Abstract
We describe here the molecular cloning and analysis of the Mr 14,000 and 16,000 outer arm dynein light chains (DLCs) from Chlamydomonas flagella. Within the outer arm, the Mr 14,000 DLC apparently is associated with the intermediate chains at the base of the soluble dynein particle; the Mr 16,000 DLC interacts directly with the a dynein heavy chain. Sequence analysis indicates that both molecules are novel members of the thioredoxin superfamily and share approximately 30% sequence identity with thioredoxin from Penicillium. Both DLCs have a perfect copy of the thioredoxin active site (WCGPCK); the Mr 16,000 DLC also contains the canonical P-loop motif (AX4GKS). There is a single gene for both DLCs within Chlamydomonas and only single messages that were upregulated more than 10-fold upon deflagellation were observed on Northern blots. Both recombinant DLCs were specifically eluted from a phenylarsine oxide matrix with beta-mercaptoethanol indicating that they contain vicinal dithiols competent to undergo reversible oxidation/reduction. Furthermore, we demonstrate that outer (but not inner) arm dynein may he purified on the basis of its affinity for phenylarsine oxide suggesting that the predicted redox-sensitive vicinal dithiols exist within the native complex.
Highlights
One of the best characterized dyneins is the outer arm from flagella of Chlamydomonas
This complex consists of three dynein heavy chains which contain the ATPase and microtubule motor domains, two intermediate chains of 76.5 and 63 kDa (IC78 and IC69,2 respectively) and 8 different light chains (DLCs) of Mr 8,000 – 22,000, several of which are present in multiple copies (reviewed in Witman et al (1994))
We demonstrate that the Mr 14,000 and 16,000 DLCs, which are associated with ICs and with the ␣ DHC, respectively, are novel members of the thioredoxin superfamily containing perfect copies of the thioredoxin active site
Summary
One of the best characterized dyneins is the outer arm from flagella of Chlamydomonas. The remaining DLCs apparently interact with the two ICs in a distinct subcomplex located at the base of the soluble dynein particle (Mitchell and Rosenbaum, 1986; Witman et al, 1991). The functional significance of the various DLCs associated with the ICs is much less clear They may be involved in interactions with other axonemal components located on or near the doublet microtubules or, as dynein arms overlap in situ, they may interact directly with the globular head domains of the adjacent dynein particle. Affinity chromatography on phenylarsine oxide indicates that both molecules are functional as thioredoxins and we demonstrate that outer arm (but not inner arm) dynein can be purified on the basis of redox-sensitive vicinal dithiols These are the first redox-sensitive proteins to be found associated with a microtubule-based molecular motor
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