Vsb1 Version 6

[Nickie Koskinen]

Thevacuole of the yeast Saccharomyces cerevisiae plays an important role in nutrient storage. Arginine, in particular, accumulates in the vacuole of nitrogen-replete cells and is mobilized to the cytosol under nitrogen starvation. The arginine import and export systems involved remain poorly characterized, however. Furthermore, how their activity is coordinated by nitrogen remains unknown. Here we characterize Vsb1 as a novel vacuolar membrane protein of the APC (amino acid-polyamine-organocation) transporter superfamily which, in nitrogen-replete cells, is essential to active uptake and storage of arginine into the vacuole. A shift to nitrogen starvation causes apparent inhibition of Vsb1-dependent activity and mobilization of stored vacuolar arginine to the cytosol. We further show that this arginine export involves Ypq2, a vacuolar protein homologous to the human lysosomal cationic amino acid exporter PQLC2 and whose activity is detected only in nitrogen-starved cells. Our study unravels the main arginine import and export systems of the yeast vacuole and suggests that they are inversely regulated by nitrogen.

The lysosome-like vacuole of the yeast Saccharomyces cerevisiae is an important storage compartment for diverse nutrients, including the cationic amino acid arginine, which accumulates at high concentrations in this organelle in nitrogen-replete cells. When these cells are transferred to a nitrogen-free medium, vacuolar arginine is mobilized to the cytosol, where it is used as an alternative nitrogen source to sustain growth. Although this phenomenon has been observed since the 1980s, the identity of the vacuolar transporters involved in the accumulation and the mobilization of arginine is not well established, and whether these processes are regulated according to nutritional cues remains unknown. In this study, we exploited in vitro and in vivo uptake assays in vacuoles to identify and characterize Vsb1 and Ypq2 as vacuolar membrane proteins mediating import and export of arginine, respectively. We further provide evidence that Vsb1 and Ypq2 are inversely regulated according to the nitrogen status of the cell. Our study sheds new light on the poorly studied topic of the diversity and metabolic control of vacuolar transporters. It also raises novel questions about the molecular mechanisms underlying their coordinated regulation and, by extension, the regulation of lysosomal transporters in human cells.

Copyright: 2020 Cools et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: M.C. was the recipient of a PhD fellowship from the Fonds pour la Formation la Recherche dans l'Industrie et dans l'Agriculture (FRIA). This work was supported by a PDR grant (nr. 746 23655065) by the Fonds National de la Recherche Scientifique (FNRS) (Fdration Wallonie-Bruxelles, Belgium), the International Brachet Foundation, and by a grant (nr. CRFF-2015-01) from the Cystinosis Research Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The vacuole of the yeast Saccharomyces cerevisiae is the counterpart of the lysosome and has proved to be a valuable model for studying this organelle [1]. The main role of the yeast vacuole, like that of lysosomes, is to carry out the degradation of proteins and other macromolecules delivered to it via the endocytic or the autophagic pathway. The released metabolites are then exported to the cytosol via diverse transporters [2]. In humans, dysfunction of a single lysosomal hydrolase or transporter can cause detrimental accumulation of non-recycled metabolites, the typical feature of lysosomal storage diseases (LSDs) [3]. One such disease, cystinosis, is caused by mutations in the CTNS gene encoding cystinosin, a lysosomal cystine exporter [4]. Patients suffering from cystinosis are treated with the aminothiol cysteamine. This molecule enters the lysosomes and reacts there with accumulated cystine, converting it to cysteine and a cysteine-cysteamine mixed disulfide. The latter compound, structurally similar to lysine, is then efficiently exported from the lysosomes via the PQLC2 cationic amino-acid exporter [5].

In yeast, the closest homologs of PQLC2 are the proteins Ypq1, 2 and 3. They localize to the vacuolar membrane via the ALP (alkaline phosphatase) traffic pathway [6] and are involved in homeostasis of cationic amino acids. Specifically, ypq1Δ and ypq2Δ deletion mutants display resistance to canavanine, a toxic analog of arginine (Arg), and the YPQ3 gene is transcriptionally repressed by excess lysine (Lys). The canavanine resistance phenotype of the ypq2Δ mutant can be suppressed by expression of PQLC2, shown to also recognize the Arg analog, suggesting that Ypq2 likewise functions as a vacuolar exporter of cationic amino acids [5]. Since this discovery, however, investigators have reported experiments on reconstituted vacuolar vesicles showing that Ypq1 and Ypq3 respectively catalyze Lys and histidine (His) uptake into the vacuole and that their activity depends on the H+ gradient established by the V-ATPase [7,8], while Ypq2 catalyzes Arg/His exchange [9]. Another study reported that Ypq1, under Lys starvation conditions, is targeted to the vacuolar lumen and degraded, a result supporting a role of Ypq1 in Lys uptake into the vacuole [10,11].

