Specificity of membrane binding of the neuronal protein NAP-22.
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 aminoterminal 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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Department of Biochemistry, McMaster University, Hamilton, ON, Canada L8N 3Z5.
Maekawa
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D000818:Animals; D001665:Binding Sites; D001923:Brain Chemistry; D002148:Calmodulin-Binding Proteins / Q000737:chemistry*; D002784:Cholesterol / Q000737:chemistry*; D003598:Cytoskeletal Proteins; D008560:Membrane Fluidity; D008561:Membrane Fusion; D008563:Membrane Lipids / Q000737:chemistry*; D021962:Membrane Microdomains / Q000737:chemistry*; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D009474:Neurons / Q000737:chemistry*; D010713:Phosphatidylcholines / Q000737:chemistry*; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D012680:Sensitivity and Specificity; D013329:Structure-Activity Relationship; D013696:Temperature
D002148:Calmodulin-Binding Proteins; D003598:Cytoskeletal Proteins; D008563:Membrane Lipids; D009419:Nerve Tissue Proteins; D010713:Phosphatidylcholines; D011994:Recombinant Proteins; C066853:Basp1 protein, rat; D002784:Cholesterol; C017251:1,2-oleoylphosphatidylcholine