Structural analysis of yeast amino acid transporters: substrate binding and substrate-induced endocytosis

Plasma membrane transport proteins play a crucial role in all cells by conferring to the cell surface a selective permeability to a wide range of ions and small molecules. The activity of these transporters is often regulated by controlling their amount at the plasma membrane, via intracellular trafficking. The recent boom in the numbers of crystallized transporters shows that many of them that belong to different functional families with little sequence similarity adopt the same structural fold implying a conserved transport mechanism. These proteins belong to the APC (Amino acid-Polyamine-organoCation) superfamily and their fold is typified by the bacterial leucine transporter LeuT. This LeuT fold is characterized by inverted structural repeats of 5 transmembrane domains that harbor the central substrate-binding site and a pseudo-symmetry axis parallel to the membrane. The yeast Saccharomyces cerevisiae possesses about 16 amino acid permeases (yAAPs) that belong to the APC superfamily and that display various substrate specificity ranges and affinities. Topological, mutational analysis and in silico data indicate that yAAPS adopt the LeuT fold.In this work we combined computational modeling and yeast genetics to study substrate binding by yAAPs and the endocytosis of these transporters in response to substrate transport. In the first part of this work, we analyzed the selective recognition of arginine by the yeast specific arginine permease, Can1. We constructed three-dimensional models of Can1 using as a template the recently resolved structure of AdiC, the bacterial arginine:agmatine antiporter, which is also a member of the APC superfamily. By comparison of the binding pockets of Can1 and Lyp1, the yeast specific lysine permease, we identified key residues that are involved in the recognition of the main and side chains of arginine. We first showed that the network of interactions of arginine in Can1 is similar to that of AdiC, and that the selective recognition of arginine is mediated by two residues: Asn 176 and Thr 456. Substituting these residues by their corresponding residues in Lyp1 converted Can1 into a specific lysine permease. In the second part of this work, we studied the regulation of two permeases, Can1 and the yeast general amino acid permease, Gap1. In the presence of their substrates, Gap1 and Can1 undergo ubiquitin-dependent endocytosis and targeting to the vacuolar lumen for degradation. We showed that this downregulation is not due to intracellular accumulation of the transported amino acids but to transport catalysis itself. By permease structural modeling, mutagenesis, and kinetic parameter analysis, we showed that Gap1 and Can1 need to switch to an intermediary conformational state and persist a minimal time in this state after binding the substrate to trigger their endocytosis. This down-regulation depends on the Rsp5 ubiquitin ligase and involves the recruitment of arrestin-like adaptors, resulting in the ubiquitylation and endocytosis of the permease.Our work shows the importance of the structural analysis of yAAPs to get further insight into the different aspects of their function and regulation. We validate the use of a bacterial APC transporter, AdiC, to construct three-dimensional models of yAAPs that can be used to guide experimental analyses and to provide a molecular framework for data interpretation. Our results contribute to a better understating of the recognition mode of amino acids by their permeases, and the regulation of this transport in response to substrate binding.Doctorat en Sciencesinfo:eu-repo/semantics/nonPublishe

Structure-Function study of the transporters and the sensor of amino acids in yeast Saccharomyces Cerevisiae

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Substrate Transport and Specificity of Amino Acid Transporters studied by Molecular Simulations

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Unveiling the mechanism of arginine transport through AdiC with molecular dynamics simulations: The guiding role of aromatic residues

Commensal and pathogenic enteric bacteria have developed several systems to adapt to proton leakage into the cytoplasm resulting from extreme acidic conditions. One such system involves arginine uptake followed by export of the decarboxylated product agmatine, carried out by the arginine/agmatine antiporter (AdiC), which thus works as a virtual proton pump. Here, using classical and targeted molecular dynamics, we investigated at the atomic level the mechanism of arginine transport through AdiC of E. coli. Overall, our MD simulation data clearly demonstrate that global rearrangements of several transmembrane segments are necessary but not sufficient for achieving transitions between structural states along the arginine translocation pathway. In particular, local structural changes, namely rotameric conversions of two aromatic residues, are needed to regulate access to both the outward- and inward-facing states. Our simulations have also enabled identification of a few residues, overwhelmingly aromatic, which are essential to guiding arginine in the course of its translocation. Most of them belong to gating elements whose coordinated motions contribute to the alternating access mechanism. Their conservation in all known E. coli acid resistance antiporters suggests that the transport mechanisms of these systems share common features. Last but not least, knowledge of the functional properties of AdiC can advance our understanding of the members of the amino acid-carbocation-polyamine superfamily, notably in eukaryotic cells.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Substrate-Induced Ubiquitylation and Endocytosis of Yeast Amino Acid Permeases

