The LC7 light chains of Chlamydomonas flagellar 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.

Figure 1.

Molecular cloning and phylogenetic analysis of LC7b. (a) Phylogenetic analysis of the LC7/Roadlock family of LCs. Distances were calculated using ClustalW. Chlamydomonas LC7b (GenBank accession no. AV387124) shares 57% identity with the Drosophila cytoplasmic dynein LC roadblock (NP523771). The family members shown include two Chlamydomonas flagellar LCs, LC7a (AF140239), and LC7b (AV387124), Drosophila Robl (NP523771), human Robl1 (BC002481), and Robl2 (NM130897), mouse Robl1 (NM025947) and Robl2 (NM029297), Ciona Ci-rbDLC (AB074929), and Caeronhabditis elegans (NM063542). Drosophila bithoraxoid (NM169732) also has been identified as a distantly related member of this family. (b) The secondary structures of LC7a and LC7b were predicted using PredictProtein. E, extended sheet; H, helix. The alignment of LC7a and LC7b was generated with ClustalW by using default parameters and shaded using BOXSHADE. Identical residues are indicated in black; similar residues in gray. The two Chlamydomonas proteins share 47% sequence identity. (c) Southern blot of Chlamydomonas wild-type genomic DNA digested with either SmaI, PvuII, PstI, or BamHI. A probe generated from the full-length LC7B coding region detected single bands in SmaI- and BamHI-digested DNA, suggesting a single gene. (d) Northern blot of total RNA isolated from nondeflagellated cells (NDF) and cells actively regenerating their flagella for 30 min (30′postDF). A faint signal at ∼1.4 kb was detected in NDF RNA and an up-regulated band of the same size was observed in 30′postDF RNA. (e) Map of the LC7B genomic region. RFLP analysis revealed that the LC7B gene maps to linkage group XXII/XXIII. The gene spans an ∼1.4-kb region and includes six exons; 5′ and 3′ untranslated regions (UTRs, dark gray), coding regions (black).

Figure 2.

Antibody specificity and localization of LC7b in axonemes. (a) MBP fusions to LC7a and LC7b were digested with Factor Xa, and the LCs separated from MBP by SDS-PAGE, stained with Coomassie Blue (top), or transferred to nitrocellulose (bottom). Blots were probed with either affinity-purified rabbit polyclonal antibody CT116 (LC7b) or R7178 (LC7a) (Bowman et al., 1999). Both antibodies are specific and only recognize their respective LCs. (b) Approximately 150 μg of wild-type (cc124) axonemes were electrophoresed in a 5–15% polyacrylamide gel. The Coomassie Blue-stained gel is shown at left. A blot of an identical sample was probed with affinity purified CT116, which recognized a single, discrete band of the appropriate molecular weight. (c) Approximately 150 μg of isolated axonemes from various strains were electrophoresed in 5–15% acrylamide gradient gels and stained with Coomassie Blue (top) or transferred to nitrocellulose and probed with CT116 (bottom). Strains used include wild-type (cc124), and mutants lacking various axonemal components: ida1 (inner arm I1), ida4 (inner arm subtypes a, c, and d from I2/3), oda1 (outer arms and docking complex), oda9 (outer arm), oda15 (LC7a null mutant), pf28pf30 (outer arm and inner arm I1), pf14 (radial spokes), and pf18 (central pair). Levels of LC7b are significantly reduced in axonemes that lack outer arms (oda1, oda9, oda15) and almost completely diminished in a mutant lacking both outer arms and inner arm I1 (pf28pf30). Molecular weight markers are indicated on the left.

Figure 3.

LC7b is a component of the outer dynein arm. (a) A 0.6 M NaCl extract of ida1 axonemes was loaded onto a 5–20% sucrose gradient either in the presence of Mg²⁺ (a) or without Mg²⁺ (b). After sedimentation under low (a) or high (b) hydrostatic pressure, fractions were electrophoresed in 5–15% acrylamide gels and either stained with Coomassie Blue (top) or blotted to detect LC7b and the docking complex protein DC2. In both a and b, positions of outer arm components are indicated to the left and outer arm docking complex components to the right. (a) In the presence of Mg²⁺, the majority of LC7b comigrated with the trimeric outer arm in fractions 3–6. DC2 sedimented in two pools: one with the outer arm (fractions 3–6) and another at 7S (fractions 10–15). In the absence of Mg²⁺, LC7b copurified with the αβ HC dimer complex (fractions 2–6), suggesting that it does not bind tightly to the γ HC which sedimented at ∼12S (fractions 7–10). Under these conditions, the majority of DC2 was found at 7S (fractions10–14), indicating that it completely dissociated from the outer arm in the absence of Mg²⁺. In both a and b, a small amount of LC7b was observed at ∼10S (fractions 10–12).

