r88; the region altered in oda6-r75 is not necessary for their assembly. Both LC2 (Mitchell and Rosenbaum, 1986) and LC9 (this study) have been shown to interact directly with IC1. Putative LC8 binding sites within both ICs are located relatively close to the WD-repeat region. The precise order of association sites on IC1 and within the oda6-r88-defective region of IC2 is speculative. Analysis of other dyneins has suggested that members of the LC7/Roadblock family interact with IC regions either N-terminal to the first WD-repeat (Susalka et al., 2002) or C-terminal of the last repeat (Hendrickson et al., 2004); due to the interlocking design of the WD-repeat β-propeller, these regions are spatially close together. Maximal distances (in angstroms) between components are based on DMP and EDC cross-linking (King et al., 1991; DiBella et al., 2001, 2004a).

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