GENETIC ANALYSIS OF COMPLEX MEMBRANE PROTEIN INSERTION

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The goal is to provide a better understanding of how membrane proteins with complex structures are inserted into the membrane. The analysis focuses on a protein of the E. coli cytoplasmic membrane which is involved in maltose uptake (MalF protein), a protein whose chain appears to span the membrane eight times, generating multiple periplasmic and and cytoplasmic domains. The first experiments aim to develop general genetic methods to identify the transmembrane structures of membrane proteins. One approach attempts to identify periplasmic domains of cytoplasmic membrane proteins as regions where the periplasmic protein alkaline phosphatase can be fused to give high enzymatic activity. A second approach attempts to identify cytoplasmic domains of membrane proteins as regions where the cytoplasmic protein beta-galactosidase can be fused to give high activity hybrid proteins, or as regions where alkaline phosphatase can be fused to give low enzymatic activity. The second set of experiments aims to select mutations altering signals of MalF protein responsible for its membrane insertion. Mutations inactivating a putative internal signal sequence of MalF protein will be identified by their failure to export a short fusion of alkaline phosphatase to MalF protein. Mutations inactivating a putative stop transfer sequence of MalF protein will be isolated by their restoration of export to the alkaline phosphatase moiety of a hybrid in which the alkaline phosphatase appears inactive due to a cytoplasmic location. These mutants should help identify the export signals in the sequence of MalF protein, and also help to distinguish between different models for the insertion of this complex membrane protein.