Figure 4.

LC7b copurifies with both the outer arms and inner arm I1. In both a and b, 0.6 M NaCl axonemal extracts were loaded onto 5–20% sucrose gradients. After sedimentation, fractions were electrophoresed in 5–15% acrylamide gels and either stained with Coomassie Blue (top) or blotted to detect LC7b. Molecular weight markers are indicated at left. (a) In oda9 extracts, the majority of LC7b comigrated with inner arm I1 (fractions 5–8). (b) In wild-type extracts, LC7b copurified with the outer arms and inner arm I1 (fractions 2–8), suggesting that it associates with both classes of arms. In both c and d, dialyzed 0.6 M NaCl axonemal extracts were subjected to anion exchange chromatography. A linear KCl gradient was used to elute the various dynein subspecies. After purification, fractions were electrophoresed in 5–15% acrylamide gels and either stained with Coomassie Blue (top) or blotted to detect LC7b. Molecular weight markers are indicated to the left and the positions of the outer arm and/or inner arm I1 ICs are indicated to the right. (c) In oda9 extracts, inner arm I1 eluted at ∼380–400 mM salt (fractions 94–99). LC7b also eluted within these same fractions. (d) In wild-type extracts, LC7b copurified with both sets of arms (fractions 90–99).

Figure 5.

Outer dynein arm LC7a is also a component of inner arm I1. (a) Approximately 150 μg of wild-type (cc124) and oda1 axonemes and (b) oda1 purified 18S dynein were electrophoresed in 5–15% gradient gels then either stained with CBB (top) or transferred to nitrocellulose (bottom) and blotted to detect LC7a using the R7178 antibody. LC7a is present in oda1 axonemes and is also a component of purified inner arm I1.

Figure 6.

LC7b cross-links to DC2 from the outer arm docking complex and the LC3 thioredoxin. Purified outer arm dynein from ida1 axonemes was cross-linked with 0 or 20 mM EDC. Samples were electrophoresed in 5–15% gradient gels and then either stained with CBB or transferred to nitrocellulose and blotted to detect various outer arm components. Molecular weight markers are indicated on the left. (a) CBB stained gel of 0 and 20 mM EDC-cross–linked dynein. (b) Blots of 0 and 20 mM EDC-cross–linked dynein probed with CT116 to detect LC7b-reactive products. Three cross-linked species of M_r ∼19,200, 27,400, and 94,700 were identified. Two additional, nonspecific bands indicated by arrows were occasionally observed with this antibody preparation. (c) Identical samples to those shown in b were probed to detect DC2-containing species. (d) Samples similar to those shown in b and c were probed to detect LC3-containing species. (e) A single lane containing the 20 mM EDC-cross–linked sample was cut lengthwise and then probed with either αDC2 (left) or CT116 (right). Realignment of both halves revealed a common reactive band detected by both antibodies. (f and g) Similar procedure as in e, however the left sides of the blots were probed with monoclonal antibodies 1878A against IC1 (f), or 1869A against IC2 (g). The M_r 94,700 band contains both LC7b and DC2 but does not include IC1 or IC2. (h) A similar blot of EDC-treated dynein was probed with antibodies against LC7b and LC3. The M_r 27,400 band contains both proteins.

Figure 7.

LC7b cross-links to IC138 from inner arm I1. Purified inner arm I1 dynein from oda1 axonemes was chemically cross-linked with 0 or 20 mM EDC. Samples were electrophoresed in 5–15% gradient gels and then either stained with CBB or transferred to nitrocellulose and blotted to identify cross-linked products. Molecular weight markers are indicated on the left. (a) CBB-stained gel of 0 and 20 mM EDC-cross–linked dynein. (b) Blots of 0 and 20 mM EDC-cross–linked dynein probed with CT116 to detect LC7b-reactive products. One major cross-linked species at M_r 133,000, a minor product at M_r 17,900 and additional very high-molecular-weight products were obtained. (c) Similar sample as in b, probed with αIC138 to detect IC138-containing species. (d) Single lane of 20 mM EDC-cross–linked dynein divided lengthwise and probed with αIC138 (left) and CT116 (right). Alignment of the blots revealed that both LC7b and IC138 recognize the same band, indicating that they are in direct contact.

