The motile beta/IC1 subunit of sea urchin sperm outer arm dynein does not form a rigor bond.
We used in vitro translocation and cosedimentation assays to study the microtubule binding properties of sea urchin sperm outer arm dynein and its beta/IC1 subunit. Microtubules glided on glass-absorbed sea urchin dynein for a period of time directly proportional to the initial MgATP2- concentration and then detached when 70-95% of the MgATP2- was hydrolyzed. Detachment resulted from MgATP2- depletion, because (a) perfusion with fresh buffer containing MgATP2- reconstituted binding and gliding, (b) microtubules glided many minutes with an ATP-regenerating system at ATP concentrations which alone supported gliding for only 1-2 min, and (c) microtubules detached upon total hydrolysis of ATP by an ATP-removal system. The products of ATP hydrolysis antagonized binding and gliding; as little as a threefold excess of ADP/Pi over ATP resulted in complete loss of microtubule binding and translocation by the beta/IC1 subunit. In contrast to the situation with sea urchin dynein, microtubules ceased gliding but remained bound to glass-absorbed Tetrahymena outer arm dynein when MgATP2- was exhausted. Cosedimentation assays showed that Tetrahymena outer arm dynein sedimented with microtubules in an ATP-sensitive manner, as previously reported (Porter, M.E., and K. A. Johnson. J. Biol. Chem. 258: 6575-6581). However, the beta/IC1 subunit of sea urchin dynein did not cosediment with microtubules in the absence of ATP. Thus, this subunit, while capable of generating motility, lacks both structural and rigor-type microtubule binding.
Moss
A G
AG
Cell Biology Group and Male Fertility Program, Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545.
Gatti
J L
JL
Witman
G B
GB
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D002499:Centrifugation, Density Gradient; D004398:Dyneins / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D008297:Male; D008870:Microtubules / Q000378:metabolism*; D009994:Osmolar Concentration; D012617:Sea Urchins; D013082:Sperm Tail / Q000201:enzymology*; D013768:Tetrahymena / Q000201:enzymology
D000255:Adenosine Triphosphate; D004398:Dyneins