Tales of tails in transporters

Abstract

Cell nutrition, detoxification, signalling, homeostasis and response to drugs, processes related to cell growth, differentiation and survival are all mediated by plasma membrane (PM) proteins called transporters. Despite their distinct fine structures, mechanism of function, energetic requirements, kinetics and substrate specificities, all transporters are characterized by a main hydrophobic body embedded in the PM as a series of tightly packed, often intertwined, α-helices that traverse the lipid bilayer in a zigzag mode, connected with intracellular or extracellular loops and hydrophilic N- and C-termini. Whereas longstanding genetic, biochemical and biophysical evidence suggests that specific transmembrane segments, and also their connecting loops, are responsible for substrate recognition and transport dynamics, emerging evidence also reveals the functional importance of transporter N- and C-termini, in respect to transport catalysis, substrate specificity, subcellular expression, stability and signalling. This review highlights selected prototypic examples of transporters in which their termini play important roles in their functioning.

Keywords: fluorescent microscopy; model fungi; molecular dynamics; mutational analysis; phylogeny; transporter structure.

Figure 1.

Cytosolic N- and C-termini play a crucial role in transporter expression, function and turnover. The figure highlights in a simplified manner how the topology of cytosolic termini alters depending on the overall conformation of the transporters (i.e. outward facing, occluded, inward facing) and this altered conformation is crucial for regulating both intramolecular events (i.e. transport activity, allosteric regulation) and intermolecular interactions (i.e. ubiquitination, endocytosis, sorting, signalling, etc.). Notice that in the outward-facing conformation the N- and C-tails are in closer contact with other domains of the transporter than in the inward-facing conformation. For more details see main text.

Figure 2.

Generalized model of transporter endocytosis highlighting the crucial role of N- and C-tails, based mostly on data concerning the FurE purine transporter, but also integrating critical findings from studies with Gap1 and Jen1 transporters (see text). The figure shows that in the outward-facing conformation the N- and C-tails are in close contact with each other and with other cytoplasmic domains of the transporter (e.g. the inner gate shown in purple), while in the inward-facing conformation the N- and C-tails separate to become more relaxed to recruit cytoplasmic effectors, such as those leading to ubiquitination and endocytosis. The figure includes a HECT-type ubiquitin ligase (Rsp5), its α-arrestin adaptor (Art) and a 14-3-3 protein that inhibits Art association with Rsp5 via phosphorylation. Upon a signal eliciting endocytosis (depicted by a blue-red star), Art is de-phosphorylated, acquires high affinity for Rsp5, which ubiquitylates Art, and the Art–Rsp5 complex is recruited to transporter tails. The relaxed topology of the cytosolic tails in the inward conformation permits more efficient recruitment of the Art–Rsp5 complex than the ‘hidden’ tails in the outward-facing conformation. The specific lysine residues (KK) and acidic motifs (e.g. EXE) necessary for ubiquitination are located either in the C-terminus (as in the figure) or in the N-terminus, but the interaction of termini is crucial for accessing these elements, so that both termini are critical for endocytosis.

Figure 3.

Structural insights into LeuT-fold transporters with emphasis on the functional role of cytosolic terminal domains. (a) LeuT dynamic topology depicting the tilt of the ‘bundle’ domain (TMS 1,2,6,7) from the outward (blue) to the inward (gold) conformation. Notice also the displacement of TMS1a and TMS5 (PDB entries 3tt1, 3tt3). (b) Interactions of the cytosolic N-tail (deep blue) with internal loops in dDAT (PDB entry 4m48). (c) Mhp1 dynamic topology showing the rocking movement of the ‘hash’ domain (TMS 3,4,8,9) from the outward (gold) to the inward (blue) conformation. Notice also the displacement of TMS5 (PDB entries 2jln, 2x79). (d) Interactions of the cytosolic N-tail (deep blue) with internal loops and TMS6 and TMS8 in Mhp1 (PDB entry 2jln). All snapshots were generated by Chimera 1.10.

Figure 4.

The multiple roles of both termini of FurE in folding, ER exit, endocytosis and substrate specificity. 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. E_N and E_C are the distal parts of the tails that interact dynamically with each other during transport, and thus recruit ubiquitination and endocytosis factors, but also the positioning of the LID, which, in turn, affects the function and specificity of the transporter. F is the part very proximal to TMS1 that is critical for the correct folding of TMS1 and of the transporter, and thus affects packaging to COPII vesicles and ER exit.