Figure 8.

Axonemes from oda15 contain randomly placed outer dynein arms. (a) Approximately 50 μg of wild-type (cc124) and oda15 (LC7a null mutant) axonemes were electrophoresed in 5–15% gradient mini gels then either stained with Coomassie Blue or transferred to nitrocellulose and blotted to detect the outer arm proteins, LC1 (using antibody R5932; Benashski et al., 1999) and LC7a (R7178). Reduced levels of LC1 were found in oda15, signifying the presence of outer arms; however, the LC7a protein was completely absent. (b) A longitudinal section of an oda15 axoneme prepared by thin section electron microscopy. The section outlined is shown at higher magnification, below, and shows stretches of outer arms (arrowheads) and randomly spaced gaps (brackets) where arms are missing. (c) Random cross sections of oda15 axonemes reveal a widely varying complement of outer arms.

Figure 9.

LC7a provides stability to the outer dynein arm. Sucrose gradient (containing Mg²⁺) fractionation of an oda15 high salt extract performed under conditions identical to those used for Figure 3a. Samples were electrophoresed in 5–15% gradient gels and either stained with Coomassie Blue (top) or blotted to detect IC140 from inner arm I1; LC8 from the outer arms, inner arm I1, and radial spokes; the outer arm LC, LC2; the outer arm γ HC; DC2 from the outer arm docking complex, and LC7b. IC140, LC8 and LC2 comigrated at 18S (fractions 6–10); however, both the γ HC and DC2 dissociated and migrated at ∼12S and ∼7S, respectively. In addition, LC7b did not cosediment with components of either the outer arms or inner arm I1, suggesting that LC7a stabilizes the association of this component with dynein.

Figure 10.

Protein interaction maps of the outer dynein arm and inner arm I1. Diagrams illustrating intradynein associations within the outer arm and inner arm I1 are shown. Experimentally determined interactions are indicated by solid connections and dashed lines represent predicted associations. Phosphorylated subunits are identified by “∼P.” Ca²⁺ binding and potential redox sites are also indicated. Within the outer arm, the three HCs (α, β, and γ) each have single LCs that are tightly associated with the stem regions of these polypeptides (LC5, LC3, and LC4, respectively; King and Patel-King, 1995a; Patel-King et al., 1996; Harrison et al., 2002). The γ HC associates with two additional copies of LC1 that bind directly to the HC motor domain and also interact with an unidentified 45-kDa axonemal protein (Benashski et al., 1999). It remains unclear whether the heavy chains interact directly with each other. The cargo-binding region of the outer arm consists of two ICs (IC1 and IC2; Pfister et al., 1982) and several LCs [LC2 (Patel-King et al., 1997; Pazour et al., 1999); LC6 and LC8 (King and Patel-King, 1995b); LC7a (Bowman et al., 1999); and LC7b] that participate in an elaborate series of interactions. This complex interacts with the N-terminal stem regions of the HCs through the ICs; however, it is possible that additional associations with this region of the motor unit exist. The outer arm seems to be linked to the docking complex (DC1-DC3; Koutoulis et al., 1997; Takada et al., 2002; Casey et al., 2003) via LC7a and LC7b. LC7b is in direct contact with DC2 and together with the γ HC, they require LC7a to form a stable association with the rest of the outer arm. Additional IC1-tubulin (indicated as TUB on the diagram) (King et al., 1991) and γ HC-tubulin (Sakakibara and Nakayama, 1998) interactions also have been identified. A novel Tctex1-related protein (labeled as LC9) also associates with the outer arm through the ICs (L. M.DiBella and S. M.King, unpublished observations). Inner arm I1 dynein contains two distinct HCs (1α and 1β), and a cargo-binding region composed of three ICs (IC110, IC138, and IC140) and several LCs (Tctex1, Tctex2b, LC8, LC7a, and LC7b). Interactions exist between 1α and 1β and cross-linking experiments have identified associations between IC110 and IC140, and IC138 and LC7b. IC138 is a key regulatory subunit of this dynein. LC7a also seems to mediate a stable interaction between LC7b and this inner arm species.