Differential Light Chain Assembly Influences Outer Arm Dynein Motor Function

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.

Systematic Comparison of in Vitro Motile Properties between Chlamydomonas Wild-type and Mutant Outer Arm Dyneins Each Lacking One of the Three Heavy Chains

“…To achieve this condition, we devised a method to specifically label light chain LC2, which is attached to the OAD tail region (25). This light chain is essential for proper OAD assembly, as evidenced by the fact that an LC2/LC10 null mutant, oda12, does not assemble functional OAD (10,26). Although oda12 lacks both LC2 and LC10, its motility defects can be rescued by transformation with a genomic fragment containing only the wild-type LC2 gene (10,11).…”

“…We were able to label OAD by introducing BCCP into a dynein subunit, LC2, by taking advantage of the availability of a mutant (oda12) that lacks LC2 and does not assemble functional OADs in the axoneme (10,26). Another important technical advancement was the development of a simple method for preparing three-headed wild-type OAD and two-headed OADs of all HC combinations.…”

“…There are several factors we must consider. First, in the axoneme, OAD is anchored to the A-tubule of the doublet through the association of multiple light/intermediate chains with tubulin (26) and is arranged unidirectionally, whereas in vitro it is arranged randomly on the glass surface through an avidin-biotin link. Second, in the axoneme, the OAD is attached to the doublet at 24-nm intervals (ϳ40 OADs are attached per micrometer), and adjacent OADs physically interact with one another (56 -59).…”

Three Members of the LC8/DYNLL Family Are Required for Outer Arm Dynein Motor Function

“…Chlamydomonas LC10, sea urchin LC4, and the human DNAL4 protein form a subgroup that is distinct from both canonical LC8/DYNLL proteins and the related Chlamydomonas outer arm component LC6. In this analysis, the Tctex1-related outer arm dynein protein LC9, which is structurally related to LC8 although it shares little sequence homology (DiBella et al, 2005;Wu et al, 2005), was used as the out-group.…”

“…In addition, a LC8-related protein termed LC6 that shares 41% sequence identity is also present (Pfister et al, 1982;King and Patel-King, 1995); intriguingly, orthologues of LC6 have yet to be identified in other organisms, suggesting that its role in outer arm function may be Chlamydomonas-specific. Lack of LC6 leads to a subtle decrease in flagellar beat frequency but does not affect outer arm dynein assembly within the axonemal superstructure (Pazour and Witman, 2000;DiBella et al, 2005). …”