Many plasma membrane transporters are downregulated by ubiquitylation, endocytosis, and delivery to the lysosome in response to various stimuli. We report here that two amino acid transporters of Saccharomyces cerevisiae, the general amino acid permease (Gap1) and the arginine-specific permease (Can1), undergo ubiquitin-dependent downregulation in response to their substrates and that this downregulation is not due to intracellular accumulation of the transported amino acids but to transport catalysis itself. Following an approach based on permease structural modeling, mutagenesis, and kinetic parameter analysis, we obtained evidence that substrate-induced endocytosis requires transition of the permease to a conformational state preceding substrate release into the cell. Furthermore, this transient conformation must be stable enough, and thus sufficiently populated, for the permease to undergo efficient downregulation. Additional observations, including the constitutive downregulation of two active Gap1 mutants altered in cytosolic regions, support the model that the substrate-induced conformational transition inducing endocytosis involves remodeling of cytosolic regions of the permeases, thereby promoting their recognition by arrestin-like adaptors of the Rsp5 ubiquitin ligase. Similar mechanisms might control many other plasma membrane transporters according to the external concentrations of their substrates.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Substrate-induced ubiquitylation and endocytosis of yeast amino acid permeases.

Many plasma membrane transporters are downregulated by ubiquitylation, endocytosis, and delivery to the lysosome in response to various stimuli. We report here that two amino acid transporters of Saccharomyces cerevisiae, the general amino acid permease (Gap1) and the arginine-specific permease (Can1), undergo ubiquitin-dependent downregulation in response to their substrates and that this downregulation is not due to intracellular accumulation of the transported amino acids but to transport catalysis itself. Following an approach based on permease structural modeling, mutagenesis, and kinetic parameter analysis, we obtained evidence that substrate-induced endocytosis requires transition of the permease to a conformational state preceding substrate release into the cell. Furthermore, this transient conformation must be stable enough, and thus sufficiently populated, for the permease to undergo efficient downregulation. Additional observations, including the constitutive downregulation of two active Gap1 mutants altered in cytosolic regions, support the model that the substrate-induced conformational transition inducing endocytosis involves remodeling of cytosolic regions of the permeases, thereby promoting their recognition by arrestin-like adaptors of the Rsp5 ubiquitin ligase. Similar mechanisms might control many other plasma membrane transporters according to the external concentrations of their substrates.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Release of arginine into the cytosol from the IF open state.

<p>(A) Number of occurrences (N_O) for finding the substrate at a certain RMSD value, computed for the carbon atoms of Arg⁺ using all tMD simulations and its crystal position in the occluded state (PDB ID: 3L1L) as a reference. The standard error is shown as bars. (B) Interactions (H-bonds, ionic and cation-π) formed between the Arg⁺ backbone and side chain and protein residues during the tMD simulations. Only interactions with an occurrence higher than 20% in at least one bin width from all 72 binding events of the 36 tMD trajectories are shown. The abbreviations used for the different interactions are listed in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160219#pone.0160219.g003" target="_blank">Fig 3</a>. The binding site region (-5 to 5 Å) is highlighted by a magenta bar. (C) Four tMD conformations highlighting the exit of arginine from the IF open state to the cytosol (1–4). The Arg⁺ side chain first leaves the primary binding site, still forming a cation-π interaction with Trp293, but from the IF side, in contrast to that formed in the OF state (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160219#pone.0160219.g005" target="_blank">Fig 5D</a>), and making an ionic bond with Glu208 (1). The cation-π interaction with Trp293 is lost and the Arg⁺ side chain interacts mostly with two distal gate residues, Tyr93 (through a cation-π interaction) and Glu208 (2). The cation-π interaction with Tyr93 serves as a hinge to pivot the Arg⁺ side chain, which then forms an ionic bond to Glu208 via its amino group (3). The backbone heads first towards the exit, with the guanidinium group making an ionic interaction with Glu208 (4). Residues making persistent interactions with Arg⁺ are shown as sticks and the protein portion shaping the OF side is shown as a white cartoon. The substrate Arg⁺ is depicted as CPK.</p

Binding of Arg⁺ to the OF open state during the tMDs.

