Cloning materials were obtained from New England Biolabs (Ipswich, MA, USA) or Thermo Scientific (Waltham, MA, USA). Eurofins Genomics (Ebersberg, Germany) provided gene sequencing service, and oligonucleotides were purchased from Integrated DNA Technologies (Leuven, Belgium). Uniformly labeled U-¹³C- D-glucose (99% purity, catalog # CLM-1396-5) and U-¹³C (98%) yeast metabolite extract from Pichia pastoris were purchased from Euriso-Top (Saarbrücken, Germany). Unless stated otherwise, all other materials were purchased from Sigma Aldrich (München, Germany).

Yeast knockout mutant strains with BY4741 (MATa; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0) background were purchased from Open Biosystems (catalog # YSC1053). Overexpression strains were generated in the same parent strain. All knockout and overexpression strains used in this study are listed in Supplementary Table S1. For transport assays, yeast pre-cultures were inoculated in 2 mL YPD medium (20 g/L glucose, 20 g/L bacto-peptone, 10 g/L yeast extract) in 24 deep-well plates and grown for 24 h at 30°C and 250 rpm. Optical density at 600 nm (OD₆₀₀) was then measured and adjusted to 0.02 in 2 mL synthetic dextrose (SD) medium (20 g/L ¹³C-glucose, 7 g/L yeast nitrogen base, histidine 2 g/L, leucine 3 g/L, methionine 2 g/L, uracil 2 g/L) and cultured for 21 h or until mid-logarithmic phase (OD₆₀₀ = 0.6 ~ 0.8). For growth profile measurements, cells from a yeast pre-culture were inoculated at OD₆₀₀ = 0.01 in 96 deep-well plates (Enzyscreen B.V., CR1424) containing 300 μL SD medium with natural glucose. The cultures were grown for 72 h at 30°C with 300 rpm agitation, and measurements were acquired every 20 min.

Escherichia coli DH5α strain was used for plasmid amplification, and successful transformants were selected on LB plates with 100 mg/mL ampicillin. Cloning was performed according to the EasyClone MarkerFree method (Jessop-Fabre et al., 2016). For the generation of transporter overexpression strains, the genes of interest were amplified from genomic DNA extracted from the wild-type BY4741 strain and cloned under the control of a constitutive TEF promoter. All genes were integrated into site XII-2 of the chromosome. Transformation of yeast was performed according to the lithium acetate method (Gietz and Woods, 2002), and clones carrying integrated genes were selected on a solid YPD medium supplemented with 200 mg/L G418. Successful integration was confirmed by colony PCR. All oligonucleotides and plasmids are listed in Supplementary Tables S2, S3, respectively.

Cloning of transporter genes for cRNA expression in Xenopus laevis oocytes (Ecocyte Bioscience, Dortmund, Germany) was completed as previously described (Wang et al., 2021). Briefly, amino acid transporter genes were first cloned into pCfB5245 plasmid encoding for T7p-β-globin 5-UTR and β-globin 3-UTR (Darbani et al., 2016). The resulting plasmids were then used as a PCR template to amplify the cassette fragment for in vitro RNA synthesis. Capped RNAs for transporter expression in oocyte cells were then synthesized with a T7 mMESSAGE mMACHINE™ kit (Ambion). The quality of the cRNAs (> 500 ng/μL) was analyzed by Agilent 2100 Bioanalyzer (Agilent Technologies) before proceeding with oocyte injection.

After reaching an OD₆₀₀ of 0.6 ~ 0.8 in SD medium, cell cultures were harvested and washed two times with 2 mL phosphate-buffered saline (PBS; 130 mM NaCl, 2.6 mM KCl, 7 mM Na₂HPO₄, 1.2 mM KH₂PO₄, pH 7.4) at 1000 g for 5 min at 23°C. The cell pellets were then resuspended in 400 μL of amino acid (AA) mix (adenine 13.3 μM, p-aminobenzoic acid 58 μM, leucine 290 μM, alanine 85.4 μM, arginine 43.6 μM, asparagine 57.5 μM, aspartic acid 57.1 μM, cysteine 62.7 μM, glutamic acid 51.7 μM, glutamine 52.0 μM, glycine 100 μM, histidine 49 μM, myo-inositol 42.2 μM, isoleucine 57.9 μM, lysine 52 μM, methionine 50.9 μM, phenylalanine 46 μM, proline 66 μM, serine 72.3 μM, threonine 63.8 μM, tryptophan 37.2 μM, tyrosine 42 μM, uracil 67.8 μM, valine 64.9 μM) and incubated for 40 min at 30°C and 300 rpm agitation.

