AN AMPHIPATHIC SEQUENCE DETERMINANT OF MEMBRANE-PROTEIN TOPOLOGY

We developed a screen involving alkaline phosphatase gene fusions to i dentify mutations altering the membrane topology of a bacterial chemor eceptor (Escherichia coli Tsr). We identified three informative classe s of mutations causing increased export of the protein's normally cyto plasmic carboxyl-terminal domain. The first class consisted of deletio ns eliminating all or most of the membrane-spanning sequence (TM2) imm ediately amino-terminal to the cytoplasmic domain. The second class co nsisted of mutations altering a highly amphipathic sequence at the beg inning of the domain. The third class of mutation was a deletion of an upstream spanning sequence (TM1). The amphipathic sequence appears to be a novel determinant of membrane topology whose function is not due to its positive residue density. The amphipathic character of the seq uence is relatively well-conserved in chemoreceptors and their relativ es. Although deletions removing the amphipathic sequence or TM1 alone caused only partial carboxyl-terminal domain export, a double mutation removing both caused efficient export. This result suggests that the two sequences function independently to promote normal membrane insert ion. The independent functioning of the two sequences may help ensure that Tsr insertion is normally a high fidelity process.