Physiological significance of amino acid recycling from vacuoles under starvation conditions

Physiological significance of amino acid recycling from vacuoles under starvation conditions | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Physiological significance of amino acid recycling from vacuoles under starvation conditions Haruto Nakajo, Takayuki Sekito, Ryogo Okamura, Shiori Nakagawa, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8111730/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Mar, 2026 Read the published version in Scientific Reports → Version 1 posted 13 You are reading this latest preprint version Abstract Avt6 is a transporter that exports acidic amino acids, such as aspartate and glutamate. We found that disrupting AVT6 in cells lacking other vacuolar amino acid exporters, including Avt3, Avt4, and Avt7, increased vacuolar neutral amino acid content. Conversely, Avt6 overexpression decreased these amino acid levels. These results suggest that Avt6 is also involved in the vacuolar export of neutral amino acids. Although avt3 ∆ avt4 ∆ avt6 ∆ avt7 ∆ cells maintained viability under nitrogen starvation as wild-type cells, protein synthesis was reduced. By further deleting LEU2 , a gene involved in leucine biosynthesis, viability was significantly reduced. Moreover, avt3 ∆ avt4 ∆ avt6 ∆ avt7 ∆ diploid cells formed fewer spores in a sporulation medium. These phenotypes overlapped with those of cells defective in macroautophagy. This study reveals an extensive redundancy of transporters in vacuolar amino acid recycling and experimentally demonstrates that the vacuolar amino acids generated by macroautophagy are utilized to manage nutrient stress. Biological sciences/Biochemistry Biological sciences/Biotechnology Biological sciences/Microbiology vacuole transporter autophagy nitrogen starvation sporulation Saccharomyces cerevisiae Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Upon shifting to nitrogen starvation, cells induce macroautophagy (hereafter autophagy) to degrade the bulk of proteins in the vacuoles/lysosomes [ 1 , 2 ]. It has been suggested that the amino acids generated inside the vacuoles/lysosomes are recycled for protein synthesis [ 3 ]. Thus, they must be exported from the vacuoles/lysosomes to the cytosol, where translation occurs. In higher eukaryotes, autophagic amino acid recycling has been suggested to be involved in not only development and differentiation [ 4 ] but also various pathophysiological roles, such as tumor progression [ 5 ]. In the budding yeast Saccharomyces cerevisiae , several vacuolar amino acid exporters have been identified. Of these, Avt3, Avt4, Avt7, Atg22, and Ypq2 are involved in the export of neutral amino acids from vacuoles in a redundant manner [ 6 – 10 ]. Avt4 also exports basic amino acids [ 7 ]. Avt6 exports acidic amino acids [ 6 , 11 ]. Avt3 and Avt4 participate in autophagic amino acid recycling, because the contents of vacuolar neutral and basic amino acids in avt3 ∆ avt4 ∆ cells are much higher compared with those in wild-type cells under nitrogen starvation [ 7 ]. The contribution of Avt7 to amino acid recycling is relatively minor, but significant for several amino acid species, such as glutamine and proline [ 8 ]. AVT6 mRNA was increased under nitrogen-starved condition [ 12 ]. Moreover, the vacuolar glutamate and aspartate content in avt6 ∆ cells was higher compared with that in wild-type cells under this condition [ 11 ]. Therefore, Avt6 also likely acts on the recycling of the vacuolar acidic amino acids generated by autophagy. Atg22 was reported to be involved in the export of tyrosine, isoleucine and leucine [ 9 ], and Ypq2 has been suggested to act as a vacuolar uniporter for arginine [ 10 ]. Since mutants that are defective in autophagy fail to survive during nitrogen starvation [ 13 ], it has been suggested that the recycling of amino acids generated by autophagy maintains protein synthesis to promote cell survival [ 14 , 15 ]. However, autophagy also degrades bulk ribosomes and various organelles, such as mitochondria and endoplasmic reticulum [ 16 – 21 ]. As a result, their degradation products, including phosphate, zinc, iron, nucleotides, lipids, and carbohydrates, are also likely to be exported from the vacuoles for recycling [ 3 , 22 , 23 ]. In addition, autophagy removes dysfunctional organelles and proteins to avoid the production of harmful products, such as reactive oxygen species, and the unwanted accumulation of proteins [ 24 , 25 ]. These suggest that the recycling of various materials other than amino acids and the quality control of organelles and proteins may also contribute to cell viability during nitrogen starvation. Therefore, to address the physiological importance of autophagic amino acid recycling, inhibiting vacuolar amino acid exporter gene function and examining the effect on cell viability will provide great insight. In this study, we determined the effect of avt3 ∆ avt4 ∆ avt6 ∆ avt7 ∆ quadruple disruption on vacuolar amino acid content and examined the resulting phenotype during nitrogen starvation. Our results suggest that Avt6 exports neutral amino acids in addition to acidic ones in a redundant manner with Avt3, Avt4, and Avt7. Moreover, the recycling of vacuolar neutral amino acids likely contributes to protein synthesis under nitrogen starvation and the efficiency of spore formation. avt3 ∆ avt4 ∆ avt6 ∆ avt7 ∆ cells partially recapitulate the phenotype of the autophagy-deficient mutant. Materials and methods Strains and media The S. cerevisiae strains used in this study are listed in Supplementary Table S1. Genes were deleted using a PCR-based method with either the kanMX , natMX , or hphMX cassette [26, 27]. For multiple gene disruption, in addition to using multiple markers, the kanMX marker was recycled using the Cre-loxP system [28]. For chromosomal tagging with HA 3 , HA 6 , or Myc 9 , PCR products amplified from the pYM-series plasmid [29] were introduced into the parent strains. S. cerevisiae cells were grown at 30°C to early logarithmic phase in YPD (1% yeast extract, 2% bacto-peptone, and 2% glucose) or SD+Cas (0.17% yeast nitrogen base w/o amino acids and ammonium sulfate, 0.5% casamino acids, 0.5% ammonium sulfate, 20 mg/L tryptophan, and 2% glucose) medium to collect cells grown under nutrient-rich conditions. For nitrogen starvation, cells grown in nutrient-rich conditions were shifted to SD-N (0.17% yeast nitrogen base w/o amino acids and ammonium sulfate and 2% glucose) and further cultured at 30°C. Plasmids Primers used to construct plasmids in this study were listed in Supplementary Table S2. pAVT6 was constructed as described previously [11]. To construct pGPD-AVT6, the AVT6 ORF was amplified by PCR using a pair of primers, AVT6 ORF F Bam HI and AVT6 ORF R Sal I, and ligated into the p416GPD plasmid [30] following restriction enzyme digestion. To construct pGPD-ATG22, the ATG22 ORF amplified by PCR using a pair of primers, ATG22 ORF F Bam HI and ATG22 ORF R Hin dIII, was inserted into the p416GPD. Amino acid analysis To prepare a vacuolar fraction, the cupric ion treatment method was used [31]. Briefly, 10 OD 660 units of cells were washed twice with an amino acid extraction (AAE) buffer (2.5 mM potassium phosphate buffer, pH 6.0, 0.6 M sorbitol, and 10 mM glucose) and incubated in AAE buffer containing 0.2 mM CuCl 2 at 30°C for 15 or 30 min for cells cultured in nutrient-rich or nitrogen-starved condition, respectively. After washing with AAE buffer, the cell pellets were resuspended in 500 µL of distilled water and boiled for 15 min. After centrifugation, the amino acid content in the supernatants was measured using an automatic amino acid analyzer (Hitachi L-8900). Western blot analysis Cell lysates (0.3 OD 660 units of cells) were analyzed using anti-HA (3F10, Roche), anti-Myc (9E10, Roche), and anti-tubulin (10G10, Wako) monoclonal antibodies. Immunodetection was performed using a chemiluminescence system (Nacalai Tesque). The relative amount of protein was determined by measuring the intensity of each band using ImageJ software (http://rsbweb.nih.gov/ij/). Quantitative real-time PCR Complementary DNA (cDNA) was synthesized using the SuperScript TM IV VILO TM Master Mix with the ezDNase TM Enzyme Kit (Thermo Fisher Scientific) from 1 µg of RNA