Cells incubated with the AA mix were transferred to a 96-well protein precipitation filter plate (Waters Corp, MA, USA), and extracellular metabolites were filtrated via positive pressure at 35 psi to a 2 mL 96 deep-well plate. The cell pellets were then washed twice with 300 μL PBS by applying positive pressure at 35 psi. To extract the intracellular metabolites, 200 μL boiling ethanol 75% (v/v) was added to the cell pellets. The filter plate was then vortexed for 1 min at 450 rpm and immediately incubated at 65°C for 15 min. Extracted intracellular metabolites were then filtered by applying positive pressure at 35 psi and collected in a 2 mL 96 deep-well plate. Residual metabolites were collected by applying three times 150 μL of boiling 75% (v/v) ethanol, positive pressure at 35 psi, and pulling together all elutions. Intracellular metabolic extracts were then dried in a vacuum Eppendorf Concentrator plus system equipped with a plate rotor for 6 h at 30°C. Dried intracellular extracts were stored at - 80°C and resuspended in 200 μL LC–MS/MS grade water before being subjected to analysis.

Oocytes were injected with 50 nL of cRNA (400 ng/μL transporter cRNA and 100 ng/μL GFP cRNA) using an automated RoboInject (Multichannel System, Germany) and incubated for 3 days at 18°C. After 3 days of incubation, GFP-positive cells were selected and subsequently used for the uptake assay. A minimum of four oocyte cells per biological replicate were pre-incubated in a Kulori buffer pH 7.4 and then transferred to ¹³C-labeled P. pastoris yeast extract pH 7.4 for 1 h at room temperature. Cells were subsequently transferred to 4°C Kulori buffer pH 7.4 to stop the assay and washed three times in fresh Kulori buffer pH 7.4 before being disrupted with 100 μL ice-cold 50% (v/v) methanol. Cell lysates were incubated for 2 h at −20°C and then spun down to remove cell debris. Finally, 70 μL from the supernatant was mixed with 50 μL water before subjecting it to LC–MS/MS analysis.

Samples were reconstituted in water (HPLC grade) and analyzed on a UHPLC-ESI-QTrap-MS (UHPLC LC20/30 series, Shimadzu and QTrap, Sciex) using an XSelect HSS T3 XP column (2.1 × 150 mm, particle size 2.5 μm, Waters) as previously described (McCloskey et al., 2015). Data was acquired using the scheduled MRM algorithm in Analyst Software (Sciex). Post-run data analysis was performed in SciexOS Software (Sciex). Compounds were quantified using ¹³C metabolically labeled internal standards from E. coli. Linear regressions for quantification were based on peak height ratios. The lower limit of quantification (LLOQ) was defined at a peak height greater than 1e3 ion counts and a signal-to-noise ratio greater than 15 (McCloskey et al., 2015).

Binary solvent gradient (A: 10 mM Tributylamine, 10 mM Acetic Acid, 5% (v/v) MeOH, 2% (v/v) Isopropanol in water; and B: Isopropanol) was applied: 0 to 5 min, isocratic 100% A; 5 to 9 min, linear ramp to 98% A, 2% B; 9 to 9.5 min, linear ramp to 94% A, 6% B; 9.5 to 11.5 min, isocratic 94% A, 6% B; 11.5 to 12 min, linear ramp to 89% A, 11% B; 12 to 13.5 min, isocratic 89% A, 11% B; 13.5 to 15.5 min, linear ramp to 72% A, 28% B; 15.5 to 16.5 min, linear ramp to 47% A, 53% B; 16.5 to 22.5 min, isocratic 47% A, 53% B; 22.5 to 23 min, linear ramp to 100% A; 23 to 27 min, isocratic 100% A. The flow rate was 0.4 mL min − 1 from 0 to 15.5 min, 0.15 mL min − 1 from 15.5 to 23 min, and 0.4 mL min − 1 from 23 to 27 min. The injection volume was 10 μL. MS data was acquired from 0.4 to 23 min (McCloskey et al., 2015).

For intracellular metabolite analysis, the spectral peak areas identified for the respective amino acids were normalized to the cell densities (OD₆₀₀) of the background strain BY4741. The normalized peak area values were further used for isotopic ratio calculations. The targeted LC–MS/MS method allowed the detection of 102 metabolites, of which 15 were amino acids. The peak areas corresponding to the uniform ¹²C compound and its ¹³C analog were acquired for each amino acid. The transport of amino acids in the intracellular extract was determined by the ¹²C/¹³C ratio, whereas the ¹³C/¹²C ratio characterized transport in the extracellular extracts. Intracellular amino acid concentrations were calculated based on total amino acid concentrations from the intracellular extract and provided that the cell density of baker’s yeast is ~1.103 g/mL (Bryan et al., 2010). Analysis of growth profiles was performed with Python version 3.10.7. Data visualization was completed with OriginPro software version 9.8.0.200. Figures 1 and 5A were created with BioRender.com.

