The lactose transport protein is a cooperative dimer with two sugar translocation pathways

The Major Facilitator Superfamily lactose transport protein (LacS) undergoe s reversible self-association in the detergent-solubilized state, and is pr esent in the membrane as a dimer. We determined the functional unit for pro ton motive force (Deltap)-driven lactose uptake and lactose/methyl-beta -D- galactopyranoside equilibrium exchange in a proteoliposomal system in which a single cysteine mutant, LacS-C67, defective in Deltap-driven uptake, was co-reconstituted with fully functional cysteine-less protein, LacS-cl. Fro m the quadratic relationship between the uptake activity and the ratio of L acS-C67/LacS-cl, we conclude that the dimeric state of LacS is required for Deltap-driven uptake. N-ethylmaleimide (NEM) treatment of proteoliposomes abolished the LacS-C67 exchange activity but left the LacS-cl unaffected. A fter NEM treatment, the exchange activity decreased linearly with increasin g ratios of LacS-C67/LacS-cl, suggesting that the monomeric state of LacS i s sufficient for this mode of transport. We propose that the two subunits o f LacS are functionally coupled in the step associated with conformational reorientation of the empty binding site, a step unique for Deltap-driven up take.