extracted by the hot phenol method. Quantitative PCR (qPCR) was performed using the QuantStudio ® 3 Real-Time PCR System (Thermo Fisher Scientific), with the cDNA as a template, gene-specific primers for each DNA target (Supplementary Table S2), and Power SYBR ® Green Master Mix (Thermo Fisher Scientific). The relative mRNA levels were quantified using the comparative Ct method (∆∆Ct method), with ACT1 serving as an internal control. In vivo protein synthesis assay ­ The labeling of cells and proteins with [ 14 C]valine and subsequent measurements of radioactivity were performed as described previously, with slight modification [14]. Briefly, [U- 14 C]valine (GE Healthcare) was added at a final concentration of 10 µM (74 kBq/ml) to cells cultured in SD-N medium and incubated at 30°C for 0, 2, and 4 min. At each time point, the reactions were stopped by adding 10 volumes of 11% (w/v) trichloroacetic acid (TCA) (for estimating [ 14 C]valine incorporation into proteins: A) or ice-cold distilled water (for estimating cellular [ 14 C]valine uptake: B). The TCA suspensions were incubated at 90°C for 10 min and cooled to 4°C. The precipitated proteins were trapped onto a 0.45 µm cellulose acetate membrane filter (Advantec, Tokyo, Japan). Cell suspensions were immediately collected using the filter. Radioactivity incorporated into the protein or whole cell was measured using a liquid scintillation counter. The ratio of [U- 14 C]valine incorporation into the proteins was calculated by the equation A/B. Determination of cell viability The cells were stained with 2 µg/ml Phloxine B and analyzed by fluorescence microscopy. Cells with bright fluorescence were counted as dead cells. Approximately 200 cells were counted in each experiment. Sporulation experiment For sporulation, diploid cells were suspended in sporulation medium (0.1% yeast extract, 0.05% glucose, 1% potassium acetate) and incubated at 27°C with vigorous shaking for 6 days. The number of packaged spores per ascus was counted for at least 200 asci under a microscope with changing z-axis position. Results and discussion Avt6 is involved in the export of neutral amino acids from vacuoles In the previous report, the vacuolar amino acid contents were apparently increased in avt3 ∆ avt4 ∆ cells [7]. Disruption of AVT7 in avt3 ∆ avt4 ∆ cells further increased their contents [8]. To assess the effect of multiple disruption of vacuolar amino acid exporters, we replaced the AVT6 ORF in the avt3 ∆ avt4 ∆ avt7 ∆ strain with the loxP-kanMX-loxP sequence to isolate the avt3 ∆ avt4 ∆ avt6∆avt7 ∆ strain. We compared the vacuolar amino acid content between the wild-type, avt6 ∆, avt3 ∆ avt4 ∆ avt7 ∆, and avt3 ∆ avt4 ∆ avt6 ∆ avt7 ∆ cells under nutrient-rich and nitrogen-starved conditions (Fig. 1A and B). avt6 ∆ cells under both conditions did not show any significant change in vacuolar amino acid content, except for a slight increase in aspartate or glutamate compared with the wild-type cells (Fig. 1A and B). Consistent with a previous report [8], the level of the various neutral amino acids in avt3 ∆ avt4 ∆ avt7 ∆ cells was greater compared with those in wild-type cells under both conditions (Fig. 1A and B). Under nitrogen-starved condition, the amount of vacuolar basic amino acids in the avt3 ∆ avt4 ∆ avt7 ∆ cells was also greater compared with that in the wild-type cells because of the lack of Avt4, whose activity is suggested to be elevated in this condition by transcriptional induction, and possibly by post-translational regulation (Fig. 1B) [12]. Moreover, by further disrupting AVT6 in avt3 ∆ avt4 ∆ avt7 ∆ cells, the amount of threonine, serine, and alanine, as well as aspartate and glutamate, was markedly increased under nutrient-rich condition (Fig. 1A). During nitrogen starvation, the effect on the vacuolar amino acid content became more evident. In addition to the above amino acids, higher amounts of glutamine, glycine, valine, and methionine were retained compared with avt3 ∆ avt4 ∆ avt7 ∆ cells (Fig. 1B). These results suggest that Avt6 is involved in the export of neutral amino acids as well as acidic ones. Under nitrogen starvation, the induction of autophagy may result in an acute and transient increase of vacuolar neutral amino acids. To efficiently recycle them, Avt6 may act in concert with Avt3, Avt4, and Avt7. Next, a vector to overexpress AVT6 , pGPD-AVT6, or an empty vector, p416GPD [30], was introduced into avt1 ∆ avt3 ∆ avt4 ∆ avt6 ∆ avt7 ∆ cells. AVT1 , which encodes a vacuolar importer of neutral amino acids [6, 32], was further deleted to assess the vacuolar amino acid export more sensitively. The AVT6 overexpression resulted in the reduction of vacuolar neutral amino acids (Fig. 1C), further supporting our notion that Avt6 is involved in the export of neutral amino acids from vacuoles. We also tested the overexpression of Atg22 to address its ability to export amino acids (Fig. 1C). The reduction of vacuolar amino acids was small, though significant for several amino acids, in cells overexpressing Atg22. Therefore, we speculate that the contribution of Atg22 to vacuolar amino acid export is rather minor compared with the Avt transporters. This is also supported by further deletion of ATG22 in avt3 ∆ avt4 ∆ avt7 ∆ cells, which did not largely elevate the vacuolar amino acid levels in both nutrient-rich and nitrogen-starved conditions (Supplementary Fig. S1A and B online). Vacuolar amino acid recycling supports protein synthesis under nitrogen starvation It has been suggested that amino acids recycled from vacuoles are used for protein synthesis under nitrogen starvation [14,15]. To address this, the level of HA 3 -tagged Arg1, an argininosuccinate synthetase in the arginine biosynthesis pathway, whose cellular level increases under nitrogen starvation in a manner dependent on autophagy [14], in avt3 ∆ avt4 ∆ avt6 ∆ avt7∆ cells (hereafter refer to avt3467 ∆ cells) was compared with that in wild-type and atg9 ∆ cells. As reported, the Arg1-HA 3 level increased under nitrogen starvation in the wild-type, but not in atg9 ∆ cells (Fig. 2A). As atg9 ∆ cells, avt3467∆ cells did not increase Arg1-HA 3 level under prolonged nitrogen starvation (Fig. 2A and B). The mRNA level of ARG1-HA 3 in avt3467∆ cells did not significantly differ from that in wild-type cells after culturing in SD-N for 24 hours, whereas it was markedly increased in atg9 ∆ cells (Fig. 2C). Therefore, the translation of ARG1-HA 3 mRNA may be reduced in avt3467∆ cells compared with wild-type cells, although the reduction may be partial compared with that in atg9 ∆ cells. We also assessed protein synthesis by estimating the amount of [ 14 C]valine incorporated into the proteins (Fig. 2D). We measured the cellular uptake and protein incorporation of [ 14 C]valine. After 3 hours of nitrogen starvation, [ 14 C]valine uptake by avt3467 ∆ cells was moderately less compared with that by wild-type cells (Fig. 2D left). During this time, [ 14 C]valine incorporation into proteins in avt3467 ∆ cells was considerably lower than that in wild-type cells (Fig. 2D middle). After 6 hours of nitrogen starvation, however, it was not significantly lower because [ 14 C]valine incorporation into proteins was further reduced in wild-type cells, but not in avt3467 ∆ cells (Fig. 2D middle). By accounting for the uptake of extracellular [ 14 C]valine (Fig. 2D left), the ratio of [ 14 C]valine incorporation into the proteins was calculated (Fig. 2D right). As in atg9 ∆ cells, the incorporation ratio was significantly lower in the avt3467 ∆ cells compared with that in wild-type cells after 3 hours of nitrogen starvation. Also after 6 hours, it tended to be lower in the avt3467 ∆ cells as well as in atg9 ∆ cells (Fig. 2D right). Although it is difficult to precisely estimate the cytosolic pool of amino acids, assuming that the amount of valine in the cytosol of avt3467 ∆ cells is at least the same or less than that of wild-type cells, these results suggest that protein synthesis in avt3467 ∆ cells under nitrogen starvation is lower compared with that in wild-type cells. Viability under nitrogen starvation is reduced by disrupting leucine biosynthesis in avt3467 ∆ cells. The recycling of amino acids generated by autophagic protein degradation has been suggested to be critical for survival by maintaining protein