MEMBRANE TOPOLOGY ANALYSIS OF ESCHERICHIA-COLI K-12 MTR PERMEASE BY ALKALINE-PHOSPHATASE AND BETA-GALACTOSIDASE FUSIONS

The mtr gene of Escherichia coli K-12 encodes an inner membrane protei n which is responsible for the active transport of tryptophan into the cell. It has been proposed that the Mtr permease has a novel structur e consisting of 11 hydrophobic transmembrane spans, with a cytoplasmic ally disposed amino terminus and a carboxyl terminus located in the pe riplasmic space (J. P. Sarsero, P. J. Wookey, P. Gollnick, C. Yanofsky , and A. J. Pittard, J, Bacteriol. 173:3231-3234, 1991). The validity of this model was examined by the construction of fusion proteins betw een the Mtr permease and alkaline phosphatase or beta-galactosidase. I n addition to the conventional methods, in which the reporter enzyme r eplaces a carboxyl-terminal portion of the membrane protein, the recen tly developed alkaline phosphatase sandwich fusion technique was utili zed, in which alkaline phosphatase is inserted into an otherwise intac t membrane protein. A cluster of alkaline phosphatase fusions to the c arboxyl-terminal end of the Mtr permease exhibited high levels of alka line phosphatase activity, giving support to the proposition of a peri plasmically located carboxyl terminus. The majority of fusion proteins produced enzymatic activities which were in agreement with the positi ons of the fusion sites on the proposed topological model of the perme ase. The synthesis of a small cluster of hybrid proteins, whose enzyma tic activity did not agree with the location of their fusion sites wit hin putative transmembrane span VIII or the preceding periplasmic loop , was not detected by immunological techniques and did not necessitate modification of the proposed model in this region. Slight alterations may need to be made in the positioning of the carboxyl-terminal end o f transmembrane span X.