Abstract
The G146V mutation in actin is dominant lethal in yeast. G146V actin filaments bind cofilin only minimally, presumably because cofilin binding requires the large and small actin domains to twist with respect to one another around the hinge region containing Gly-146, and the mutation inhibits that twisting motion. A number of studies have suggested that force generation by myosin also requires actin filaments to undergo conformational changes. This prompted us to examine the effects of the G146V mutation on myosin motility. When compared with wild-type actin filaments, G146V filaments showed a 78% slower gliding velocity and a 70% smaller stall force on surfaces coated with skeletal heavy meromyosin. In contrast, the G146V mutation had no effect on either gliding velocity or stall force on myosin V surfaces. Kinetic analyses of actin-myosin binding and ATPase activity indicated that the weaker affinity of actin filaments for myosin heads carrying ADP, as well as reduced actin-activated ATPase activity, are the cause of the diminished motility seen with skeletal myosin. Interestingly, the G146V mutation disrupted cooperative binding of myosin II heads to actin filaments. These data suggest that myosin-induced conformational changes in the actin filaments, presumably around the hinge region, are involved in mediating the motility of skeletal myosin but not myosin V and that the specific structural requirements for the actin subunits, and thus the mechanism of motility, differ among myosin classes.
Highlights
The roles of conformational changes of actin in myosin motility are unclear
We previously reported that the G146V mutation in actin inhibits the binding of cofilin, presumably by perturbing the twisting motion between the large and small domains, as Gly146 is situated within the hinge region between the two domains, and cofilin prefers to bind to the twisted conformation [14]
We found that the gliding velocity and stall force of skeletal heavy meromyosin (sk HMM) were significantly reduced by the G146V mutation, presumably reflecting the weaker affinity of the mutant filaments for the myosin heads carrying ADP
Summary
The roles of conformational changes of actin in myosin motility are unclear. Results: A G146V mutation in actin, which perturbed its conformation, impaired force generation by myosin II, but not by myosin V. A number of studies have suggested that force generation by myosin requires actin filaments to undergo conformational changes This prompted us to examine the effects of the G146V mutation on myosin motility. Interaction between G146V Mutant Actin Filaments and Myosin ability to move on surfaces of skeletal heavy meromyosin (sk HMM), they bind to HMM and stimulate its ATPase activity This suggests that the flexibility of the actin filaments and their capacity for conformational changes are important determinants of myosin motility. Neither the gliding velocity nor the stall force for myosin V was detectably affected by the mutation These results suggest that the importance of the actin subunit conformation for motility is myosin class-specific
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