Membrane topology of recombinant rat liver microsomal glutathione transferase expressed in E. coli.
Rat liver microsomal glutathione transferase is a mammalian membrane protein that can be successfully expressed in Escherichia coli in an enzymatically active form. The protein does not form inclusion bodies and is recovered in the membrane fraction. The membrane topology of recombinant rat liver microsomal glutathione transferase expressed in E. coli was investigated by comparing the proteolytic cleavage products from intact and permeabilized spheroplasts. It was shown that lysine-4 of 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 orientation in E. coli spheroplasts as compared to liver microsomes. This fact enables us to make topology experiments that were previously not possible. Intact spheroplasts treated with pronase yielded a cleavage pattern consistent with two additional exposed segments closer to the C-terminus. Thus a polytopic model is suggested for the membrane association of microsomal glutathione transferase.
Raza
H
H
Division of Biochemical Toxicology, Karolinska Institutet, Stockholm, Sweden.
Weinander
R
R
Ekström
L
L
Morgenstern
R
R
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology*; D004926:Escherichia coli; D005982:Glutathione Transferase / Q000032:analysis* / Q000737:chemistry; D008862:Microsomes, Liver / Q000201:enzymology*; D011402:Pronase / Q000378:metabolism; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry; D013104:Spheroplasts / Q000201:enzymology*; D014357:Trypsin / Q000378:metabolism
D011994:Recombinant Proteins; D005982:Glutathione Transferase; D014357:Trypsin; D011402:Pronase