M. Sahintoth et al., PROPERTIES OF PERMEASE DIMER, A FUSION PROTEIN CONTAINING 2 LACTOSE PERMEASE MOLECULES FROM ESCHERICHIA-COLI, Proceedings of the National Academy of Sciences of the United Statesof America, 91(12), 1994, pp. 5421-5425
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.