Distinguishing between endogenous and exogenous metabolites is one of the main challenges in establishing an assay for transporter identification. Studies on intracellular metabolic fluxes have successfully taken advantage of stable isotope tracing for mapping biosynthetic pathways and distinguishing endogenous from exogenous metabolites (Zamboni et al., 2009). Utilizing a similar strategy, we decided to culture the yeast transporter mutants with uniformly labeled U-¹³C-glucose until reaching almost complete isotopic enrichment of endogenous metabolites after 21 h of cultivation (Wu et al., 2005; Figure 1).

After cultivation, the ¹³C labeled yeast cells were subjected to transport assay with a mixture of 20 proteinogenic unlabeled amino acids prior to targeted LC–MS/MS analysis (McCloskey et al., 2015). Analysis of both the intra- and extracellular metabolite extracts allowed us to monitor the alterations in the isotopologues ratio that served as an indicator of both transport occurrences and the operational direction of the transporter. Five S. cerevisiae amino acid transporters were selected to assess our method’s accuracy: Agp1, Can1, Gap1, Lyp1, and Mup1. All five transporters belong to the APC (amino acid-polyamine-organocation) family of transporters, according to the Transporter Classification Database TCDB 2.A.3 (Saier et al., 2021). Our criteria for transporter selection were based on the proteins’ localization at the plasma membrane and their known substrate ranges fitting to the metabolic extract. Agp1 and Gap1 represent amino acid transporters with a broad substrate specificity (more than seven amino acids), while Can1, Lyp1, and Mup1 have a relatively narrow range (less than four) of transported amino acids (Table 1).

Amino acid homeostasis is tightly regulated in living organisms (Magasanik and Kaiser, 2002; Ljungdahl, 2009; Ljungdahl and Daignan-Fornier, 2012). Therefore, we first aimed to evaluate the effect of the deletion or overexpression of the five transporters on the total intracellular amino acid concentrations. In parallel, to test the viability of the transporter mutant strains, a growth assay for 72 h was completed.

Hierarchical cluster analysis revealed two distinct groups of transporter mutants based on their average amino acid concentrations. One cluster consisted of transporter overexpression and another of deletion mutant strains (Figure 2).

The cluster of strains overexpressing Lyp1, Gap1, and Can1 had minor negative to no effect on the total amino acid concentrations. In contrast, the strain overexpressing the broad-range Agp1 transporter had the highest average intracellular concentration of the 13 analyzed amino acids compared to the wild-type strain. Among others, a substantial increase in concentrations was observed for Asn-L (0.67 ± 0.14 mM), Phe-L (1.32 ± 0.26 mM), Glu-L (84.71 ± 10.34 mM), Trp-L (0.19 ± 0.04 mM) and Tyr (4.14 ± 0.63 mM) (Figure 2). Intriguingly, overexpression of the narrow-range Mup1 permease led to similar results with increased concentrations for 11 amino acids. Therefore, it was unsurprising that Mup1 and Agp1 overexpression strains clustered together based on their amino acid profiles.

Deletion of the lysine permease Lyp1 had almost no effect on the amino acid profile of the strain, except for a slight increase in Met-L (1.17 fold), Phe-L (1.26 fold), and Trp (1.27 fold) and a decrease in His-L (0.2 fold) concentrations. Therefore, the Lyp1 mutant strain clustered together with the background strain. In comparison, deletion of the Agp1 permease led to a significant decrease in the intracellular concentrations of Asn-L, Gln-L, Glu-L, Thr-L, and Tyr-L. A significant reduction in Asn-L, Gln-L, His-L, and Thr-L concentrations was also observed for the Gap1 deletion mutant. Except for His-L, a similar reduction in the three amino acid concentrations was observed upon deletion of the Can1 transporter. The amino acid profile of the Mup1 deletion mutant placed it furthest away from the rest of the transporter deletion mutants. Despite having a minor effect on 11 of the amino acids, deletion of Mup1 had a positive effect on Orn (1.57 fold increase) and a significant increase in Ser-L (0.41 ± 0.03 mM) concentrations (Figure 2).

Except for Agp1, the observed fluctuations in the amino acid pools of the yeast transporter mutants did not affect the cells’ growth profiles (Supplementary Figure S1). Overexpression of Agp1 led to retarded growth of the yeast cells with a maximum OD₆₀₀ of 2.4 after 31 h. In contrast, the corresponding deletion mutant showed a slight growth improvement (~1.2 fold) compared to the background strain.

