REVERSIBLE TOPOLOGY OF A BIFUNCTIONAL TRANSMEMBRANE PROTEIN DEPENDS UPON THE CHARGE BALANCE AROUND ITS TRANSMEMBRANE DOMAIN

Hybrid genes were constructed to express bifunctional hybrid proteins in which staphyloccal nuclease A with or without an amino-terminal OmpA signal sequence was fused with TEM beta-lactamase (at the carboxyl terminal side) using the signal peptide of the major outer membrane lipoprotein of Escherichia call as an internal linker. The hybrid proteins were found to be inserted in the membrane. Orientation of the hybrid protein with the OmpA signal peptide showed that the nuclease was translocated into the periplasm and the beta-lactamase remained in the cytoplasm. This indicates that the cleavable OmpA signal peptide served as a secretory signal for nuclease and the internal lipoprotein signal served as the transmembrane anchor. In the absence of the OmpA signal sequence the topology of the hybrid protein was reversed indicating that the internal lipoprotein signal peptide initially served as the signal peptide for the secretion of the carboxy terminal beta-lactamase domain across the membrane and subsequently as a membrane anchoring signal. The role of charged amino acids in the translocation and transmembrane orientation of membrane proteins was also analysed by introducing charged amino acids to either or bath sides of the internal lipoprotein signal sequence in the bifunctional hybrid proteins In the absence of the amino-terminal signal sequence. Introduction of two lysine residues at the carboxy-terminal side of the internal signal sequence reversed the topology of the transmembrane protein by translocating the aminoterminal nuclease domain across the membrane, leaving the carboxyl terminal beta-lactamase domain in the cytoplasm. When three more lysine residues were added to the amino-terminal side of the internal signal sequence of the same construct the membrane topology flipped back to the original orientation. A similar reversion of the topology could be obtained by introducing negatively charged residues at the amino-terminal side of the internal signal sequence. Present results demonstrate for the first time that a bifunctional transmembrane protein can be engineered to assume either of the two opposite orientations and that charge balance around the transmembrane domain is a major factor in controlling the topology of a transmembrane protein.