Cytosolic termini of the FurE transporter regulate endocytosis, pH-dependent gating and specificity

FurE, a member of the NCS1 family, is an Aspergillus nidulans transporter specific for uracil, allantoin and uric acid. Recently we showed that C- or N-terminally truncated FurE versions are blocked for endocytosis and, surprisingly, show modified substrate specifities. Bifluorescence complementation assays and genetic analyses supported that the C- and N-termini interact dynamically and through this interaction regulate selective substrate translocation. Here we functionally dissect and delimit distinct motifs crucial for endocytosis, transport activity, substrate specificity and folding, in both cytosolic termini of FurE. Subsequently, we obtain novel genetic and in silico evidence supporting that the molecular dynamics of specific N- and C-terminal regions affect allosterically the gating mechanism responsible for substrate selection, via pH-dependent interactions with other internal cytosolic loops and membrane lipids. Our work shows that elongated cytoplasmic termini, acquired through evolution mostly in eukaryotic transporters, provide novel specific functional roles.