These findings suggested that the five transporters, to varying degrees, contribute to yeast’s amino acid homeostasis. Since the determined amino acid concentrations are based on total intracellular amounts, it was not possible to account for whether the observed differences were due to transport events or the deregulation of the amino acid biosynthetic pathways. Hence, it was necessary to differentiate between de novo synthesized and exogenously supplied amino acids to investigate the reason for the observed differences.

To investigate further to what extent uptake and excretion contributed to the observed differences in the amino acid concentrations, we continued with isotopic labeling of the yeast endometabolites followed by a transport assay.

Principal component analysis (PCA) models of both the intracellular and the extracellular extracts revealed distinguishable differences (Figure 3). Furthermore, the distribution on PC1 and PC2 score plot showed the relative grouping of the biological replicates for most yeast transporter mutants.

The analysis of intracellular extracts found that all overexpression strains, except Agp1, had minimal changes in amino acid profiles (Figure 3A). Agp1 overexpression resulted in a different transport profile due to a positive correlation with Arg-L and Citr-L. Gap1 deletion mutant had the highest amino acid dissimilarity and was defined by seven amino acids. Can1 deletion mutant negatively correlated to Arg-L, confirming its role as an arginine transporter. The Mup1 deletion mutant showed an altered amino acid uptake profile for two of the biological replicates.

PCA analysis of extracellular extracts revealed clear differences between transporter overexpression and deletion mutants compared to intracellular data (Figure 3B). Agp1, Can1, Gap1, and Mup1 deletions showed a positive correlation with Arg-L, Gln-L, Ser-L, Tyr-L, and Met-L quadrant, implying reduced uptake and possible excretion of these amino acids. Lyp1 deletion had minimal impact on the yeast mutant amino acid profile, while the Gap1 overexpression strain grouped with the reference strain. The rest of the overexpression strains were distributed toward the plot’s periphery. Can1, Mup1, and Lyp1 overexpression led to negatively correlated extracellular amino acid profiles with the respective deletion variants, as anticipated. Agp1 overexpression stood out, displaying a unique scatter pattern with covaried Asn-L, Phe-L, Thr-L, and Glu-L, hinting at potential amino acid excretion involvement.

The contribution of amino acids to the PCA model and their correlation with transport was revealed by their coefficients and position on the loading plot (Supplementary Table S5 and Figures 3C,D). The intracellular extracts showed that Arg-L had the strongest impact on the second principal component, and Thr-L, Met-L, Phe-L, and Tyr-L had a positive correlation, indicating interdependent transport. Extracellular amino acids fell into two independent groups, with Phe-L, Thr-L, Asn-L, and Glu-L in one group and Arg-L, Gln-L, Ser-L, Tyr-L, and Met-L in another. Cit-L and Asp-L were transported differently from other amino acids. Arg-L had the strongest impact on PC2.

Altogether, PCA analyses of intra- and extracellular metabolite extracts revealed distinct correlations among the five transporter deletion and overexpression mutants. These correlations reflected the substrate preference for each of the five amino acid carriers.

To understand the distinct transport patterns observed in PCA analysis, we examined relative isotopologue ratios in intra- and extracellular metabolite extracts for each amino acid. For quantifying transport levels, we compared ¹³C and ¹²C isotope ratios of transporter deletion mutants and overexpressing strains to the wild-type under the same conditions (Figure 4).

Agp1 overexpression led to significant intracellular accumulation of Arg-L, Glu-L, Thr-L, and Met-L. Notably, Agp1 overexpression also resulted in increased extracellular Glu-L and Thr-L levels. In addition, the Agp1 deletion mutant showed high intracellular Phe-L, Tyr-L, and Met-L, while extracellular amino acid levels remained unaffected.

As anticipated, Can1 deletion reduced intracellular Arg-L levels and increased them in the extracellular fraction. Overexpression of Can1 had the inverse effect on the extracellular Arg-L levels and no significant change in the amino acid endometabolite content. Unexpectedly, deleting Can1 resulted in elevated intracellular levels of Asn-L, Asp-L, Glu-L, Phe-L, Thr-L, Tyr-L, and Met-L.