synthesis under nitrogen starvation [14,15]. We determined whether avt3467 ∆ cells show reduced viability under nitrogen starvation. After 3 days of culture in SD-N, atg9 ∆ cells exhibited reduced viability compared with wild-type cells (Fig. 3A). In contrast, avt3467 ∆ cells maintained their viability as wild-type cells, even after prolonged starvation (Fig. 3A; see after 19 days in SD-N). Amino acids from vacuoles are catabolized to produce glutamate and aspartate, which are then converted into other amino acids for the efficient use of limited nitrogen [33]. Therefore, the viability of avt3467 ∆ cells may be maintained by the de novo synthesis of neutral amino acids from basic and acidic amino acids exported by other vacuolar amino acid transporters. To address this, we evaluated the deletion of other genes, LEU2 , ARO8 / ARO9 , or THR4 , in the biosynthesis of either leucine, aromatic amino acids, or threonine, respectively, in the avt3467 ∆ strain. After culturing for 7 days in SD-N, the viability of avt3467 ∆ leu2 ∆ cells was significantly decreased compared with leu2 ∆ cells (Fig. 3B and Supplementary Fig. S2 online), which suggests that amino acid recycling from vacuoles contributes to the maintenance of viability. Conversely, avt3467 ∆ aro8 ∆ aro9 ∆ and avt3467 ∆ thr4 ∆ survived as aro8 ∆ aro9 ∆ and thr4 ∆ cells, respectively (Fig. 3B). Therefore, the degree of the defect in recycling from the vacuoles may vary depending on amino acid species. It is also possible that the unknown transporter(s) may export neutral amino acids. In support of this, vacuolar neutral amino acids were still decreased upon shifting to nitrogen starvation in avt3467 ∆ cells (Fig 1A and B). In addition, there may be a threshold amount of amino acids that determines the yield of the translation product and the cell fate as its consequence. It should be noted that the increase in the vacuolar leucine content in avt3467 ∆ cells was not remarkable compared with the contents of the other neutral amino acids (Fig. 1A and B). Some intermediate metabolites resulting from LEU2 deletion may cause the loss of viability of avt3467∆ cells. We also cannot exclude the possibility that the recycling of degradation products other than amino acids, such as nucleic acids, metal ions, phosphates, lipids, and/or sugars, may contribute to cell viability under nitrogen starvation. Vacuolar amino acid recycling is required for efficient spore formation Yeast diploid cells sporulate in a manner dependent on autophagy to survive nutrient-harsh conditions [13]. Therefore, we hypothesized that sporulation would be impaired in avt3467∆ diploid cells. After 6 days of nitrogen starvation with acetate as a carbon source, approximately half of the ascus from the wild-type diploids contained four spores (tetrads), whereas atg9 ∆ diploids did not form any spores (Fig. 4A). In avt3467∆ diploid cells, the ascus containing four spores was decreased, whereas those containing two or one spore (dyads or monads, respectively) was increased (Fig. 4A). The spore number was restored by introducing plasmids to express AVT3 , AVT4 , and AVT6 (Supplementary Fig. S3A online). The spore number per ascus decreases when carbon sources are limited, which is regulated by the induction of the structural meiotic spindle pole body components, Mpc54, Mpc70, and Spo74 [34]. We examined the induction of Myc 9 -tagged Mpc54 in diploid cells under sporulation condition. Myc 9 -Mpc54 was transiently increased in wild-type cells, but was nearly undetectable in atg9 ∆ cells (Fig. 4B). In avt3467 ∆ diploid cells, the increase in Myc 9 -Mpc54 levels was lower compared with that in wild-type cells (Fig. 4B). This is consistent with the observed reduction in the spore number. Introducing the plasmids to express AVT3 , AVT4, and AVT6 restored the induction of Myc 9 -Mpc54 (Supplementary Fig. S3B online). These suggest that the spore number also appears to be determined by the amount of amino acids recycled from the vacuoles. Upon shifting to sporulation condition, diploid cells initially induce the master transcription factor, Ime1, which is followed by the induction of Ndt80, a transcription factor acting at the middle phase of sporulation, which binds the MPC54 promoter as determined by high-throughput ChIP sequencing [35]. HA 6 -tagged Ime1 was normally induced in atg9 ∆ and avt3467 ∆ cells, similar to the wild-type cells (Fig. 4C). In contrast, Ndt80-HA 6 was almost not detectable in atg9 ∆ cells and partially reduced in avt3467∆ diploid cells (Fig. 4C). Therefore, the expression of sporulation-related factors may be attenuated during the course of sporulation in avt3467∆ diploid cells. We speculated that the shortage of amino acids to support protein synthesis may reduce the production of sporulation-related factors. Interestingly, atg9 ∆ cells induced Ime1 as wild-type cells. Therefore, autophagy may not be required for the onset of sporulation, but play a role in completing the process. Our results suggest that the amino acids recycled from vacuoles are required for carrying out the sporulation process after the trigger event. The significance of amino acid recycling in coping with starvation should be further explored in future studies. The effect of further disrupting genes encoding other vacuolar transporters involved in exporting amino acids and/or the other degradation products should be examined. Identifying such transporters essential for the survival and/or sporulation in avt3467 ∆ cells will give us a great advance for understanding the physiology of autophagy. Abbreviations AVT, amino acid vacuolar transport; HA, hemagglutinin Declarations Data availability All data generated or analyzed during this study are included in this published article (and its Supplementary Information files). Acknowledgements We thank the Advanced Research Support Center (ADRES) in Ehime University for determining amino acid contents. Funding This work was supported by Grants-in-Aid for Scientific Research (C) from JSPS (grant numbers 21K05383 and 25K09043 to M.K., 21K05507 and 24K08834 to T.S.) and Grant-in-Aid for Scientific Research on Innovative Areas from JSPS (grant number 22H04650 to T.S.), and funding for Women Scientists provided from Japan Society for Bioscience, Biotechnology, and Agrochemistry (JSBBA) (to M.K.). Author contributions M.K. and T.S. conceived and designed experiments; H.N. and T.S. performed experiments, analyzed data, and prepared figures; , R.O., S.N., N.H., Y.Y., W.Y., N.O., T.K., S.A., N.S., and K.A. performed experiments; H.N., M.K., and T.S. wrote the manuscript. Competing interests The authors declare no competing interests. Supplementary information The online version contains supplementary materials. References Klionsky, D. J. Autophagy: from phenomenology to molecular understanding in less than a decade. Nat . Rev . Mol . Cell Biol . 8, 931-937 (2007). Ohsumi, Y. Historical landmarks of autophagy research. Cell Res . 24, 9-23 (2014). Parzych, K. R. & Klionsky, D. J. Vacuolar hydrolysis and efflux: current knowledge and unanswered questions. Autophagy 15, 212-227 (2019). Mizushima, N. & Levine, B. Autophagy in mammalian development and differentiation. Nat . Cell Biol . 12, 823-830 (2010). Galluzzi, L. et al. Autophagy in malignant transformation and cancer progression. 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08:41:54","extension":"html","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":121702,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8111730/v1/2dd8322d630faf0f262140ad.html"},{"id":96977072,"identity":"37c2770e-47d1-4edb-9d07-c3b8f64f799d","added_by":"auto","created_at":"2025-11-28 08:41:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":141186,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChange in the vacuolar amino acid content by deletion or overexpression of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAVT6 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egene.