Chlamydomonas flagellar outer row dynein assembly protein ODA7 interacts with both outer row and I1 inner row dyneins

Abstract

We previously found that a mutation at the ODA7 locus in Chlamydomonas prevents axonemal outer row dynein assembly by blocking association of heavy chains and intermediate chains in the cytoplasm. We have now cloned the ODA7 locus by walking in the Chlamydomonas genome from nearby molecular markers, confirmed the identity of the gene by rescuing the mutant phenotype with genomic clones, and identified the ODA7 gene product as a 58-kDa leucine-rich repeat protein unrelated to outer row dynein LC1. Oda7p is missing from oda7 mutant flagella but is present in flagella of other outer row or inner row dynein assembly mutants. However, Oda7 levels are greatly reduced in flagella that lack both outer row dynein and inner row I1 dynein. Biochemical fractionation and rebinding studies support a model in which Oda7 participates in a previously uncharacterized structural link between inner and outer row dyneins.

Fig. 1

Chromosome walk to identify the ODA7 locus. Recombinants between ARG7 and ODA7 on the right arm of linkage group (L.G.) I were tested for segregation of markers PBT302, CNC41, Gbp1, and CNA73. In (A) the number of recombinants in each interval is shown below a map of this region of L.G. I and indicates that PBT302 is the molecular marker closest to ODA7. In (B) three PBT302-selected BACs are shown below a diagram of sequence scaffolds 41 and 139, as well as the locations of marker CNC41, the ODA7 locus, and BAC clone 17H5 which spans the ODA7 locus. An enlarged diagram of BAC 17H5 (C) shows the location of presumed transcription units (arrows), including ODA7. Lines below the 17H5 diagram indicate sequence regions spanned by genomic lambda and plasmid clones that were selected with a probe from the ODA7 coding region and found to complement the oda7 mutation.

Fig. 2

Sequence analysis of the Oda7 protein. Six leucine rich repeats (LRR) appear near the amino terminus of Oda7 (A). All six repeats contain elements typical of SDS22 family LRR units (B), including alternating alpha helix-beta sheet regions and an LRR cap sequence (underlined). Residues in the SDS22-family consensus pattern are shown in bold. Sequence comparison with other LRR proteins that were selected from phylogenetically diverse organisms indicate that most organisms contain a single Oda7 homolog, and that Oda7 homologs group in a separate subfamily from dynein light chain 1 (LC1) and protein phosphatase regulatory subunit SDS22, as illustrated by an unrooted tree (C). For (C), alignments were generated with ClustalW using the following sequences: Urchin LC1, BBA24184; Giardia LC1, XP_767743; Canine LC1, XP_853805; Human LC1, AAQ11377; Xenopus LC1, AAH82218; Fly LC1, NP_610483; Chlamy LC1, AAD41040; Cerevisiae SDS, P36047; Pombe SDS, S43988; Fly SDS, AAM50611; Human SDS, AAD26610; Mouse SDS, BAE26253; Chlamy SDS, C_1490024 (JGI database v2.0); Giardia Oda7, EAA42565; Fly Oda7, AAN11119; Malaria Oda7, AAX86879; Zebrafish Oda7, AAH45963; Human Oda7, AAH24009; Mouse Oda7, AAH50751; Chlamy Oda7, ABI63572.

Fig. 3

Distribution of Oda7 protein in flagellar assembly mutants. A, an affinity-purified antibody to Oda7 recognizes a ca. 58 kDa flagellar protein. After fractionation of flagella (F) with NP40 into insoluble axoneme (A) and soluble membrane/matrix (M), most of Oda7 becomes soluble. B, immunoblots of whole flagella from wild type (WT) and outer row dynein assembly mutants (oda strain) shows that Oda7 is only missing from oda7 flagella. C. Immunoblots of flagella from representative inner row dynein mutants (ida strains), all of which retain Oda7. The ida7 strain consistently shows a slight reduction in Oda7 levels. D, comparison of Oda7 in flagella that lack only I1 inner row dynein (ida7), or both outer row dynein and I1 dynein (WS4). The WS4 strain also contains a suppressor mutation (ssh1), which has no apparent affect on Oda7 assembly.

Fig. 4

Salt-extracted Oda7 sediments as a large complex. A, axonemes, created by treating flagella with 1.0 % OG, were extracted with the indicated concentrations of NaCl. The gel and immunoblot of extracted pellets show that most of the Oda7p is removed by 0.6 M NaCl treatment. B, sedimentation of a 0.6 M NaCl extract on a sucrose gradient reveals co-sedimentation of Oda7 with I1 inner arm proteins. Blots show distribution of Oda7p and I1 subunit IC140. IC1 and IC2 are outer row dynein intermediate chains, IC138/140 and IC97 are I1 inner row dynein intermediate chains.

Fig. 5

Sedimentation properties of Oda7 in 0.6 M NaCl extracts of flagella that lack outer row dynein (A), I1 inner row dynein (B), or both dyneins (C). A, in the absence of outer row dynein, Oda7 co-sediments with I1 dynein. B, when I1 dynein is absent, Oda7 sediments in two peaks, one at the top of the gradient and one distributed over several fractions. C, when both dyneins are missing, most of the residual Oda7 sediments near the top of the gradient.

Fig. 6

Sedimentation under non-standard conditions. 0.6 M NaCl extracts of OG-demembranated axonemes were sedimented on sucrose gradients at 181,000 × g for 4.5 hr to maintain outer row dynein as a single complex. A, Oda7 co-sediments with outer row dynein in ida7 extracts (absence of I1 dynein). B, Oda7 co-sediments with I1 dynein in wild type extracts. Outer row dynein docking complex subunits (DC1, DC2) co-sediment with outer row dynein intermediate chains (IC1, IC2) and light chains (LC).

Fig. 7

Re-association of Oda7 protein with oda7 axonemes. Axonemes from oda7 flagella were incubated in the presence of a 0.6 M NaCl extract (HSE) of wild type axonemes (+) or in buffer alone (−) and centrifuged. A, pellets analyzed by SDS-PAGE and immunoblot show the rebinding of both outer row dynein (IC2) and Oda7 to levels similar to those in wild type axonemes (WT AX). B, thin section EM shows restoration of outer row dynein structures. Bar in B = 100 nm.