Conversely, yeast strains overexpressing Can1 exhibited reduced extracellular content of Glu-L, Phe-L, Ser-L, Thr-L, and Tyr-L, along with a notable decrease in Phe-L, Tyr-L, and Met-L in the endometabolite analysis. Although Lyp1 and Mup1 are narrow-range transporters, the Lyp1 deletion mutant had surprisingly increased Phe-L, Tyr-L, and Met-L intracellularly and elevated Thr-L and Tyr-L extracellularly. Lyp1 overexpression decreased extracellular Glu-L, Phe-L, Thr-L, and Tyr-L and had no effect on the amino acids intracellular content. For the Mup1 overexpression strain, decreased extracellular Arg-L, Asn-L, Phe-L, Ser-L, Thr-L, and Tyr-L levels were observed. Overexpression of Mup1 also decreased intracellular Phe-L and Tyr-L content. Moreover, Mup1 deletion raised extracellular Arg-L, Ser-L, and Gln-L while inversely affecting Phe-L and Thr-L outside the cells. Additionally, Mup1 deletion led to higher intracellular Asn-L, Phe-L, Thr-L, Tyr-L, and Met-L. Deletion of Gap1, a broad-range amino acid permease, had a substantial impact on nearly all amino acid levels. Intriguingly, Gap1 deletion significantly increased intracellular levels of Asn-L, Asp-L, Glu-L, Phe-L, Thr-L, Tyr-L, and Met-L. The notable decrease in Glu-L and Phe-L observed in exometabolite analysis was consistent with the intracellular findings. However, Gap1 deletion had the opposite effect on Arg-L, causing decreased intracellular levels and increased extracellular content. Gap1 overexpression also affected amino acid transport, albeit to a lesser extent than the deletion mutant variant. Gap1 overexpression strain showed elevated Phe-L, Thr-L, Tyr-L, and Met-L in the yeast endometabolite extracts, while Arg-L levels were reduced. Extracellular extracts of the Gap1 overexpression strain revealed increased Arg-L but significantly lower amounts of Phe-L, Thr-L, and Tyr-L.

Overall, the five amino acid transporters exhibited distinct uptake and excretion patterns for the 11 amino acids. However, due to S. cerevisiae amino acid transporters’ promiscuity and overlapping functions, the observed transport patterns needed further validation. Therefore, we next set up an uptake assay in Xenopus oocytes expressing the single amino acids transporters. In this way, we aimed to omit background transport activity and investigate the single transporter uptake profile.

Xenopus oocytes have proven to be a valuable heterologous system for characterizing transporter proteins from a wide range of organisms (Miller and Zhou, 2000; Wagner et al., 2000). Motivated by their well-described low background transport activity, we employed this system to express the five S. cerevisiae amino acid carriers. To achieve this, we injected oocytes with specific cRNA encoding each of the five S. cerevisiae transporters, supplemented with cRNA encoding free GFP to control for potential endogenous transporter-mediated amino acid uptake. The transport assay involved incubating the injected oocytes with a uniformly labeled ¹³C yeast extract derived from P. pastoris, followed by comprehensive LC–MS/MS analysis of the extracted metabolite contents (Figure 5A).

From the intracellular extracts of the injected oocytes, we successfully detected ten ¹³C-labeled amino acids, namely Arg-L, Asn-L, Asp-L, Cit, Cys-L, Gln-L, Glu-L, His-L, Met-L, and Thr-L (Figure 5B). However, due to the detection of fewer than three biological replicates, Asp-L and Glu-L were excluded from further data analysis (Supplementary Figure S4).

In the control group of oocytes injected with GFP, we observed uptake of ¹³C-labeled amino acids solely for Gln-L (1608.64 ± 501.22 AU) and Citr (396.49 ± 53.23 AU). The remaining six amino acids in the GFP control extract were below the detection limit of our instrument. Consequently, we attributed the increased intracellular levels of the detected amino acids, excluding Gln-L and Citr, to uptake by the amino acid carriers.

Expression of Agp1 led to the notable accumulation of Arg-L, consistent with the uptake assay performed in S. cerevisiae. As expected, oocytes injected with Can1 exhibited increased levels of Arg-L. Moreover, Can1 facilitated the import of Asn-L and Cys-L. Similar uptake profiles were observed for Lyp1, resulting in the intracellular accumulation of Arg-L and Asn-L but not Cys-L.

Consistent with previously reported substrate specificities, Mup1 mediated the uptake of Met-L. Interestingly, Mup1 also facilitated the accumulation of Asn-L, His-L, and Thr-L in the oocyte cells. Although we observed Citr-L uptake by Mup1, it did not reach statistical significance compared to the control strain.

Collectively, our results from the Xenopus oocyte uptake assay demonstrated uptake profiles by the five transporters that partially aligned with our findings in the yeast transport assay (Table 2). However, two transporters, Gap1 and Mup1, exhibited altered uptake profiles between the two assays.