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

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

Tales of tails in transporters

“…This is in line with some other cases, where mutations blocking ubiquitination and endocytotic turnover are located not only in the terminal segment that includes the ubiquitination-specific Lys residues and a nearby arrestin target, but also in the opposite cytosolic terminus. This is nicely exemplified in studies concerning the Fur purine-related transporters of A. nidulans ([16,17]; figure 2). In this case, relatively short specific truncations or triple Ala substitutions in either terminus of the FurE transporter block its turnover.…”

“…Unlike the N-terminus, the C-tail of members of NCS1 does not include any ubiquitously conserved motif. However, the acidic sequence E/D- X -E-E has been shown to be absolutely essential for endocytic regulation via specific ubiquitination of the purine-related FurE transporter of the fungus Aspergillus nidulans [17]. Versions of such short poly-acidic motifs are present in the tails of different transporters, but their positioning related to the last TMS or the C-end differs.…”

“…In the purine-related UapA and FurE transporters of A. nidulans a short acidic motif ( E/D -X- E - E; see table 1) located at the cytosolic C-terminus was shown to be essential for ubiquitination and/or endocytosis [17,39]. In both UapA and FurE, this acidic motif is located immediately next to the last TMS and 23–25 residues upstream from the Lys residues modified by ubiquitination.…”

“…loss of uric acid transport) of N- or C-terminal mutants are located at a tentative external gate, along the substrate translocation path, or in flexible loops that act as dynamic hinges during transport catalysis [16,17]. Additionally, MD and further mutational analysis supported that specific polar residues of the N-terminus (Asn24, Asp26 and Asp28) interact dynamically with residues of several internal cytosolic loops of FurE, and this interaction controls the opening and closing of the outer and inner gates [17]. Given that the positioning of the N-terminus also depends on its interaction with the C-terminus, both termini are thus involved in a dynamic molecular cross-talk with the internal loops of FurE (figure 4).…”

“…The figure is based mostly on data concerning the FurE transporter, which contains a LeuT-like fold. Details are described in the text and in [17]. LID stands for the central part of the N-tail that is specifically involved in interactions with several other cytosolic internal loops and thus allosterically regulates the functioning of the outer and inner gates and substrate specificity.…”