Cargo Activation of Full Length Myosin Va by Melanophilin Observed at the Single Molecule Level

Abstract

Full-length myosin Va (myoVa) is auto-inhibited via a motor domain-globular tail interaction, unlike the truncated constitutively active myoVa-HMM. One potential mechanism to activate the full-length motor is cargo binding to the tail, which would compete with the head-tail interaction and trigger the molecule to extend and be activated for transport.In the absence of cargo, it was recently shown that full-length myoVa has two modes of interaction with actin in the presence of MgATP (Armstrong et al.). Most motors bind to actin but do not move, while the remainder show processive motion, but with a variable stepping pattern and altered gating. Here we investigate how binding of melanophilin (Mlph), which links the melanocyte-specific isoform of myoVa to the Rab27a(GTP)-melanosome complex, affects the properties of myoVa at the single-molecule level.In the absence of Mlph at 150mM KCl, a subset of Quantum dot labeled full-length myoVa moved at a median velocity of 566nm/s with the variable stepping pattern previously described, suggesting altered gating under these conditions. Addition of Mlph recruited 7-times more motors to move processively, consistent with a simple model of cargo activation. The myoVa-Mlph complex also showed increased run lengths, with many traveling to the ends of the actin filament. In the presence of Mlph, myoVa moved much more slowly (median velocity=76nm/s), leading to longer travel times on actin. When Mlph was bound to the motor the step sizes were normally distributed around 60 ± 14nm (SD) steps. Therefore, while myoVa moves more slowly along actin in the presence of the cargo adapter protein Mlph, it covers a greater distance with a more uniform and efficient stepping pattern. This slower processive movement could potentially facilitate binding of the Rab27a(GTP)-melanosome complex.