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

Abstract

The Major Facilitator Superfamily lactose transport protein (LacS) undergoes reversible self-association in the detergent-solubilized state, and is present in the membrane as a dimer. We determined the functional unit for proton motive force (Dp)-driven lactose uptake and lactose/methyl-b-D-galactopyranoside equilibrium exchange in a proteoliposomal system in which a single cysteine mutant, LacS-C67, defective in Dp-driven uptake, was co-reconstituted with fully functional cysteine-less protein, LacS-cl. From the quadratic relationship between the uptake activity and the ratio of LacS-C67/LacS-cl, we conclude that the dimeric state of LacS is required for Dp-driven uptake. N-ethylmaleimide (NEM) treatment of proteoliposomes abolished the LacS-C67 exchange activity but left the LacS-cl unaffected. After NEM treatment, the exchange activity decreased linearly with increasing ratios of LacS-C67/LacS-cl, suggesting that the monomeric state of LacS is suf®cient for this mode of transport. We propose that the two subunits of LacS are functionally coupled in the step associated with conformational reorientation of the empty binding site, a step unique for Dp-driven uptake.

Identification and Functional Impact of Homo-oligomers of the Human Proton-coupled Folate Transporter

“…This was further tested by co-expressing P425R and WT hPCFTs in defined molar ratios, with levels of surface mutant and WT proteins measured by surface biotinylation and deglycosylation so that mutant and WT forms could be simultaneously detected on Western blots. In these experiments, when net transport was plotted versus the fraction of surface WT hPCFT (34,(51)(52)(53), a distinct positive non-linearity was detected, strongly implying functional "rescue" of the mutant monomer, independent of its intracellular trafficking but rather mediated allosterically via monomer interactions across the oligomer interface(s). Thus, functional cooperation between hPCFT monomers in facilitating transport of folate substrates appears likely.…”

“…However, if there is functional interaction between hPCFT monomers, total transport activity should increase or decrease quadratically with the increasing fraction of active hPCFT. These methods are completely analogous to those previously described for numerous other oligomeric transporters, including hRFC (34,(51)(52)(53)(54).…”

“…If hPCFT monomers function independently (i.e. monomeric hPCFT is the "minimal functional unit"), transport activity should closely reflect the amounts of WT hPCFT and increase linearly with increasing ratios of WT to total surface hPCFT forms (34,(51)(52)(53)(54). However, if there is functional interaction between hPCFT monomers, total transport activity should increase or decrease quadratically with the increasing fraction of active hPCFT.…”

“…Co-expression of WT and Mutant hPCFT Monomers-Coexpression of WT and inactive mutant protein monomers within the same cell is a powerful approach to study potential functionally important monomer interactions within homooligomeric membrane transporters (34,40,51,53). As long as transfection efficiencies are carefully controlled for (e.g.…”

“…(34,(51)(52)(53)(54). However, if interactions (positive or negative) occur between WT and mutant monomers, the results should conform to a distinctly non-linear (quadratic) pattern, depending on whether the effect of WT/mutant co-association is dominant-positive or -negative and on the oligomerization state (dimer, trimer, etc.)…”