<p>(A) Number of occurrences (N_O) for finding the substrate at a certain RSMD value, computed for the carbon atoms of Arg⁺ using all 6 tMD trajectories (each containing two AdiC monomers) and its crystal position in the OF open crystal structure (PDB ID 3OB6; Monomer A) as a reference. An RMSD value of zero Å corresponds to a perfect match between the MD conformation and the targeted crystal position of arginine. The standard error is shown as black bars. (B) Interactions (H bonds, ionic and cation-π interactions) formed between the Arg⁺ backbone or side chain and protein residues during its migration down to the binding site are shown, along with the occurrence of observing the center of mass of Arg⁺ at a certain position along the main axis of the transporter between the external medium and the binding site, as depicted by a bar graph representation. Only interactions with an occurrence higher than 20% in at least one bin width from all 12 binding events of the 6 tMD trajectories are shown. The abbreviations used for the different interactions are: ionic for ionic interaction, HB for H bond, HB_H2O for water-mediated H bond, and Cat-π for cation π interaction. The binding site region (-5 to 5 Å) is highlighted by a magenta bar. (C) Four tMD conformations of AdiC highlighting Arg⁺ migration (1–4). The Arg⁺ side chain first forms a cation-π interaction with Phe350 (1). It then moves further down to form a sandwich configuration between the Phe350 and Trp202 side chains (2). Following this configuration, Arg⁺ slides down towards the binding site, forming a cation-π interaction with Trp202 (3). Arg⁺ leaves Trp202 to form a cation-π interaction with Trp293, which shapes the bottom of the binding site. Residues making persistent interactions with Arg⁺ are shown as sticks and the protein portion shaping the OF side is shown as a white cartoon. The Arg⁺ substrate is depicted as CPK.</p

Transition from the occluded to the IF open substrate-bound state.

<p>(A) The profile of the funnel radius as a function of position along the main axis of the transporter, averaged over the last 0.1 ns of one tMD simulation is shown with (red) and without (violet) the inclusion of Trp293 in the targeted atom ensemble (see text). The standard error is shown as bars. The radius is also depicted for the IF open GadC (4DJI, green) and the occluded AdiC (3L1L, cyan) crystal structures. (B) Interactions (H bonds, ionic and cation-π) formed by the Arg⁺ backbone or side chain with protein residues during all tMD trajectories. Only interactions with an occurrence higher than 20% in at least one bin width from all 24 binding events of the 12 tMD trajectories are shown. The abbreviations used for the different interactions are listed in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160219#pone.0160219.g002" target="_blank">Fig 2</a>. The binding site region (-5 to 5 Å) is highlighted by a magenta bar in (A) and (B). (C) (left) Number of occurrences (N_O) for finding the substrate at a certain RMSD value, computed for the carbon atoms of Arg⁺ using all tMD simulations and its crystal position in the occluded state (PDB ID: 3L1L) as a reference (left). The standard error is shown as bars. Two different Arg⁺ positions (labelled (1) and (2)) are depicted. The position (1) (in yellow CPK) corresponds to the position with the highest occurrence and the (2) (in magenta CPK) features Arg⁺ with its sidechain reoriented towards Glu208. Surrounding binding site residues are shown as sticks (atom type colored). These two positions are marked with their number on the RMSD plot.</p

Arg⁺ bound to the OF open state after the binding stage and during the relaxation MDs.

<p>(A) Number of occurrences (N_O) for finding the substrate at a certain RSMD value, computed for the carbon atoms of Arg⁺ using all MD relaxation simulations and its crystal position in the OF open state (PDB ID: 3OB6) as a reference (left). The standard error is shown as bars. A global and a local maxima are found (labeled (1) and (2)), which describe the most populated Arg⁺ positions as depicted on the right: the major peak corresponds (1) to a binding position shown as balls-and-sticks, similar to that in the crystal structure (PDB ID 3OB6, monomer A); the minor peak (2), shown as sticks, indicates a different position, in which the substrate is rotated relative to position (1). Regions surrounding the binding site (white ribbons) as well as a few binding site residues (atom type colored sticks) are also shown. (B) Interactions (H bonds, ionic and cation-π) formed during the relaxation MD simulations between the Arg⁺ backbone or side chain and protein residues in the binding site (the abbreviations used for the different interactions are listed in the legend of Fig 3). Only interactions with an occurrence higher than 20% in at least one bin width from all 12 binding events of the 6 tMD trajectories are shown. The binding site region (-5 to 5 Å) is highlighted by a magenta bar. The interactions formed in the OF open substrate-bound crystal structure (PDB ID: 3OB6 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160219#pone.0160219.ref015" target="_blank">15</a>]) are indicated by asterisks.</p