Effects of mixed proximal and distal topogenic signals on the topological sensitivity of a membrane protein to the lipid environment.

The final topology of membrane proteins is thought to be dictated primarily by the encoding sequence. However, according to the Charge Balance Rule the topogenic signals within nascent membrane proteins are interpreted in agreement with the Positive Inside Rule as influenced by the protein phospholipid environment. The role of long-range protein-lipid interactions in establishing a final uniform or dual topology is unknown. In order to address this role, we determined the positional dependence of the potency of charged residues as topological signals within Escherichia coli sucrose permease (CscB) in cells in which the zwitterionic phospholipid phosphatidylethanolamine (PE), acting as topological determinant, was either eliminated or tightly titrated. Although the position of a single or paired oppositely charged amino acid residues within an extramembrane domain (EMD), either proximal, central or distal to a transmembrane domain (TMD) end, does not appear to be important, the oppositely charged residues exert their topogenic effects separately only in the absence of PE. Thus, the Charge Balance Rule can be executed in a retrograde manner from any cytoplasmic EMD or any residue within an EMD most likely outside of the translocon. Moreover, CscB is inserted into the membrane in two opposite orientations at different ratios with the native orientation proportional to the mol % of PE. The results demonstrate how the cooperative contribution of lipid-protein interactions affects the potency of charged residues as topological signals, providing a molecular mechanism for the realization of single, equal or different amounts of oppositely oriented protein within the same membrane.