This study aims to further characterize the molecular determinants of Arg transport (export and import) across the yeast vacuolar membrane and to assess whether they are under nitrogen control. We report that Ypq2, the yeast homolog of human PQLC2, plays a particularly important role in recycling vacuolar Arg stores to the cytosol under nitrogen starvation. We further characterize Vsb1/Ygr125w as a novel vacuolar membrane protein playing an essential role in accumulation of Arg and other cationic amino acids within the vacuole. Finally, we provide evidence that Ypq2 and Vsb1 are inversely regulated according to the nitrogen supply conditions.

To study the transport of amino acids into and out of the yeast vacuole, we considered isolated intact vacuoles to be the most appropriate material, as they retain their sap and metabolite content, including amino acids, and should be more similar to the vacuoles naturally present in cells than the reconstituted vacuolar vesicles used in many previous works. Our first step was thus to implement and adapt methods for isolating intact vacuoles and measuring the uptake of radiolabeled amino acids into them. To assess the purity and integrity of our isolated vacuoles and to readily measure their internal pH, we isolated cells stably expressing the vacuolar lumen protein Sna3 fused to pHluorin, the vacuolar membrane protein Sna4 fused to DsRed, or both (Fig 1A). Microscopy analysis of cells expressing both constructs showed, as expected, localization of Sna3-pHluorin to the lumen and of Sna4-DsRed to the peripheral membrane of the vacuole (Fig 1B) [23,24]. The method of Cabrera and Ungermann for isolating intact vacuoles was then applied to these cells [25]. Isolated DsRed-labeled vacuoles appeared intact, as all of them also displayed luminal pHluorin labeling, i.e. they obviously had not ruptured and should have conserved their sap (Fig 1B). Vacuoles labeled only with Sna3-pHluorin were then isolated and incubated with the lipophilic red fluorescent dye FM4-64, which should stain all isolated membranes. All the isolated compartments (stained in red) were found to display luminal pHluorin green fluorescence, confirming that the isolated compartments visible under the microscope were vacuoles only (Fig 1B).

(A) Left. Sketch of the DNA cassette integrated into the UGA1 locus and expressing the SNA3-PHLUORIN and SNA4-DsRED hybrid genes under the control of the TPI1 (T) or PGK1 (P) gene promoter, respectively. Strains having integrated this cassette were selected for resistance to geneticin (G418R) and loss of the ability to use GABA as a nitrogen source (uga1Δ phenotype). Right. Schematic representation of Sna3-pHluorin and Sna4-DsRed located, respectively, at the peripheral membrane and in the lumen of the vacuole. Sna3 is a transmembrane protein targeted to the vacuole via the multivesicular-body (MVB) pathway. Scissors represent vacuolar proteases. (B) Cells expressing Sna3-pHluorin and/or Sna4-DsRed and samples of vacuoles isolated from them were examined by epifluorescence microscopy. In whole cells, Sna3-pHluorin and Sna4-DsRed correctly label the vacuolar lumen and limiting membrane, respectively. Isolated vacuoles exhibit the same labeling. Vacuoles isolated from cells only expressing Sna3-pHluorin were labeled with FM4-64 (10 μM). (C) The V-ATPase is active in isolated vacuoles. The pH of vacuoles isolated from cells expressing Sna3-pHluorin was measured after addition of ATP (4 mM), nigericin (6.5 μM), or bafilomycin A (6.5 μM) (n = 2). (D) Time course of [3H]-L-tyrosine (50 μM) uptake into vacuoles isolated from the w-t strain. Vacuoles were pre-incubated for 8 minutes with or without ATP (4 mM) before adding the radiolabeled amino acid (n = 2). (E) Accumulation of 3H-tyrosine (50 μM) was measured in vacuoles from the w-t or the avt1Δ mutant after a 4-minute incubation. The vacuoles were pre-incubated with ATP (4 mM), and with or without nigericin (6.5 μM) or concanamycin A (6.5 μM) (n = 4). For all experiments, error bars represent the SD.

The vacuolar Avt1 transporter is reported to catalyze tyrosine uptake. This transport activity, measured on vacuolar vesicles, depends on the H+ gradient established by the V-ATPase [15]. Using intact vacuoles instead of vacuolar vesicles, we likewise measured ATP-dependent tyrosine uptake (Fig 1D). This uptake activity was lost if nigericin or a V-ATPase inhibitor was also added to the transport reaction (Fig 1E). Nor was it detected in vacuoles isolated from the avt1Δ mutant (Fig 1E).

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