The unique insert at the end of the myosin VI motor is the sole determinant of directionality.

Myosin VI moves toward the pointed (minus) end of actin filaments, the reverse direction of other myosin classes. The myosin VI structure demonstrates that a unique insert at the end of the motor repositions its lever arm and is at least in part responsible for the reversal of directionality. However, it has been proposed that there must be additional modifications within the motor that contribute to its large step size and to the reversal of directionality. To ascertain the inherent directionality of the motor core, we attached the myosin V lever arm to myosin VI, with and without the unique insert. If the insert was maintained, the motor moved toward the minus end of actin filaments, but if removed, movement was redirected toward the plus end. Single-molecule studies revealed that further adaptations within the motor increase the magnitude and variability of the plus-end directed converter movements, and unexpectedly provide the source of the highly variable myosin VI step size. Thus, the unique insert is necessary and sufficient to reverse an inherently plus-end directed myosin.