bMany 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.

Background: Can1 is a yeast plasma membrane permease catalyzing specific uptake of arginine. Results: Two residues in the binding pocket of Can1 affect its selectivity. Conclusion: Subtle amino acid changes can convert Can1 to a lysine-specific permease. Significance: Understanding, at the molecular level, the translocation mechanism(s) of yeast amino acid transporters has bearings on our knowledge of other transporters featuring the same fold.

Background: Prolonged exposure to elevated free fatty acids induces β-cell failure (lipotoxicity) and contributes to the pathogenesis of type 2 diabetes. In vitro exposure of β-cells to the saturated free fatty acid palmitate is a valuable model of lipotoxicity, reproducing features of β-cell failure observed in type 2 diabetes. In order to map the β-cell response to lipotoxicity, we combined RNA-sequencing of palmitate-treated human islets with iTRAQ proteomics of insulin-secreting INS-1E cells following a time course exposure to palmitate. Results: Crossing transcriptome and proteome of palmitate-treated β-cells revealed 85 upregulated and 122 downregulated genes at both transcript and protein level. Pathway analysis identified lipid metabolism, oxidative stress, amino-acid metabolism and cell cycle pathways among the most enriched palmitate-modified pathways. Palmitate induced gene expression changes compatible with increased free fatty acid mitochondrial import and βoxidation, decreased lipogenesis and modified cholesterol transport. Palmitate modified genes regulating endoplasmic reticulum (ER) function, ER-to-Golgi transport and ER stress pathways. Furthermore, palmitate modulated cAMP/protein kinase A (PKA) signaling, inhibiting expression of PKA anchoring proteins and downregulating the GLP-1 receptor. SLC7 family amino-acid transporters were upregulated in response to palmitate but this induction did not contribute to β-cell demise. To unravel critical mediators of lipotoxicity upstream of the palmitate-modified genes, we identified overrepresented transcription factor binding sites and performed network inference analysis. These identified LXR, PPARα, FOXO1 and BACH1 as key transcription factors orchestrating the metabolic and oxidative stress responses to palmitate.

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.

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