title: Synthetic competition between cytoplasmic folding and translocation of a soluble membrane protein domain.
abstract: In wild-type strains of Escherichia coli, alkaline phosphatase (AP), either when present as a soluble protein or when fused to a membrane protein, is only active after translocation to the periplasm. In thioredoxin reductase (trxB) mutants, however, cytoplasmically localized AP can form disulphide bonds and can reach an active conformation. Once it has folded in the cytoplasm, it can no longer be translocated. On the other hand, when AP is fused to periplasmic domains of a membrane protein, translocation can be more rapid than folding. Thus, expressing hybrids of AP and integral membrane proteins in a trxB mutant generates competition between folding of AP in the cytoplasm and its translocation to the periplasm. The cellular localization of AP can be monitored in phosphoserine phosphatase (serB) mutants causing auxotrophy for L-serine. Cytoplasmically but not periplasmically localized AP can compensate for the lack of SerB, leading to growth on indicator plates. As expected, when AP was fused to cytoplasmic domains of membrane proteins, serB-mediated auxotrophy was abolished. Surprisingly, AP fusions to periplasmic domains exhibited a non-uniform response pattern. Fusions that translocate AP rapidly did not complement the SerB defect, while those that export AP only slowly could do so. The usefulness of these strains for studying a variety of aspects related to membrane protein biogenesis is discussed.
authors.affiliation: Fakultät für Biologie, Universität Konstanz, Germany.
authors.forename: K
authors.initials: K
authors.lastname: Uhland
authors.forename: T
authors.initials: T
authors.lastname: Zander
authors.forename: M
authors.initials: M
authors.lastname: Ehrmann
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D003593:Cytoplasm / Q000737:chemistry*; D004789:Enzyme Activation / Q000502:physiology*; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology*; D066298:In Vitro Techniques; D009154:Mutation
chemical_list: D001425:Bacterial Outer Membrane Proteins; D000469:Alkaline Phosphatase