No abstract

19FurE, a member of the NCS1 family, is an Aspergillus nidulans transporter specific 20 for uracil, allantoin and uric acid. Recently we showed that C-or N-terminally 21 truncated FurE versions are blocked for endocytosis and, surprisingly, show modified 22 substrate specifities. Bifluorescence complementation assays and genetic analyses 23 supported that the C-and N-termini interact dynamically and through this interaction 24 regulate selective substrate translocation. Here we functionally dissect and delimit 25 distinct motifs crucial for endocytosis, transport activity, substrate specificity and 26 folding, in both cytosolic termini of FurE. Subsequently, we obtain novel genetic and 27 in silico evidence supporting that the molecular dynamics of specific N-and C-28 terminal regions affect allosterically the gating mechanism responsible for substrate 29 selection, via pH-dependent interactions with other internal cytosolic loops and 30 membrane lipids. Our work shows that elongated cytoplasmic termini, acquired 31 through evolution mostly in eukaryotic transporters, provide novel specific functional 32 roles. 33 Key words: Aspergillus nidulans/fungi/transport/folding/membrane lipids/allosteric 34 35 36 3 Introduction 37 38Transporters are membrane proteins that mediate the import and export of nutrients, 39 metabolites, signaling molecules or drugs in and out of cells, and are thus essential for 40 cell communication and life. Despite their evolutionary, structural and functional 41 differences all transporters use an alternating-access mechanism where a substrate 42 binding site, in allosteric co-operation with distinct gating domains, alternates 43 between multiple conformations for receiving and delivering specific substrate(s) 44 from one side of the membrane to the other. This basic mechanism, carried out by 45