Lipids and Topological “rules” of Membrane Protein Assembly: Testing the Generality of Net Charge Balance Rule

Transmembrane domain (TMD) orientation within some membrane proteins is dependent on membrane lipid composition. When the lactose permease (LacY) is assembled in Escherichia coli membranes lacking the major phospholipid phosphatidylethanolamine (PE), the N-terminal TMD bundle is inverted with respect to the C-terminal TMD bundle and the plane of the membrane. This inversion is dependent on the interfacial net positive charge of the protein, the net negative charge of the membrane and a TMD of low hydrophobicity, acting as a molecular hinge between the two halves of the protein by exiting the membrane to the periplasm. Homologous E. coli sucrose permease (CscB) and non-homologous phenylalanine permease (PheP) were investigated to generalize these original observations.CscB function, like that of LacY, is dependent on the presence of PE but topological dependence on membrane lipid composition is less sensitive and only becomes evident after significant changes in the net positive charge of the cytoplasmic surface of the N-terminal bundle. The first cytoplasmic domains of PheP, which are misoriented in PE-lacking cells, have a net negative charge. Decreasing the negative charge density of the extramembrane domains flanking the N-terminal TMD hairpin of PheP favors a reorientation of the N-terminus and adjacent hairpin to their native orientation in the absence of PE as predicted by the above net charge balance rule.Polytopic membrane proteins containing competing opposite charges within their cytoplasmic domains may share a common mechanism for topogenesis dependent on PE. However the degree of sensitivity to phospholipid composition appears to be sequence-specific and might be a result of conformational flexibility, topological preference of individual domains or the availability of mechanical hinge region. Supported by NIH grant R37-GM20478.