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e and \u003cstrong\u003eB\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eWild-type (WT), \u003cem\u003eavt6\u003c/em\u003e∆, \u003cem\u003eavt3\u003c/em\u003e∆\u003cem\u003eavt4\u003c/em\u003e∆\u003cem\u003eavt7\u003c/em\u003e∆ (\u003cem\u003eavt347\u003c/em\u003e∆), or \u003cem\u003eavt3\u003c/em\u003e∆\u003cem\u003eavt4\u003c/em\u003e∆\u003cem\u003eavt6\u003c/em\u003e∆\u003cem\u003eavt7\u003c/em\u003e∆ (\u003cem\u003eavt3467\u003c/em\u003e∆) cells grown in SD+Cas (A) or cultured in SD-N (B) were treated with CuCl\u003csub\u003e2\u003c/sub\u003e as described in “Materials and methods” to extract vacuolar amino acids. (\u003cstrong\u003eC\u003c/strong\u003e) \u003cem\u003eavt1\u003c/em\u003e∆\u003cem\u003eavt3\u003c/em\u003e∆\u003cem\u003eavt4\u003c/em\u003e∆\u003cem\u003eavt6\u003c/em\u003e∆\u003cem\u003eavt7\u003c/em\u003e∆ cells carrying an empty vector (E.V.), overexpressing \u003cem\u003eAVT6\u003c/em\u003e (\u003cem\u003eAVT6 \u003c/em\u003eO.E.), or overexpressing \u003cem\u003eATG22\u003c/em\u003e (\u003cem\u003eATG22 \u003c/em\u003eO.E.) were cultured in SD+Cas, and the vacuolar amino acids were extracted as in (A). The level of amino acids was measured with an amino acid analyzer. The results are means ± standard deviation (SD) of three independent experiments. A Student’s \u003cem\u003et\u003c/em\u003e-test between \u003cem\u003eavt3\u003c/em\u003e∆\u003cem\u003eavt4\u003c/em\u003e∆\u003cem\u003eavt7\u003c/em\u003e∆ and \u003cem\u003eavt3\u003c/em\u003e∆\u003cem\u003eavt4\u003c/em\u003e∆\u003cem\u003eavt6\u003c/em\u003e∆\u003cem\u003eavt7\u003c/em\u003e∆ cells (A and B), or between cells with an E.V. and \u003cem\u003eAVT6 \u003c/em\u003eor \u003cem\u003eATG22 \u003c/em\u003eoverexpression (C), was used for statistical analysis. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, Student’s \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8111730/v1/6029a102d6649a4d9b291a6d.png"},{"id":96977073,"identity":"1c466dce-2154-4ddb-8fe8-812019205f88","added_by":"auto","created_at":"2025-11-28 08:41:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":209728,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of vacuolar amino acid recycling on protein synthesis. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Wild-type, \u003cem\u003eatg9\u003c/em\u003e∆, and \u003cem\u003eavt3467\u003c/em\u003e∆ cells expressing HA\u003csup\u003e3\u003c/sup\u003e-tagged Arg1 were cultured in SD-N for the indicated hours, and lysates were prepared. Arg1-HA\u003csup\u003e3\u003c/sup\u003e was detected by western blot analysis using anti-HA antibody. Tubulin was detected as a loading control. (\u003cstrong\u003eB\u003c/strong\u003e) The relative intensities of the Arg1-HA\u003csup\u003e3\u003c/sup\u003e bands in the wild-type cells and those in \u003cem\u003eatg9∆ \u003c/em\u003eor \u003cem\u003eavt3467\u003c/em\u003e∆ cells at the indicated time points were plotted. Data are means ± SD of four independent experiments. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, Student’s \u003cem\u003et\u003c/em\u003e-test. (\u003cstrong\u003eC\u003c/strong\u003e) Total RNA extracted from wild-type, \u003cem\u003eatg9\u003c/em\u003e∆, and \u003cem\u003eavt3467\u003c/em\u003e∆ cells cultured in SD-N for 24 h was subjected to qPCR analysis using \u003cem\u003eARG1\u003c/em\u003e-specific primers. \u003cem\u003eARG1\u003c/em\u003e mRNA values were normalized to actin (\u003cem\u003eACT1\u003c/em\u003e) mRNA and plotted relative to that of wild-type cells. Data were means ± SD of three independent experiments. **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, Student’s \u003cem\u003et\u003c/em\u003e-test. (D) Wild-type, \u003cem\u003eatg9\u003c/em\u003e∆, and \u003cem\u003eavt3467\u003c/em\u003e∆ cells were cultured in SD-N for 3 and 6 h. [\u003csup\u003e14\u003c/sup\u003eC]valine was added to the culture, and the uptake of [\u003csup\u003e14\u003c/sup\u003eC]valine into the cells (left) and the protein incorporation of [\u003csup\u003e14\u003c/sup\u003eC]valine (middle) were measured. The valine incorporation ratio into the proteins (right) was calculated as described in “Materials and methods”. Data are means ± SD of three independent experiments. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, Student’s \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8111730/v1/cbaf15817e0de2fedcee9602.png"},{"id":96977100,"identity":"706d60c5-b1ba-43d2-9323-a422b902bebb","added_by":"auto","created_at":"2025-11-28 08:41:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":130374,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eViability under nitrogen starvation. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Wild-type (WT), \u003cem\u003eatg9\u003c/em\u003e∆ and \u003cem\u003eavt3467∆ \u003c/em\u003ecells were cultured in SD-N for the indicated days. Dead cells stained with Phloxine B were counted under a microscope. The data represent the average of three independent experiments and the bars indicate SD. (\u003cstrong\u003eB\u003c/strong\u003e) \u003cem\u003eavt3467∆ \u003c/em\u003ecells blocked the biosynthesis of leucine, aromatic amino acids, or threonine by deleting \u003cem\u003eLEU2\u003c/em\u003e, \u003cem\u003eARO8\u003c/em\u003e/\u003cem\u003eARO9\u003c/em\u003e, or \u003cem\u003eTHR4\u003c/em\u003e, respectively, were cultured in SD-N for 7 days, and their viabilities were examined as in (A). Data are means ± SD of three independent experiments. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, Student’s \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8111730/v1/22cb6702bc016d0c4d221e28.png"},{"id":97138442,"identity":"70408742-7e69-4c87-915e-d8cf030d0cd7","added_by":"auto","created_at":"2025-12-01 09:58:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":490056,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInvolvement of vacuolar amino acid export in sporulation. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Wild-type (WT), \u003cem\u003eatg9\u003c/em\u003e∆, and \u003cem\u003eavt3467\u003c/em\u003e∆ diploid cells were cultured in sporulation medium for 6 days. The number of spores in the ascus was counted under the microscope. Data are means ± SD of three independent experiments. (\u003cstrong\u003eB\u003c/strong\u003e) Left: Lysates from wild-type (WT), \u003cem\u003eatg9\u003c/em\u003e∆, and \u003cem\u003eavt3467\u003c/em\u003e∆ diploid cells were prepared after culture in sporulation medium for the indicated times. Mpc54-Myc\u003csup\u003e9\u003c/sup\u003e in the lysates was detected by western blot analysis using anti-Myc antibody. Tubulin was detected as a loading control. The asterisk indicates a nonspecific band. Experiments were performed in three independent replicates with essentially identical results, and representative results are shown. Right: After 24 h in sporulation medium, the relative intensities of the Mpc54-Myc\u003csup\u003e9\u003c/sup\u003e band normalized by that of tubulin are shown as the relative ratio to the control (WT). Data are means ± SD of three independent experiments. \u0026nbsp;(*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. control, Student’s \u003cem\u003et\u003c/em\u003e-test).\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eC\u003c/strong\u003e) Ime1-HA\u003csup\u003e6 \u003c/sup\u003e(top) and Ndt80-HA\u003csup\u003e6\u003c/sup\u003e (bottom) were detected by western blot analysis and analyzed as in (B).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8111730/v1/427552d8aa8d1bbbd28d7ed6.png"},{"id":104250765,"identity":"b8c30edc-4449-4e9f-ba0e-53513cfaf120","added_by":"auto","created_at":"2026-03-09 16:08:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1866947,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8111730/v1/05c73183-e59a-49c4-bd10-e520e55f9a24.pdf"},{"id":96977074,"identity":"969d4bed-a479-4545-bfee-7ce33f216044","added_by":"auto","created_at":"2025-11-28 08:41:54","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":3455197,"visible":true,"origin":"","legend":"","description":"","filename":"SupportinginformationSciRep.docx","url":"https://assets-eu.researchsquare.com/files/rs-8111730/v1/e7b8f12ab3c238d801b00231.