Properties of the full-length heavy chains ofTetrahymena ciliary outer arm dynein separated by urea treatment

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Abstract
An important challenge is to understand the functional specialization of dynein heavy chains. The ciliary outer arm dynein from Tetrahymena thermophila is a heterotrimer of three heavy chains, called α, β and γ. In order to dissect the contributions of the individual heavy chains, we used controlled urea treatment to dissociate Tetrahymena outer arm dynein into a 19S β/γ dimer and a 14S α heavy chain. The three heavy chains remained full‐length and retained MgATPase activity. The β/γ dimer bound microtubules in an ATP‐sensitive fashion. The isolated α heavy chain also bound microtubules, but this binding was not reversed by ATP. The 19S β/γ dimer and the 14S α heavy chain could be reconstituted into 22S dynein. The intact 22S dynein, the 19S β/γ dimer, and the reconstituted dynein all produced microtubule gliding motility. In contrast, the separated α heavy chain did not produce movement under a variety of conditions. The intact 22S dynein produced movement that was discontinuous and slower than the movement produced by the 19S dimer. We conclude that the three heavy chains of Tetrahymena outer arm dynein are functionally specialized. The α heavy chain may be responsible for the structural binding of dynein to the outer doublet A‐tubule and/or the positioning of the β/γ motor domains near the surface of the microtubule track. Cell Motil. Cytoskeleton 58:30–38, 2004.