Myo1e binds anionic phospholipids with high affinity.
Myo1e is a single-headed motor protein that has been shown to play roles in clathrin-mediated endocytosis in HeLa cells and podocyte function in the kidney. The myo1e C-terminal tail domain includes a basic region that is required for localization to clathrin-coated vesicles and contains a putative pleckstrin-homology (PH) domain that has been shown to play a role in phospholipid binding in other myosin-I proteins. We used sedimentation assays, stopped-flow fluorescence, and fluorescence microscopy to determine the membrane binding affinities, kinetics, and in vivo localization of fluorescently labeled recombinant myo1e-tail constructs. We found that the myo1e tail binds tightly to large unilamellar vesicles (LUVs) containing physiological concentrations of the anionic phospholipids phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) or phosphatidylserine. The rate of myo1e attachment to LUVs nears the diffusion limit while the calculated rate of detachment from LUVs is slow (k(diss) ≤ 0.4 s(-1)). Mutation of conserved residues in the myo1e PH domain has little effect on lipid binding in vitro or membrane localization in vivo. Soluble inositol phosphate headgroups, such as inositol 1,4,5-trisphosphate, can compete with PtdIns(4,5)P(2) for binding, but the apparent affinity for the soluble inositol phosphate is substantially lower than that for PtdIns(4,5)P(2). These results suggest that myo1e binds lipids through nonspecific electrostatic interactions rather than a stereospecific protein-phosphoinositide interaction.
Feeser
Elizabeth A
EA
Pennsylvania Muscle Institute and Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
Ignacio
Cherry Mae G
CM
Krendel
Mira
M
Ostap
E Michael
EM
D001665:Binding Sites; D006801:Humans; D008081:Liposomes / Q000378:metabolism; D024461:Myosin Type I / Q000378:metabolism*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D011485:Protein Binding; D055672:Static Electricity
D008081:Liposomes; D018129:Phosphatidylinositol Phosphates; D010718:Phosphatidylserines; D010743:Phospholipids; C511011:MYO1E protein, human; D024461:Myosin Type I