Kinetics of the Interaction of myo1c with Phosphoinositides

myo1c is a single-headed myosin that dynamically links membranes to the actin cytoskeleton. A putative pleckstrin homology domain has been identified in the myo1c tail that binds phosphoinositides and soluble inositol phosphates with high affinity. However, the kinetics of association and dissociation and the influence of phospholipid composition on the kinetics have not been determined. Stopped-flow spectroscopy was used to measure the binding and dissociation of a recombinant myo1c construct containing the tail and regulatory domains (myo1c^IQ-tail) to and from 100-nm diameter large unilamellar vesicles (LUVs). We found the time course of association of myo1c^IQ-tail with LUVs containing 2% phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) followed a two-exponential time course, and the rate of the predominant fast phase depended linearly upon the total lipid concentration. The apparent second-order rate constant was approximately diffusion-limited. Increasing the molar ratio of anionic phospholipid by adding phosphatidylserine, additional PtdIns(4,5)P₂, or by situating PtdIns(4,5)P₂ in a more physiologically relevant lipid background increased the apparent association rate constant less than 2-fold. myo1c^IQ-tail dissociated from PtdIns(4,5)P₂ at a slower rate (2.0 s⁻¹) than the pleckstrin homology domain of phospholipase C-δ (13 s⁻¹). The presence of additional anionic phospholipid reduced the myo1c^IQ-tail dissociation rate constant >50-fold but marginally changed the dissociation rate of phospholipase C-δ, suggesting that additional electrostatic interactions in myo1c^IQ-tail help to stabilize binding. Remarkably, high concentrations of soluble inositol phosphates induce dissociation of myo1c^IQ-tail from LUVs, suggesting that phosphoinositides are able to bind to and dissociate from myo1c^IQ-tail as it remains bound to the membrane.Myosin-I isoforms are low molecular weight members of the myosin superfamily that link cell membranes with the actin cytoskeleton and play crucial roles driving a diverse array of dynamic membrane processes (1–5). Cell biological studies have shown that myosin-I isoforms localize and fractionate with cell membranes (2, 6), and biochemical experiments have shown myosin-I isoforms bind directly to lipid membranes (7–10). Thus, a key property of some myosin-I isoforms is their ability to bind membranes.myo1c is a widely expressed vertebrate myosin-I isoform that has roles in a variety of important membrane events, including insulin-stimulated fusion of vesicles containing glucose transporter-4 with the plasma membrane (2, 11), compensatory endocytosis following regulated exocytosis (12), and tensioning of mechano-sensitive ion channels (3). The mechanisms of myo1c targeting and anchoring to specific regions on the membrane to support these functions are not well understood. However, evidence is building that myo1c targeting requires direct binding of myo1c to phosphoinositides in cell membranes (13–16).We have shown that binding of myo1c to membranes is mediated by a putative pleckstrin homology (PH)³ domain in its tail that binds phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) and other phosphoinositides with high affinity (K_d <0.5 μm in terms of accessible phosphoinositide concentration) (13). myo1c also binds soluble inositol phosphates (e.g. inositol 1,4,5-trisphosphate (InsP₃)) with similar affinity. Point mutations of amino acids known to be essential for phosphoinositide binding in other PH domains inhibit myo1c binding to PtdIns(4,5)P₂ in vitro, and these mutations disrupt membrane localization in vivo (13). The affinity of myo1c for PtdIns(4,5)P₂-containing membranes is increased by the presence of additional anionic phospholipids in the membrane. This increased affinity may be due to nonspecific electrostatic interactions between the anionic phospholipids and positively charged regions within the myo1c tail or regulatory domain (13, 17), which is similar to what has been found for the guanine nucleotide exchange factor, ARNO (18). However, high affinity membrane binding via these nonspecific electrostatic interactions (i.e. binding in the absence of PtdIns(4,5)P₂) requires the membrane composition to contain a nonphysiological mole fraction (e.g. >40% phosphatidylserine) of anionic phospholipids (13, 14).Because phosphoinositide binding is important for the cellular localization and function of myo1c (13), it is important to determine the physical constants that define this interaction. Determining the kinetics of membrane attachment will provide insight into the relationship between membrane attachment and actin attachment lifetimes and will also provide details about the role of anionic lipids in regulating membrane attachment. Therefore, we used stopped-flow kinetics to measure the in vitro association and dissociation kinetics of myo1c from LUVs as a function of phosphoinositide composition and anionic charge.