Abstract
This study examines the steady state activity and in vitro motility of single-headed (S1) and double-headed (HMM) myosin VI constructs within the context of two putative modes of regulation. Phosphorylation of threonine 406 does not alter either the rate of actin filament sliding or the maximal actin-activated ATPase rate of S1 or HMM constructs. Thus, we do not observe any regulation of myosin VI by phosphorylation within the motor domain. Interestingly, in the absence of calcium, the myosin VI HMM construct moves in an in vitro motility assay at a velocity that is twice that of S1 constructs, which may be indicative of movement that is not based on a "lever arm" mechanism. Increasing calcium above 10 microm slows both the rate of ADP release from S1 and HMM actomyosin VI and the rates of in vitro motility. Furthermore, high calcium concentrations appear to uncouple the two heads of myosin VI. Thus, phosphorylation and calcium are not on/off switches for myosin VI enzymatic activity, although calcium may alter the degree of processive movement for myosin VI-mediated cargo transport. Lastly, calmodulin mutants reveal that the calcium effect is dependent on calcium binding to the N-terminal lobe of calmodulin.
Highlights
Myosin VI was the first myosin demonstrated to move toward the pointed (Ϫ) end of an actin filament [1]
In the absence of calcium, the myosin VI HMM construct moves in an in vitro motility assay at a velocity that is twice that of S1-like myosin VI constructs (S1) constructs, which may be indicative of movement that is not based on a “lever arm” mechanism
Under our culture conditions, the S1 and HMM myosin VI constructs expressed in SF9 cells are consistently isolated in a highly phosphorylated state that can be subsequently dephosphorylated by -phosphatase
Summary
To resolve the apparent contradictions pertaining to the mechanism and regulation of movement of myosin VI on actin filaments, we examined the in vitro motility and solution kinetics of both single- and double-headed myosin VI with different levels of phosphorylation and at different calcium concen-. We examined the site of calcium action using calmodulin mutants with either N- or C-terminal calcium binding eliminated
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