The α subunit of sea urchin sperm outer arm dynein mediates structural and rigor binding to microtubules

Abstract

Glass-adsorbed intact sea urchin outer arm dynein and its β/IC1 subunit supports movement of microtubules, yet does not form a rigor complex upon depletion of ATP (16). We show here that rigor is a feature of the isolated intact outer arm, and that this property subfractionates with its α heavy chain. Intact dynein mediates the formation of ATP-sensitive microtubule bundles, as does the purified α heavy chain, indicating that both particles are capable of binding to microtubules in an ATP-sensitive manner. In contrast, the β/IC1 subunit does not bundle microtubules. Bundles formed with intact dynein are composed of ribbon-like sheets of parallel microtubules that are separated by 54 nm (center-to-center) and display the same longitudinal repeat (24 nm) and cross-sectional geometry of dynein arms as do outer doublets in situ. Bundles formed by the α heavy chain are composed of microtubules with a center-to-center spacing of 43 nm and display infrequent, fine cross-bridges. In contrast to the bridges formed by the intact arm, the links formed by the α subunit are irregularly spaced, suggesting that binding of the α heavy chain to the microtubules is not cooperative. Cosedimentation studies showed that: (a) some of the intact dynein binds in an ATP-dependent manner and some binds in an ATP-independent manner; (b) the β/IC1 subunit does not cosediment with microtubules under any conditions; and (c) the α heavy chain cosediments with microtubules in the absence or presence of MgATP2-. These results suggest that the structural binding observed in the intact arm also is a property of its α heavy chain. We conclude that whereas forcegeneration is a function of the β/IC1 subunit, both structural and ATP-sensitive (rigor) binding of the arm to the microtubule are mediated by the α subunit.