MEMBRANE TOPOLOGY OF RECOMBINANT RAT-LIVER MICROSOMAL GLUTATHIONE TRANSFERASE EXPRESSED IN ESCHERICHIA-COLI

Rat liver microsomal glutathione transferase is a mammalian membrane p rotein that can be successfully expressed in Escherichia coli in an en zymatically active form. The protein does not form inclusion bodies an d is recovered in the membrane fraction. The membrane topology of reco mbinant rat liver microsomal glutathione transferase expressed in E. c oli was investigated by comparing the proteolytic cleavage products fr om intact and permeabilized spheroplasts. It was shown that lysine-4 o f microsomal glutathione transferase is directed towards the outside, whereas lysine-41 faces the inside of the E. coli inner membrane. This shows that microsomal glutathione transferase has an inside-out orien tation in E. coli spheroplasts as compared to liver microsomes. This f act enables us to make topology experiments that were previously not p ossible. Intact spheroplasts treated with pronase yielded a cleavage p attern consistent with two additional exposed segments closer to the C -terminus. Thus a polytopic model is suggested for the membrane associ ation of microsomal glutathione transferase. (C) 1996 Academic Press, Inc.