Kinetics Of Myo1c Association To And Dissociation From Phosphoinositide-containing Vesicles

Abstract

Myo1c is a single-headed unconventional myosin that associates with negatively-charged lipids through electrostatic interactions. A putative pleckstrin homology (PH) domain has been identified in the myo1c tail that binds PI(4,5)P2 with high affinity. However, the kinetics of association and dissociation, as well as the influence of membrane phospholipid composition and Mg2+ on the kinetics, remain unknown. Stopped-flow measurements were made using the increase in light scattering that occurred upon myo1c-tail binding to 100 nm diameter large unilamellar vesicles (LUVs).We found that the association of myo1c-tail with phosphatidylcholine (PC) LUVs containing 2% PI(4,5)P2 followed a 2-exponential time-course. The rate of the predominant, fast phase depended linearly upon the total lipid concentration. The apparent second order rate constant in the absence of Mg2+ was approximately diffusion-limited, indicating that no conformational change occurs upon binding. The molar ratio of anionic lipid was increased by adding phosphatidylserine (PS) or additional PI(4,5)P2 to LUVs or by situating PI(4,5)P2 in a more physiologically relevant lipid background (phosphatidylethanolamine, PC, PS, phosphatidylinositol, and sphingomyelin). None of these conditions increased the apparent association rate constant much more than two-fold. Dissociation of myo1c-tail was measured by chasing with excess inositol 1,4,5-trisphosphate (InsP3). The presence of additional anionic phospholipid reduced the observed dissociation rate constant by orders of magnitude (3.2 s-1 vs. 0.03 s-1). This suggests that once myo1c-tail interacts via its putative PH domain with PI(4,5)P2, additional electrostatic interactions between positively-charged regions of the tail and negatively-charged lipids help to stabilize binding. The presence of Mg2+, known to interact with polyvalent anions, did not alter these trends, though it did increase the dissociation rate for all lipid compositions. Finally, we measured the dissociation rate of myo1c-tail from InsP3 and found that it approximates the dissociation rate from PI(4,5)P2.