Modulation of cytoplasmic dynein ATPase activity by the accessory subunits.
The microtubule-based motor molecule cytoplasmic dynein has been proposed to be regulated by a variety of mechanisms, including phosphorylation and specific interaction with the organelle-associated complex, dynactin. In this study, we examined whether the intermediate chain subunits of cytoplasmic dynein are involved in modulation of ATP hydrolysis, and thereby affect motility. Treatment of testis cytoplasmic dynein under hypertonic salt conditions resulted in separation of the intermediate chains from the remainder of the dynein molecule, and led to a 4-fold enhancement of ATP hydrolysis. This result suggests that the accessory subunits act as negative regulators of dynein heavy chain activity. Comparison of ATPase activities of dyneins with differing intermediate chain isoforms showed significant differences in basal ATP hydrolysis rates, with testis dynein 7-fold more active than dynein from brain. Removal of the intermediate chain subunits led to an equalization of ATPase activity between brain and testis dyneins, suggesting that the accessory subunits are responsible for the observed differences in tissue activity. Finally, our preparative procedures have allowed for the identification and purification of a 1:1 complex of dynein with dynactin. As this interaction is presumed to be mediated by the dynein intermediate chain subunits, we now have defined experimental conditions for further exploration of dynein enzymatic and motility regulation.
Kini
A R
AR
Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois, USA.
Collins
C A
CA
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D001923:Brain Chemistry; D000072159:Dynactin Complex; D004398:Dyneins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D004789:Enzyme Activation; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D008297:Male; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000378:metabolism; D011193:Potassium Iodide / Q000494:pharmacology; D021122:Protein Subunits; D051381:Rats; D017207:Rats, Sprague-Dawley; D013737:Testis / Q000737:chemistry / Q000378:metabolism
D000072159:Dynactin Complex; D007527:Isoenzymes; D008869:Microtubule-Associated Proteins; D021122:Protein Subunits; D011193:Potassium Iodide; D000255:Adenosine Triphosphate; D004398:Dyneins