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Physiological significance of amino acid recycling from vacuoles under starvation conditions","fulltext":[{"header":"Introduction","content":"\u003cp\u003eUpon shifting to nitrogen starvation, cells induce macroautophagy (hereafter autophagy) to degrade the bulk of proteins in the vacuoles/lysosomes [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. It has been suggested that the amino acids generated inside the vacuoles/lysosomes are recycled for protein synthesis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Thus, they must be exported from the vacuoles/lysosomes to the cytosol, where translation occurs. In higher eukaryotes, autophagic amino acid recycling has been suggested to be involved in not only development and differentiation [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] but also various pathophysiological roles, such as tumor progression [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In the budding yeast \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e, several vacuolar amino acid exporters have been identified. Of these, Avt3, Avt4, Avt7, Atg22, and Ypq2 are involved in the export of neutral amino acids from vacuoles in a redundant manner [\u003cspan additionalcitationids=\"CR7 CR8 CR9\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Avt4 also exports basic amino acids [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Avt6 exports acidic amino acids [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Avt3 and Avt4 participate in autophagic amino acid recycling, because the contents of vacuolar neutral and basic amino acids in \u003cem\u003eavt3\u003c/em\u003e∆\u003cem\u003eavt4\u003c/em\u003e∆ cells are much higher compared with those in wild-type cells under nitrogen starvation [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The contribution of Avt7 to amino acid recycling is relatively minor, but significant for several amino acid species, such as glutamine and proline [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. \u003cem\u003eAVT6\u003c/em\u003e mRNA was increased under nitrogen-starved condition [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Moreover, the vacuolar glutamate and aspartate content in \u003cem\u003eavt6\u003c/em\u003e∆ cells was higher compared with that in wild-type cells under this condition [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Therefore, Avt6 also likely acts on the recycling of the vacuolar acidic amino acids generated by autophagy. Atg22 was reported to be involved in the export of tyrosine, isoleucine and leucine [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], and Ypq2 has been suggested to act as a vacuolar uniporter for arginine [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSince mutants that are defective in autophagy fail to survive during nitrogen starvation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], it has been suggested that the recycling of amino acids generated by autophagy maintains protein synthesis to promote cell survival [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. However, autophagy also degrades bulk ribosomes and various organelles, such as mitochondria and endoplasmic reticulum [\u003cspan additionalcitationids=\"CR17 CR18 CR19 CR20\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. As a result, their degradation products, including phosphate, zinc, iron, nucleotides, lipids, and carbohydrates, are also likely to be exported from the vacuoles for recycling [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In addition, autophagy removes dysfunctional organelles and proteins to avoid the production of harmful products, such as reactive oxygen species, and the unwanted accumulation of proteins [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. These suggest that the recycling of various materials other than amino acids and the quality control of organelles and proteins may also contribute to cell viability during nitrogen starvation. Therefore, to address the physiological importance of autophagic amino acid recycling, inhibiting vacuolar amino acid exporter gene function and examining the effect on cell viability will provide great insight.\u003c/p\u003e\u003cp\u003eIn this study, we determined the effect of \u003cem\u003eavt3\u003c/em\u003e∆\u003cem\u003eavt4\u003c/em\u003e∆\u003cem\u003eavt6\u003c/em\u003e∆\u003cem\u003eavt7\u003c/em\u003e∆ quadruple disruption on vacuolar amino acid content and examined the resulting phenotype during nitrogen starvation. Our results suggest that Avt6 exports neutral amino acids in addition to acidic ones in a redundant manner with Avt3, Avt4, and Avt7. Moreover, the recycling of vacuolar neutral amino acids likely contributes to protein synthesis under nitrogen starvation and the efficiency of spore formation. \u003cem\u003eavt3\u003c/em\u003e∆\u003cem\u003eavt4\u003c/em\u003e∆\u003cem\u003eavt6\u003c/em\u003e∆\u003cem\u003eavt7\u003c/em\u003e∆ cells partially recapitulate the phenotype of the autophagy-deficient mutant.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eStrains and media\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eS. cerevisiae\u0026nbsp;\u003c/em\u003estrains used in this study are listed in Supplementary Table S1. Genes were deleted using a PCR-based method with either the \u003cem\u003ekanMX\u003c/em\u003e, \u003cem\u003enatMX\u003c/em\u003e, or \u003cem\u003ehphMX\u0026nbsp;\u003c/em\u003ecassette [26, 27]. For multiple gene disruption, in addition to using multiple markers, the \u003cem\u003ekanMX\u003c/em\u003e marker was recycled using the Cre-loxP system [28]. For chromosomal tagging with HA\u003csup\u003e3\u003c/sup\u003e, HA\u003csup\u003e6\u003c/sup\u003e, or Myc\u003csup\u003e9\u003c/sup\u003e, PCR products amplified from the pYM-series plasmid [29] were introduced into the parent strains.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eS. cerevisiae\u003c/em\u003e cells were grown at 30\u0026deg;C to early logarithmic phase in YPD (1% yeast extract, 2% bacto-peptone, and 2% glucose) or SD+Cas (0.17% yeast nitrogen base w/o amino acids and ammonium sulfate, 0.5% casamino acids, 0.5% ammonium sulfate, 20 mg/L tryptophan, and 2% glucose) medium to collect cells grown under nutrient-rich conditions. For nitrogen starvation, cells grown in nutrient-rich conditions were shifted to SD-N (0.17% yeast nitrogen base w/o amino acids and ammonium sulfate and 2% glucose) and further cultured at 30\u0026deg;C.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlasmids\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrimers used to construct plasmids in this study were listed in Supplementary Table S2. pAVT6 was constructed as described previously [11]. To construct pGPD-AVT6, the\u003cem\u003e\u0026nbsp;AVT6\u0026nbsp;\u003c/em\u003eORF was amplified by PCR using a pair of primers, \u003cem\u003eAVT6\u003c/em\u003e ORF F \u003cem\u003eBam\u003c/em\u003eHI and \u003cem\u003eAVT6\u003c/em\u003e ORF R \u003cem\u003eSal\u003c/em\u003eI, and ligated into the p416GPD plasmid [30] following restriction enzyme digestion. To construct pGPD-ATG22, the \u003cem\u003eATG22\u0026nbsp;\u003c/em\u003eORF amplified by PCR using a pair of primers, \u003cem\u003eATG22\u0026nbsp;\u003c/em\u003eORF F \u003cem\u003eBam\u003c/em\u003eHI and \u003cem\u003eATG22\u0026nbsp;\u003c/em\u003eORF R \u003cem\u003eHin\u003c/em\u003edIII, was inserted into the p416GPD. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAmino acid analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo prepare a vacuolar fraction, the cupric ion treatment method was used [31]. Briefly, 10 OD\u003csub\u003e660\u003c/sub\u003e units of cells were washed twice with an amino acid extraction (AAE) buffer (2.5 mM potassium phosphate buffer, pH 6.0, 0.6 M sorbitol, and 10 mM glucose) and incubated in AAE buffer containing 0.2 mM CuCl\u003csub\u003e2\u003c/sub\u003e at 30\u0026deg;C for 15 or 30 min for cells cultured in nutrient-rich or nitrogen-starved condition, respectively. After washing with AAE buffer, the cell pellets were resuspended in 500 \u0026micro;L of distilled water and boiled for 15 min. After centrifugation, the amino acid content in the supernatants was measured using an automatic amino acid analyzer (Hitachi L-8900).\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell lysates (0.3 OD\u003csub\u003e660\u003c/sub\u003e units of cells) were analyzed using anti-HA (3F10, Roche), anti-Myc (9E10, Roche), and anti-tubulin (10G10, Wako) monoclonal antibodies. Immunodetection was performed using a chemiluminescence system (Nacalai Tesque). The relative amount of protein was determined by measuring the intensity of each band using ImageJ software (http://rsbweb.nih.gov/ij/).