Quantitative analysis of the binding of ezrin to large unilamellar vesicles containing phosphatidylinositol 4,5 bisphosphate

被引:48
作者
Blin, Guillaume [1 ,2 ]
Margeat, Emmanuel [3 ,4 ]
Carvalho, Kevin [1 ,2 ]
Royer, Catherine A. [3 ,4 ]
Roy, Christian [1 ,2 ]
Picart, Catherine [1 ,2 ]
机构
[1] Univ Montpellier 2, CNRS, DIMNP, Montpellier 05, France
[2] Univ Montpellier I, CNRS, DIMNP, Montpellier 05, France
[3] Inst Natl Sante & Rech Med, Unite 554, Montpellier, France
[4] Univ Montpellier, Ctr Natl Rech Sci, UMR 5048, Ctr Biochim Struct, F-34059 Montpellier, France
关键词
D O I
10.1529/biophysj.107.110213
中图分类号
Q6 [生物物理学];
学科分类号
071011 ;
摘要
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 similar to 5 mu M 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 similar to 30 nm(2)) 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.
引用
收藏
页码:1021 / 1033
页数:13
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