Myosin-V (MyoV) is a ubiquitous motor protein that transports an astonishingly diverse set of cargos on the actin network in eukaryotes. Phosphorylation-dependent processes often regulate MyoV-mediated cargo transport, molecular details of which remain largely unknown. We previously showed that phosphorylation regulates MyoV’s switching from microtubules onto actin filaments, not its motor activity. Regulation of switching at reconstituted microtubule-actin-crossings in fact sufficed to recapitulate the MyoVa-driven redistribution of pigment-organelles in amphibian melanophores. However, in those cells, MyoVa also encounter many actin-actin crossings. Here, we show that isolated MyoVa motors switch with equal... More
Myosin-V (MyoV) is a ubiquitous motor protein that transports an astonishingly diverse set of cargos on the actin network in eukaryotes. Phosphorylation-dependent processes often regulate MyoV-mediated cargo transport, molecular details of which remain largely unknown. We previously showed that phosphorylation regulates MyoV’s switching from microtubules onto actin filaments, not its motor activity. Regulation of switching at reconstituted microtubule-actin-crossings in fact sufficed to recapitulate the MyoVa-driven redistribution of pigment-organelles in amphibian melanophores. However, in those cells, MyoVa also encounter many actin-actin crossings. Here, we show that isolated MyoVa motors switch with equal probabilities at reconstituted actin-actin-crossings. Under the control of its adaptor-protein melanophilin (Mlph), however, the motor differentiates between the actin filaments at crossing points in a phosphorylation-regulated manner. Whereas phosphorylation of Mlph forced about ∼2/3 of MyoVa to ignore the intersections, dephosphorylation completely reversed this behavior and forced ∼2/3 to switch. We show that the filament-binding domain (FBD) of Mlph controls this switching behavior. This property evolved in amphibians, but not in the early vertebrate zebrafish. By protein engineering, we demonstrate that changes of a few residues are sufficient to impart actin-binding capability onto the zebrafish Mlph. We thus unmask the molecular beginnings of dual filament binding in Mlph that allow it to control the switching behavior of MyoVa at cytoskeletal crossings. We therefore propose a direct link between intracellular phosphorylation activity and the adaptor-protein, not to regulate MyoVa activity, but to navigate the motor through the entire cytoskeletal maze for correct positioning of cargo.
