This Article Full Text Full Text (PDF) All Versions of this Article: biophysj.107.110213v1 94/3/1021    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Blin, G. Articles by Picart, C. Search for Related Content PubMed PubMed Citation Articles by Blin, G. Articles by Picart, C.

Quantitative Analysis of the Binding of Ezrin to Large Unilamellar Vesicles Containing Phosphatidylinositol 4,5 Bisphosphate

Guillaume Blin ^*, Emmanuel Margeat , Kévin Carvalho ^*, Catherine A. Royer , Christian Roy ^* and Catherine Picart ^*

^* DIMNP, Universités Montpellier II et I, CNRS, Montpellier cedex 05, France; and Institut National de la Sante et de la Recherche Medicale Unité 554, and Université Montpellier, Centre National de Recherche Scientifique, UMR 5048, Centre de Biochimie Structurale, Montpellier, France

Correspondence: Address reprint requests to Catherine Picart, E-mail: catherine.picart{at}univ-montp2.fr.

The plasma membrane-cytoskeleton interface is a dynamic structure participating in a variety of cellular events. Among the proteins involved in the direct linkage between the cytoskeleton and the plasma membrane is the ezrin/radixin/moesin (ERM) family. The FERM (4.1 ezrin/radixin/moesin) domain in their N-terminus contains a phosphatidylinositol 4,5 bisphosphate (PIP₂) (membrane) binding site whereas their C-terminus binds actin. In this work, our aim was to quantify the interaction of ezrin with large unilamellar vesicles (LUVs) containing PIP₂. For this purpose, we produced human recombinant ezrin bearing a cysteine residue at its C-terminus for subsequent labeling with Alexa488 maleimide. The functionality of labeled ezrin was checked by comparison with that of wild-type ezrin. The affinity constant between ezrin and LUVs was determined by cosedimentation assays and fluorescence correlation spectroscopy. The affinity was found to be 5 µM for PIP₂-LUVs and 20- to 70-fold lower for phosphatidylserine-LUVs. These results demonstrate, as well, that the interaction between ezrin and PIP₂-LUVs is not cooperative. Finally, we found that ezrin FERM domain (area of 30 nm²) binding to a single PIP₂ can block access to neighboring PIP₂ molecules and thus contributes to lower the accessible PIP₂ concentration. In addition, no evidence exists for a clustering of PIP₂ induced by ezrin addition.