Functional reconstitution of Chlamydomonas outer dynein arms from alpha-beta and gamma subunits: requirement of a third factor.

Abstract

Abstract. The outer dynein arm of Ch/an~omonas flagella, when isolated under Mg2+-free conditions, tends to dissociate into an 11 to 12S particle (12S dynein) containing the ~ heavy chain and a 21S particle (called 18S dynein) containing the ol and/3 heavy chains. We show here that functional outer arms can be reconstituted by the addition of 12S and 18S dyneins to the axonemes of the outer armless mutants odal-oda6. A third factor that sediments at ~7S is required for efficient reconstitution of the outer arms on the axonemes of odal and oda3. However, this factor is not necessary for reconstitution on the axonemes of oda2, oda4, oda5, and oda6. SDS-PAGE analysis indicates that the axonemes of the former two mutants lack a ,~70-kD polypeptide that is present in those of the other mutants as well as in the 7S fraction from the wild-type extract. Furthermore, electron micrographs of axonemal cross sections revealed that the latter four mutants, but not odal or oda3, have small pointed structures on the outer doublets, at a position in cross section where outer arms normally occur. We suggest that the 7S factor constitutes the pointed structure on the outer doublets and facilitates attachment of the outer arm. The discovery of this structure raises a new question as to how the attachment site for the outer arm dynein is determined within the axoneme.

The Outer Dynein Arm-Docking Complex: Composition and Characterization of a Subunit (Oda1) Necessary for Outer Arm Assembly

“…Figure 1, A and B). The M r ϳ70,000 polypeptide previously had been identified as a component of the ODA-DC (Takada and Kamiya, 1994). The present results provide evidence that the factor contains additional proteins of M r ϳ105,000 and ϳ25,000.…”

“…The ODA-DC previously was shown to be absent from axonemes of the outer dynein armless mutants oda1 and oda3, but to be present and functional in axonemes of mutants oda2, oda4, oda5, and oda6 (Takada and Kamiya, 1994). The complex also was shown to be present in 0.6 M KCl extracts of oda6 axonemes, and to sediment at 7S in Mg 2ϩ -free sucrose density gradients.…”

“…These results confirm that the three putative ODA-DC polypeptides occur together in a complex in wild-type axonemes. Moreover, because a functional ODA-DC can be isolated from the 0.6 M KCl extract of wild-type axonemes (Takada and Kamiya, 1994), it is likely that these three polypeptides are sufficient for ODA-DC function and constitute the entire ODA-DC.…”

“…When the Chlamydomonas outer arm dynein is removed from the wild-type axoneme by extraction with 0.6 M KCl in the absence of Mg 2ϩ , it dissociates into a single-headed ␥ subunit (containing the ␥ DHC and two LCs) that sediments as a 12S particle, and a twoheaded ␣␤ subunit (containing the ␣ and ␤ DHCs, the two ICs, and all the remaining LCs) that sediments as a 21S particle (Piperno and Luck, 1979;Pfister et al, 1982;King and Witman, 1989). When these two particles were purified and added back to axonemes of the outer armless mutants oda2, oda4, oda5, or oda6, outer arms were reconstituted in their correct positions on the doublet microtubules (Takada and Kamiya, 1994). In contrast, if the subunits were added back to axonemes of the outer armless mutants oda1 or oda3, very few arms rebound.…”

“…These findings strongly suggested that the projection facilitates attachment of the dynein to the doublet microtubule, and that the 7S factor is the solubilized form of the projection. Partial purification of the factor and comparison of extracts from oda mutant axonemes showed that the factor was correlated with a polypeptide of M r ϳ70,000 (Takada and Kamiya, 1994). Because the factor can assemble onto the doublet microtubules in the absence of the outer dynein arms in vivo, and is necessary for binding of the arms to the microtubules, it has been termed the outer dynein armdocking complex (ODA-DC).…”