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative real-time PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eComplementary DNA (cDNA) was synthesized using the SuperScript\u003csup\u003eTM\u0026nbsp;\u003c/sup\u003eIV VILO\u003csup\u003eTM\u003c/sup\u003e Master Mix with the ezDNase\u003csup\u003eTM\u003c/sup\u003e Enzyme Kit (Thermo Fisher Scientific) from 1 \u0026micro;g of RNA extracted by the hot phenol method. Quantitative PCR (qPCR) was performed using the QuantStudio\u003csup\u003e\u0026reg;\u003c/sup\u003e3 Real-Time PCR System (Thermo Fisher Scientific), with the cDNA as a template, gene-specific primers for each DNA target (Supplementary Table S2), and Power SYBR\u003csup\u003e\u0026reg;\u0026nbsp;\u003c/sup\u003eGreen Master Mix (Thermo Fisher Scientific). The relative mRNA levels were quantified using the comparative Ct method (∆∆Ct method), with \u003cem\u003eACT1\u003c/em\u003e serving as an internal control.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vivo\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;protein synthesis assay \u0026shy;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe labeling of cells and proteins with [\u003csup\u003e14\u003c/sup\u003eC]valine and subsequent measurements of radioactivity were performed as described previously, with slight modification [14]. Briefly, [U-\u003csup\u003e14\u003c/sup\u003eC]valine (GE Healthcare) was added at a final concentration of 10 \u0026micro;M (74 kBq/ml) to cells cultured in SD-N medium and incubated at 30\u0026deg;C for 0, 2, and 4 min. At each time point, the reactions were stopped by adding 10 volumes of 11% (w/v) trichloroacetic acid (TCA) (for estimating [\u003csup\u003e14\u003c/sup\u003eC]valine incorporation into proteins: A) or ice-cold distilled water (for estimating cellular [\u003csup\u003e14\u003c/sup\u003eC]valine uptake: B). The TCA suspensions were incubated at 90\u0026deg;C for 10 min and cooled to 4\u0026deg;C. The precipitated proteins were trapped onto a 0.45 \u0026micro;m cellulose acetate membrane filter (Advantec, Tokyo, Japan). Cell suspensions were immediately collected using the filter. Radioactivity incorporated into the protein or whole cell was measured using a liquid scintillation counter.\u0026nbsp;The ratio of [U-\u003csup\u003e14\u003c/sup\u003eC]valine incorporation into the proteins was calculated by the equation A/B.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of cell viability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cells were stained with 2 \u0026micro;g/ml Phloxine B and analyzed by fluorescence microscopy. Cells with bright fluorescence were counted as dead cells. Approximately 200 cells were counted in each experiment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSporulation experiment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor sporulation, diploid cells were suspended in sporulation medium (0.1% yeast extract, 0.05% glucose, 1% potassium acetate) and incubated at 27\u0026deg;C with vigorous shaking for 6 days. The number of packaged spores per ascus was counted for at least 200 asci under a microscope with changing z-axis position.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cp\u003e\u003cstrong\u003eAvt6 is involved in the export of neutral amino acids from vacuoles\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the previous report, the vacuolar amino acid contents were apparently increased in \u003cem\u003eavt3\u003c/em\u003e∆\u003cem\u003eavt4\u003c/em\u003e∆ cells [7]. Disruption of \u003cem\u003eAVT7\u0026nbsp;\u003c/em\u003ein \u003cem\u003eavt3\u003c/em\u003e∆\u003cem\u003eavt4\u003c/em\u003e∆ cells further increased their contents [8]. To assess the effect of multiple disruption of vacuolar amino acid exporters, we replaced the \u003cem\u003eAVT6\u003c/em\u003e ORF in the \u003cem\u003eavt3\u003c/em\u003e∆\u003cem\u003eavt4\u003c/em\u003e∆\u003cem\u003eavt7\u003c/em\u003e∆ strain with the \u003cem\u003eloxP-kanMX-loxP\u0026nbsp;\u003c/em\u003esequence to isolate the \u003cem\u003eavt3\u003c/em\u003e∆\u003cem\u003eavt4\u003c/em\u003e∆\u003cem\u003eavt6∆avt7\u003c/em\u003e∆\u003cem\u003e\u0026nbsp;\u003c/em\u003estrain. We compared the vacuolar amino acid content between the wild-type, \u003cem\u003eavt6\u003c/em\u003e∆, \u003cem\u003eavt3\u003c/em\u003e∆\u003cem\u003eavt4\u003c/em\u003e∆\u003cem\u003eavt7\u003c/em\u003e∆, and \u003cem\u003eavt3\u003c/em\u003e∆\u003cem\u003eavt4\u003c/em\u003e∆\u003cem\u003eavt6\u003c/em\u003e∆\u003cem\u003eavt7\u003c/em\u003e∆ cells under nutrient-rich and nitrogen-starved conditions (Fig. 1A and B).\u003cem\u003e\u0026nbsp;avt6\u003c/em\u003e∆ cells under both conditions did not show any significant change in vacuolar amino acid content, except for a slight increase in aspartate or glutamate compared with the wild-type cells (Fig. 1A and B). Consistent with a previous report [8], the level of the various neutral amino acids in \u003cem\u003eavt3\u003c/em\u003e∆\u003cem\u003eavt4\u003c/em\u003e∆\u003cem\u003eavt7\u003c/em\u003e∆ cells was greater compared with those in wild-type cells under both conditions (Fig. 1A and B). Under nitrogen-starved condition, the amount of vacuolar basic amino acids in the \u003cem\u003eavt3\u003c/em\u003e∆\u003cem\u003eavt4\u003c/em\u003e∆\u003cem\u003eavt7\u003c/em\u003e∆ cells was also greater compared with that in the wild-type cells because of the lack of Avt4, whose activity is suggested to be elevated in this condition by transcriptional induction, and possibly by post-translational regulation (Fig. 1B) [12]. Moreover, by further disrupting \u003cem\u003eAVT6\u0026nbsp;\u003c/em\u003ein \u003cem\u003eavt3\u003c/em\u003e∆\u003cem\u003eavt4\u003c/em\u003e∆\u003cem\u003eavt7\u003c/em\u003e∆ cells, the amount of threonine, serine, and alanine, as well as aspartate and glutamate, was markedly increased under nutrient-rich condition (Fig. 1A). During nitrogen starvation, the effect on the vacuolar amino acid content became more evident. In addition to the above amino acids, higher amounts of glutamine, glycine, valine, and methionine were retained compared with \u003cem\u003eavt3\u003c/em\u003e∆\u003cem\u003eavt4\u003c/em\u003e∆\u003cem\u003eavt7\u003c/em\u003e∆ cells (Fig. 1B). These results suggest that Avt6 is involved in the export of neutral amino acids as well as acidic ones. Under nitrogen starvation, the induction of autophagy may result in an acute and transient increase of vacuolar neutral amino acids. To efficiently recycle them, Avt6 may act in concert with Avt3, Avt4, and Avt7. Next, a vector to overexpress \u003cem\u003eAVT6\u003c/em\u003e, pGPD-AVT6, or an empty vector, p416GPD [30], was introduced into \u003cem\u003eavt1\u003c/em\u003e∆\u003cem\u003eavt3\u003c/em\u003e∆\u003cem\u003eavt4\u003c/em\u003e∆\u003cem\u003eavt6\u003c/em\u003e∆\u003cem\u003eavt7\u003c/em\u003e∆ cells.\u003cem\u003e\u0026nbsp;AVT1\u003c/em\u003e, which encodes a vacuolar importer of neutral amino acids [6, 32], was further deleted to assess the vacuolar amino acid export more sensitively. The \u003cem\u003eAVT6\u003c/em\u003e overexpression resulted in the reduction of vacuolar neutral amino acids (Fig. 1C), further supporting our notion that Avt6 is involved in the export of neutral amino acids from vacuoles. We also tested the overexpression of Atg22 to address its ability to export amino acids (Fig. 1C). The reduction of vacuolar amino acids was small, though significant for several amino acids, in cells overexpressing Atg22. Therefore, we speculate that the contribution of Atg22 to vacuolar amino acid export is rather minor compared with the Avt transporters. This is also supported by further deletion of \u003cem\u003eATG22\u0026nbsp;\u003c/em\u003ein \u003cem\u003eavt3\u003c/em\u003e∆\u003cem\u003eavt4\u003c/em\u003e∆\u003cem\u003eavt7\u003c/em\u003e∆ cells, which did not largely elevate the vacuolar amino acid levels in both nutrient-rich and nitrogen-starved conditions (Supplementary Fig. S1A and B online). