Acanthamoeba Myosin IC Colocalizes with Phosphatidylinositol 4,5-Bisphosphate at the Plasma Membrane Due to the High Concentration of Negative Charge

Hanna Brzeska,Kae-Jung Hwang,Edward D Korn

doi:10.1074/jbc.m804828200

Abstract

The tail of Acanthamoeba myosin IC (AMIC) has a basic region (BR), which contains a putative pleckstrin homology (PH) domain, followed by two Gly/Pro/Ala (GPA)-rich regions separated by a Src homology 3 (SH3) domain. Cryoelectron microscopy had shown that the tail is folded back on itself at the junction of BR and GPA1, and nuclear magnetic resonance spectroscopy indicated that the SH3 domain may interact with the putative PH domain. The BR binds to acidic phospholipids, and the GPA region binds to F-actin. We now show that the folded tail does not affect the affinity of AMIC for acidic phospholipids. AMIC binds phosphatidylinositol 4,5-bisphosphate (PIP2) with high affinity (approximately 1 microm), but binding is not stereospecific. When normalized to net negative charge, AMIC binds with equal affinity to phosphatidylserine (PS) and PIP2. This and other data show that the putative PH domain of AMIC is not a typical PIP2-specific PH domain. We have identified a 13-residue sequence of basic-hydrophobic-basic amino acids within the putative PH domain that may be a major determinant of binding of AMIC to acidic phospholipids. Despite the lack of stereospecificity, AMIC binds 10 times more strongly to vesicles containing 5% PIP2 plus 25% PS than to vesicles containing only 25% PS, suggesting that AMIC may be targeted to PIP2-enriched regions of the plasma membrane. In agreement with this, AMIC colocalizes with PIP2 at dynamic, protrusive regions of the plasma membrane. We discuss the possibility that AMIC binding to PIP2 may initiate the formation of a multiprotein complex at the plasma membrane.

Highlights

Class-I myosins, single-headed myosins with globular tails, are widely expressed in a variety of organisms, including yeast, amoebas, and humans
We initially asked if Acanthamoeba myosin IC (AMIC) binds PIP2 with higher affinity than it binds other acidic phospholipids, if binding to phospholipids is affected by interactions between the basic region and Src homology 3 (SH3) domain of AMIC, and if the putative pleckstrin homology (PH) domain in the basic region is important for the binding of AMIC to acidic phospholipids
We first studied the binding of AMIC to acidic phospholipids in vitro

Summary

Introduction

Class-I myosins, single-headed myosins with globular tails, are widely expressed in a variety of organisms, including yeast, amoebas, and humans. The head domains of class-I myosins are highly homologous to other myosins, but their tail domains are unique. The head domains of all class-I myosins that have been tested bind F-actin, express actin-activated Mg-ATPase activity, and, with the neck domains, function as an actin-dependent motor. Their tail domains can be divided into two categories: short and long. NMR studies of expressed AMIC tail and tail subdomains, combined with homology modeling, showed the presence of a putative PH domain within the basic region [10]. NMR data and homology modeling indicate that the putative PH domain of AMIC has the required ␤-strands (Fig. 1) but not the ␣-helix. In contrast to related studies, we used full-length as well as truncated myosin

Methods

Results

Conclusion