PROPERTIES OF PERMEASE DIMER, A FUSION PROTEIN CONTAINING 2 LACTOSE PERMEASE MOLECULES FROM ESCHERICHIA-COLI

An engineered fusion protein containing two tandem lactose permease mo lecules (permease dimer) exhibits high transport activity and is used to test the phenomenon of negative dominance. Introduction of the muta tion Glu-325 --> Cys into either the first or the second half of the d imer results in a 50% decrease in activity, whereas introduction of th e mutation into both halves of the dimer abolishes transport. Lactose transport by permease dimer is completely inactivated by N-ethylmalein ide; however, 40-45% activity is retained after N-ethylmaleimide treat ment when either the first or the second half of the dimer is replaced with a mutant devoid of cysteine residues. The observations demonstra te that both halves of the fusion protein are equally active and sugge st that each half may function independently. To test the possibility that oligomerization between dimers might account for the findings, a permease diner was constructed that contains two different deletion mu tants that complement functionally when expressed as untethered molecu les. Because this construct does not catalyze lactose transport to any extent whatsoever, it is unlikely that the two halves of the dimer in teract or that there is an oligomeric interaction between dimers. The approach is consistent with the contention that the functional unit of lactose permease is a monomer.