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVacuolar amino acid recycling supports protein synthesis under nitrogen starvation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIt has been suggested that amino acids recycled from vacuoles are used for protein synthesis under nitrogen starvation [14,15]. To address this, the level of HA\u003csup\u003e3\u003c/sup\u003e-tagged Arg1, an argininosuccinate synthetase in the arginine biosynthesis pathway, whose cellular level increases under nitrogen starvation in a manner dependent on autophagy [14], in \u003cem\u003eavt3\u003c/em\u003e∆\u003cem\u003eavt4\u003c/em\u003e∆\u003cem\u003eavt6\u003c/em\u003e∆\u003cem\u003eavt7∆\u003c/em\u003e cells (hereafter refer to \u003cem\u003eavt3467\u003c/em\u003e∆ cells) was compared with that in wild-type and \u003cem\u003eatg9\u003c/em\u003e∆ cells. As reported, the Arg1-HA\u003csup\u003e3\u003c/sup\u003e level increased under nitrogen starvation in the wild-type, but not in \u003cem\u003eatg9\u003c/em\u003e∆ cells (Fig. 2A). As \u003cem\u003eatg9\u003c/em\u003e∆ cells,\u003cem\u003e\u0026nbsp;avt3467∆\u003c/em\u003e cells did not increase Arg1-HA\u003csup\u003e3\u003c/sup\u003e level under prolonged nitrogen starvation (Fig. 2A and B). The mRNA level of \u003cem\u003eARG1-HA\u003csup\u003e3\u003c/sup\u003e\u0026nbsp;\u003c/em\u003ein \u003cem\u003eavt3467∆\u003c/em\u003e cells did not significantly differ from that in wild-type cells after culturing in SD-N for 24 hours, whereas it was markedly increased in \u003cem\u003eatg9\u003c/em\u003e∆ cells (Fig. 2C). Therefore, the translation of \u003cem\u003eARG1-HA\u003csup\u003e3\u003c/sup\u003e\u0026nbsp;\u003c/em\u003emRNA may be reduced in \u003cem\u003eavt3467∆\u003c/em\u003e cells compared with wild-type cells, although the reduction may be partial compared with that in \u003cem\u003eatg9\u003c/em\u003e∆ cells. We also assessed protein synthesis\u003cem\u003e\u0026nbsp;\u003c/em\u003eby estimating the amount of [\u003csup\u003e14\u003c/sup\u003eC]valine incorporated into the proteins (Fig. 2D). We measured the cellular uptake and protein incorporation of [\u003csup\u003e14\u003c/sup\u003eC]valine.\u0026nbsp;After 3 hours of nitrogen starvation, [\u003csup\u003e14\u003c/sup\u003eC]valine uptake by \u003cem\u003eavt3467\u003c/em\u003e∆ cells was moderately less compared with that by wild-type cells (Fig. 2D left). During this time, [\u003csup\u003e14\u003c/sup\u003eC]valine incorporation into proteins in \u003cem\u003eavt3467\u003c/em\u003e∆ cells was considerably lower than that in wild-type cells (Fig. 2D middle). After 6 hours of nitrogen starvation, however, it was not significantly lower because [\u003csup\u003e14\u003c/sup\u003eC]valine incorporation into proteins was further reduced in wild-type cells, but not in \u003cem\u003eavt3467\u003c/em\u003e∆ cells (Fig. 2D middle). By accounting for the uptake of extracellular [\u003csup\u003e14\u003c/sup\u003eC]valine (Fig. 2D left), the ratio of [\u003csup\u003e14\u003c/sup\u003eC]valine incorporation into the proteins was calculated (Fig. 2D right). As in \u003cem\u003eatg9\u003c/em\u003e∆ cells, the incorporation ratio was significantly lower in the \u003cem\u003eavt3467\u003c/em\u003e∆ cells compared with that in wild-type cells after 3 hours of nitrogen starvation. Also after 6 hours, it tended to be lower in the \u003cem\u003eavt3467\u003c/em\u003e∆ cells as well as in \u003cem\u003eatg9\u003c/em\u003e∆ cells (Fig. 2D right). Although it is difficult to precisely estimate the cytosolic pool of amino acids, assuming that the amount of valine in the cytosol of \u003cem\u003eavt3467\u003c/em\u003e∆ cells is at least the same or less than that of wild-type cells, these results suggest that protein synthesis in \u003cem\u003eavt3467\u003c/em\u003e∆ cells under nitrogen starvation is lower compared with that in wild-type cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eViability under nitrogen starvation is reduced by disrupting leucine biosynthesis in\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eavt3467\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e∆ cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe recycling of amino acids generated by autophagic protein degradation has been suggested to be critical for survival by maintaining protein synthesis under nitrogen starvation [14,15]. We determined whether \u003cem\u003eavt3467\u003c/em\u003e∆ cells show reduced viability under nitrogen starvation. After 3 days of culture in SD-N, \u003cem\u003eatg9\u003c/em\u003e∆ cells exhibited reduced viability compared with wild-type cells (Fig. 3A). In contrast, \u003cem\u003eavt3467\u003c/em\u003e∆ cells maintained their viability as wild-type cells, even after prolonged starvation (Fig. 3A; see after 19 days in SD-N). Amino acids from vacuoles are catabolized to produce glutamate and aspartate, which are then converted into other amino acids for the efficient use of limited nitrogen [33]. Therefore, the viability of \u003cem\u003eavt3467\u003c/em\u003e∆ cells may be maintained by the\u003cem\u003e\u0026nbsp;de novo\u0026nbsp;\u003c/em\u003esynthesis of neutral amino acids from basic and acidic amino acids exported by other vacuolar amino acid transporters. To address this, we evaluated the deletion of other genes,\u003cem\u003e\u0026nbsp;LEU2\u003c/em\u003e, \u003cem\u003eARO8\u003c/em\u003e/\u003cem\u003eARO9\u003c/em\u003e, or \u003cem\u003eTHR4\u003c/em\u003e, in the biosynthesis of either leucine, aromatic amino acids, or threonine, respectively, in the \u003cem\u003eavt3467\u003c/em\u003e∆ strain. After culturing for 7 days in SD-N, the viability of \u003cem\u003eavt3467\u003c/em\u003e∆\u003cem\u003eleu2\u003c/em\u003e∆ cells was significantly decreased compared with \u003cem\u003eleu2\u003c/em\u003e∆ cells (Fig. 3B and Supplementary Fig. S2 online), which suggests that amino acid recycling from vacuoles contributes to the maintenance of viability. Conversely, \u003cem\u003eavt3467\u003c/em\u003e∆\u003cem\u003earo8\u003c/em\u003e∆\u003cem\u003earo9\u003c/em\u003e∆ and \u003cem\u003eavt3467\u003c/em\u003e∆\u003cem\u003ethr4\u003c/em\u003e∆ survived as \u003cem\u003earo8\u003c/em\u003e∆\u003cem\u003earo9\u003c/em\u003e∆ and \u003cem\u003ethr4\u003c/em\u003e∆ cells, respectively (Fig. 3B). Therefore, the degree of the defect in recycling from the vacuoles may vary depending on amino acid species. It is also possible that the unknown transporter(s) may export neutral amino acids. In support of this, vacuolar neutral amino acids were still decreased upon shifting to nitrogen starvation in \u003cem\u003eavt3467\u003c/em\u003e∆ cells (Fig 1A and B). In addition, there may be a threshold amount of amino acids that determines the yield of the translation product and the cell fate as its consequence. It should be noted that the increase in the vacuolar leucine content in \u003cem\u003eavt3467\u003c/em\u003e∆ cells was not remarkable compared with the contents of the other neutral amino acids (Fig. 1A and B). Some intermediate metabolites resulting from \u003cem\u003eLEU2\u0026nbsp;\u003c/em\u003edeletion may cause the loss of viability of \u003cem\u003eavt3467∆\u0026nbsp;\u003c/em\u003ecells. We also cannot exclude the possibility that the recycling of degradation products other than amino acids, such as nucleic acids, metal ions, phosphates, lipids, and/or sugars, may contribute to cell viability under nitrogen starvation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVacuolar amino acid recycling is required for efficient spore formation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYeast diploid cells sporulate in a manner dependent on autophagy to survive nutrient-harsh conditions [13]. Therefore, we hypothesized that sporulation would be impaired in \u003cem\u003eavt3467∆\u003c/em\u003e diploid cells. After 6 days of nitrogen starvation with acetate as a carbon source, approximately half of the ascus from the wild-type diploids contained four spores (tetrads), whereas \u003cem\u003eatg9\u003c/em\u003e∆ diploids did not form any spores (Fig. 4A). In \u003cem\u003eavt3467∆\u003c/em\u003e diploid cells, the ascus containing four spores was decreased, whereas those containing two or one spore (dyads or monads, respectively) was increased (Fig. 4A). The spore number was restored by introducing plasmids to express \u003cem\u003eAVT3\u003c/em\u003e, \u003cem\u003eAVT4\u003c/em\u003e, and \u003cem\u003eAVT6\u003c/em\u003e (Supplementary Fig. S3A\u003cem\u003e\u0026nbsp;\u003c/em\u003eonline). The spore number per ascus decreases when carbon sources are limited, which is regulated by the induction of the structural meiotic spindle pole body components, Mpc54, Mpc70, and Spo74 [34]. We examined the induction of Myc\u003csup\u003e9\u003c/sup\u003e-tagged Mpc54 in diploid cells under sporulation condition. Myc\u003csup\u003e9\u003c/sup\u003e-Mpc54 was transiently increased in wild-type cells, but was nearly undetectable in \u003cem\u003eatg9\u003c/em\u003e∆ cells (Fig. 4B). In \u003cem\u003eavt3467\u003c/em\u003e∆ diploid cells, the increase in Myc\u003csup\u003e9\u003c/sup\u003e-Mpc54 levels was lower compared with that in wild-type cells (Fig. 4B). This is consistent with the observed reduction in the spore number. Introducing the plasmids to express \u003cem\u003eAVT3\u003c/em\u003e, \u003cem\u003eAVT4,\u0026nbsp;\u003c/em\u003eand \u003cem\u003eAVT6\u003c/em\u003e restored the induction of Myc\u003csup\u003e9\u003c/sup\u003e-Mpc54 (Supplementary Fig. S3B online). These suggest that the spore number also appears to be determined by the amount of amino acids recycled from the vacuoles.