Myo1c binds tightly and specifically to phosphatidylinositol 4,5-bisphosphate and inositol 1,4,5-trisphosphate

David E. Hokanson and
E. Michael Ostap*

Pennsylvania Muscle Institute and Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6085

Edited by Edward D. Korn, National Institutes of Health, Bethesda, MD, and approved January 3, 2006 (received for review July 6, 2005)

Abstract

Myosin-I is the single-headed member of the myosin superfamily that associates with acidic phospholipids through its basic tail domain. Membrane association is essential for proper myosin-I localization and function. However, little is known about the physiological relevance of the direct association of myosin-I with phospholipids or about phospholipid headgroup-binding specificity. To better understand the mechanism of myosin-I–membrane association, we measured effective dissociation constants for the binding of a recombinant myo1c tail construct (which includes three IQ domains and bound calmodulins) to large unilamellar vesicles (LUVs) composed of phosphatidylcholine and various concentrations of phosphatidylserine (PS) or phosphatidylinositol 4,5-bisphosphate (PIP₂). We found that the myo1c-tail binds tightly to LUVs containing >60% PS but very weakly to LUVs containing physiological PS concentrations (<40%). The myo1c tail and not the IQ motifs bind tightly to LUVs containing 2% PIP₂. Additionally, we found that the myo1c tail binds to soluble inositol-1,4,5-trisphosphate with nearly the same affinity as to PIP₂ in LUVs, suggesting that myo1c binds specifically to the headgroup of PIP₂. We also show that a GFP-myosin-I-tail chimera expressed in epithelial cells is transiently localized to regions known to be enriched in PIP₂. Our results suggest that myo1c does not bind to physiological concentrations of PS but rather binds tightly to PIP₂.

Footnotes

*To whom correspondence should be addressed. E-mail: ostap{at}mail.med.upenn.edu
Author contributions: D.E.H. and E.M.O. designed research, performed research, contributed new reagents/analytic tools, analyzed data, and wrote the paper.
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations:

Abbreviations:

InsP3,

inositol 1,4,5-trisphosphate;

LUV,

large unilamellar vesicle;

PIP2,

phosphatidylinositol 4,5-bisphosphate;

PH,

pleckstrin homology;

PLCδ-PH,

PH domain of phospholipase-Cδ;

PC,

phosphatidylcholine;

PS,

phosphatidylserine.