REQUIREMENTS FOR TRANSLOCATION OF PERIPLASMIC DOMAINS IN POLYTOPIC MEMBRANE-PROTEINS

Periplasmic domains of cytoplasmic membrane proteins require export si gnals for proper translocation. These signals were studied by using a MalF-alkaline phosphatase fusion in a genetic selection that allowed t he isolation of mislocalization mutants. In the original construct, al kaline phosphatase is fused to the second periplasmic domain of the me mbrane protein, and its activity is thus confined exclusively to the p eriplasm. Mutants that no longer translocated alkaline phosphatase wer e selected by complementation of a serB mutation. A total of 11 deleti ons in the amino terminus were isolated, all of which spanned at least the third transmembrane segment. This domain immediately precedes the periplasmic domain to which alkaline phosphatase was fused. Our resul ts obtained in vivo support the model that amino-terminal membrane-spa nning segments are required for translocation of large periplasmic dom ains. In addition, we found that the inability to export the alkaline phosphatase domain could be suppressed by a mutation, prlA4, in the se cretion apparatus.