\u003c/p\u003e\n\u003cp\u003eUpon shifting to sporulation condition, diploid cells initially induce the master transcription factor, Ime1, which is followed by the induction of Ndt80, a transcription factor acting at the middle phase of sporulation, which binds the \u003cem\u003eMPC54\u0026nbsp;\u003c/em\u003epromoter as determined by high-throughput ChIP sequencing [35]. HA\u003csup\u003e6\u003c/sup\u003e-tagged Ime1 was normally induced in \u003cem\u003eatg9\u003c/em\u003e∆ and \u003cem\u003eavt3467\u003c/em\u003e∆ cells, similar to the wild-type cells (Fig. 4C). In contrast, Ndt80-HA\u003csup\u003e6\u003c/sup\u003e was almost not detectable in \u003cem\u003eatg9\u003c/em\u003e∆ cells and partially reduced in \u003cem\u003eavt3467∆\u003c/em\u003e diploid cells (Fig. 4C). Therefore, the expression of sporulation-related factors may be attenuated during the course of sporulation in \u003cem\u003eavt3467∆\u003c/em\u003e diploid cells. We speculated that the shortage of amino acids to support protein synthesis may reduce the production of sporulation-related factors. Interestingly, \u003cem\u003eatg9\u003c/em\u003e∆ cells induced Ime1 as wild-type cells. Therefore, autophagy may not be required for the onset of sporulation, but play a role in completing the process. Our results suggest that the amino acids recycled from vacuoles are required for carrying out the sporulation process after the trigger event.\u003c/p\u003e\n\u003cp\u003eThe significance of amino acid recycling in coping with starvation should be further explored in future studies. The effect of further disrupting genes encoding other vacuolar transporters involved in exporting amino acids and/or the other degradation products should be examined. Identifying such transporters essential for the survival and/or sporulation in \u003cem\u003eavt3467\u003c/em\u003e∆ cells will give us a great advance for understanding the physiology of autophagy.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAVT, amino acid vacuolar transport;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHA, hemagglutinin\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article (and its Supplementary Information files).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Advanced Research Support Center (ADRES) in Ehime University for determining amino acid contents.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Grants-in-Aid for Scientific Research (C) from JSPS (grant numbers 21K05383 and 25K09043 to M.K., 21K05507 and 24K08834 to T.S.) and Grant-in-Aid for Scientific Research on Innovative Areas from JSPS (grant number 22H04650 to T.S.), and funding for Women Scientists provided from Japan Society for Bioscience, Biotechnology, and Agrochemistry (JSBBA) (to M.K.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.K. and T.S. conceived and designed experiments; H.N. and T.S. performed experiments, analyzed data, and prepared figures; , R.O., S.N., N.H., Y.Y., W.Y., N.O., T.K., S.A., N.S., and K.A. performed experiments; H.N., M.K., and T.S. wrote the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe online version contains supplementary materials.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKlionsky, D. 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Gene regulatory network plasticity predates a switch in function of a conserved transcription regulator. \u003cem\u003eElife\u003c/em\u003e\u003cstrong\u003e 6,\u003c/strong\u003e e23250 (2017).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"vacuole, transporter, autophagy, nitrogen starvation, sporulation, Saccharomyces cerevisiae","lastPublishedDoi":"10.21203/rs.3.rs-8111730/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8111730/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAvt6 is a transporter that exports acidic amino acids, such as aspartate and glutamate. We found that disrupting \u003cem\u003eAVT6\u003c/em\u003e in cells lacking other vacuolar amino acid exporters, including Avt3, Avt4, and Avt7, increased vacuolar neutral amino acid content. Conversely, Avt6 overexpression decreased these amino acid levels. These results suggest that Avt6 is also involved in the vacuolar export of neutral amino acids. Although \u003cem\u003eavt3\u003c/em\u003e∆\u003cem\u003eavt4\u003c/em\u003e∆\u003cem\u003eavt6\u003c/em\u003e∆\u003cem\u003eavt7\u003c/em\u003e∆ cells maintained viability under nitrogen starvation as wild-type cells, protein synthesis was reduced. By further deleting \u003cem\u003eLEU2\u003c/em\u003e, a gene involved in leucine biosynthesis, viability was significantly reduced. Moreover, \u003cem\u003eavt3\u003c/em\u003e∆\u003cem\u003eavt4\u003c/em\u003e∆\u003cem\u003eavt6\u003c/em\u003e∆\u003cem\u003eavt7\u003c/em\u003e∆ diploid cells formed fewer spores in a sporulation medium. These phenotypes overlapped with those of cells defective in macroautophagy. This study reveals an extensive redundancy of transporters in vacuolar amino acid recycling and experimentally demonstrates that the vacuolar amino acids generated by macroautophagy are utilized to manage nutrient stress.\u003c/p\u003e","manuscriptTitle":"Physiological significance of amino acid recycling from vacuoles under starvation conditions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-28 08:41:47","doi":"10.21203/rs.3.rs-8111730/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-15T13:49:34+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-12T23:05:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-12T19:37:49+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-09T14:39:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"333070903851364674304086783482915181286","date":"2025-11-22T13:49:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"300465261588786187487844350925165407993","date":"2025-11-22T13:31:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"65618806613812400254008206356156048108","date":"2025-11-22T12:59:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"148699570791793281385444232013045446940","date":"2025-11-21T11:06:53+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-20T18:01:10+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-11-20T17:01:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-15T06:42:15+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-15T06:39:58+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-11-14T07:08:04+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"556beade-4737-498d-900b-8f529b3aba8f","owner":[],"postedDate":"November 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":58435992,"name":"Biological sciences/Biochemistry"},{"id":58435993,"name":"Biological sciences/Biotechnology"},{"id":58435994,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-03-09T16:03:53+00:00","versionOfRecord":{"articleIdentity":"rs-8111730","link":"https://doi.org/10.1038/s41598-026-42129-3","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-03-05 15:59:44","publishedOnDateReadable":"March 5th, 2026"},"versionCreatedAt":"2025-11-28 08:41:47","video":"","vorDoi":"10.1038/s41598-026-42129-3","vorDoiUrl":"https://doi.org/10.1038/s41598-026-42129-3","workflowStages":[]},"version":"v1","identity":"rs-8111730","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8111730","identity":"rs-8111730","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}