The LC7 Light Chains ofChlamydomonasFlagellar Dyneins Interact with Components Required for Both Motor Assembly and Regulation

Abstract

Members of the LC7/Roadblock family of light chains (LCs) have been found in both cytoplasmic and axonemal dyneins. LC7a was originally identified within Chlamydomonas outer arm dynein and associates with this motor's cargo-binding region. We describe here a novel member of this protein family, termed LC7b that is also present in the Chlamydomonas flagellum. Levels of LC7b are reduced approximately 20% in axonemes isolated from strains lacking inner arm I1 and are approximately 80% lower in the absence of the outer arms. When both dyneins are missing, LC7b levels are diminished to <10%. In oda9 axonemal extracts that completely lack outer arms, LC7b copurifies with inner arm I1, whereas in ida1 extracts that are devoid of I1 inner arms it associates with outer arm dynein. We also have observed that some LC7a is present in both isolated axonemes and purified 18S dynein from oda1, suggesting that it is also a component of both the outer arm and inner arm I1. Intriguingly, in axonemal extracts from the LC7a null mutant, oda15, which assembles approximately 30% of its outer arms, LC7b fails to copurify with either dynein, suggesting that it interacts with LC7a. Furthermore, both the outer arm gamma heavy chain and DC2 from the outer arm docking complex completely dissociate after salt extraction from oda15 axonemes. EDC cross-linking of purified dynein revealed that LC7b interacts with LC3, an outer dynein arm thioredoxin; DC2, an outer arm docking complex component; and also with the phosphoprotein IC138 from inner arm I1. These data suggest that LC7a stabilizes both the outer arms and inner arm I1 and that both LC7a and LC7b are involved in multiple intradynein interactions within both dyneins.

Cooperative binding of the outer arm-docking complex underlies the regular arrangement of outer arm dynein in the axoneme

“…The ODA-DC molecules cooperatively bind to the doublet in an end-toend manner and establish a 24-nm periodicity. Subsequently, OAD binds to the outer doublet through its association with the ODA-DCs on the doublet, and this interaction is stabilized through the direct interaction of IC1 with the microtubule, thereby establishing the 24-nm periodicity in the OAD arrangement (24,25,29,30).…”

“…Defects in CCDC114 are known to cause PCD and the patients lack outer dynein arms, suggesting that DC2 functions in OAD docking in humans also (22,23). The ODA-DC binds OAD via at least three interactions: one between DC1 and the OAD intermediate chain IC1, one between DC1 and the other OAD intermediate chain IC2, and one between DC2 and the OAD light chain LC7b (24,25). However, we still do not understand the architecture of the ODA-DC and the detailed manner of its binding to the doublet microtubules, which will be essential for understanding the molecular basis of OAD periodicity.…”

Differential Light Chain Assembly Influences Outer Arm Dynein Motor Function

“…After demembranation with 1% IGEPAL CA-630 (replaces Nonidet P-40), dynein was extracted from the resulting axonemes by treatment with 0.6 M NaCl. The outer arm and associated docking complex were then purified by sucrose density gradient centrifugation in the presence of Mg 2ϩ at low hydrostatic pressure (Takada et al, 1992;DiBella et al, 2004a). Samples were routinely electrophoresed in 5-15% acrylamide SDS gradient gels or 12.5% acrylamide slab gels and either stained with Coomassie blue or transferred to nitrocellulose for immunoblotting.…”

“…Other antibodies used in this study were blot-purified rabbit polyclonal antibodies R5932 (vs. LC1; Benashski et al, 1999), R5391 (vs. LC2;Patel-King et al, 1997), R4930 (vs. LC3;Patel-King et al, 1996), CT61 (vs. LC4; M. Sakato and King, unpublished data), R4929 (vs. LC5;Patel-King et al, 1996), R4928 (vs. LC6; King and Patel-King, 1995), R7178 (vs. LC7a;Bowman et al, 1999), CT116 (vs. LC7b;DiBella et al, 2004a), R4058 (vs. LC8; King and Patel-King, 1995), and anti-DC 2 (Wakabayashi et al, 2001), and mouse monoclonal antibodies 12␥B (vs. ␥ HC; King et al, 1985), 1878A (vs. IC1; King et al, 1986), and 1869A (vs. IC2; King et al, 1985). Antibody reactivity was detected using peroxidase-conjugated secondary antibodies and an enhanced chemiluminescent substrate (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom).…”

“…Cross-linking with EDC was performed essentially as described previously (King et al, 1991;DiBella et al, 2004a). Isolated axonemes and purified dynein samples in 30 mM HEPES, pH 7.4, 5 mM MgSO 4 , 0.5 mM EDTA, and 25 mM KCl (HMEK) buffer were treated with either 0 or 20 mM EDC in 100 mM HEPES, pH 7.4, for 60 min at room temperature.…”