Pre-Insertion Topology of Transmembrane Proteins is Highly Plastic and Can Be Controlled by a Single C-Terminal Residue

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The mechanism by which helical membrane proteins are inserted into the cellular membrane remains unclear. It is known that membrane proteins are inserted co-translationally into the lipid bilayer and that positively charged residues in the loop regions of TM proteins are important topological determinants. However, it is unclear whether those charges act strictly locally-- affecting only the nearest transmembrane helices-- or act globally-- affecting the topology of the entire protein. We have found that the topology of an Escherichia coli inner membrane protein with four or five transmembrane helices can be controlled by a single positively charged residue placed in different locations throughout the protein, including the very C-terminus. Addition of a charged, cleavable signal peptide to the N-terminus of the protein indicates that topology does not change after insertion into the membrane. These observation point to an unanticipated plasticity in pre-insertion membrane protein topology.