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Specificity of Membrane Binding of the Neuronal Protein NAP-22

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Abstract

NAP-22, a major protein of neuronal rafts is known to preferentially bind to membranes containing cholesterol. In this work we establish the requirements for membrane binding of NAP-22. We find that other sterols can replace cholesterol to promote binding. In addition, bilayers containing phosphatidylethanolamine bind NAP-22 in the absence of cholesterol. Thus, there is not a specific interaction of NAP-22 with cholesterol that determines its binding to membranes. Addition of a mol fraction of phosphatidylserine of 0.05 to membranes of phosphatidylcholine and cholesterol enhances the membrane binding of NAP-22. The dependence of binding on the mol fraction of phosphatidylserine indicates that NAP-22 binds to membranes with its amino-terminal segment closer to the membrane than the remainder of the protein. We have also determined which segments of NAP-22 are required for membrane binding. A non-myristoylated form binds only weakly to membranes. Truncating the protein from 226 amino acids to the myristoylated amino-terminal 60 amino acids does not prevent binding to membranes in a cholesterol-dependent manner, but this binding is of weaker affinity. However, myristoylation is not sufficient to promote binding to cholesterol-rich domains. An N-terminal 19-amino-acid, myristoylated peptide binds to membranes but without requiring specific lipids. Thus, the remainder of the protein contributes to the lipid specificity of the membrane binding of NAP-22.

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Acknowledgements

This work was supported by grant MT-7654 from the Canadian Institutes of Health Research and a Grant-in-Aid for Scientific Research on Priority Areas (14014227) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Richard M. Epand is a Senior Investigator of the Canadian Institutes of Health Research.

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Authors and Affiliations

  1. Department of Biochemistry, McMaster University, Hamilton, ON L8N 3Z5, Canada

    R. F. Epand & R. M. Epand

  2. Department of Bioinformation, Department of Life Science, Graduate School of Science and Technology, Kobe-University, Rokkodai, Nada-ku, Kobe 657-8501, Japan

    S. Maekawa

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  1. R. F. Epand
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  2. S. Maekawa
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Correspondence to R. M. Epand.

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Epand, R., Maekawa, S. & Epand, R. Specificity of Membrane Binding of the Neuronal Protein NAP-22 . J. Membrane Biol. 193, 171–176 (2003). https://doi.org/10.1007/s00232-003-2015-y

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  • DOI: https://doi.org/10.1007/s00232-003-2015-y

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