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. 2025 Jun 26;42(8-10):161–168. doi: 10.1002/yea.4003

Cooperative Function of Atg8‐ and TORC1‐Mediated Activities in Yeast

Yumiko Oba 1,2, Miyuki Higuchi 1, Naoka Takahashi 3, Haruko Katsuta 3, Naoki Koike 3, Takashi Ushimaru 3, Yoko Kimura 1,3,✉
PMCID: PMC12511908  PMID: 40568955

ABSTRACT

The target of rapamycin complex 1 (TORC1) protein kinase plays an important role in regulating various cellular activities in response to nutrient availability. In this study, an autophagy‐related protein 8 (atg8) mutant of Saccharomyces cerevisiae was highly sensitive to cellular processes in which TORC1 activity was inhibited by rapamycin treatment or by a mutated allele of KOG1 which encodes a subunit of TORC1. Atg8 exhibits both lipidation‐dependent and ‐independent activities, each involving distinct factors. Lipidation of Atg8 is necessary for autophagy and functions with autophagy‐related proteins like Atg7, whereas the lipidation‐independent activities of Atg8 require Hfl1. The atg7Δhfl1Δ double mutant exhibited defects for the impaired TORC1 activities, suggesting that both lipidation‐dependent and ‐independent functions of Atg8 are required for survival during impaired TORC1 activity. Moreover, atg8Δ and atg7Δhfl1Δ mutants exhibited sensitivity to metal ion Zn2+ during low‐dose rapamycin treatment. The results suggest that Atg8‐mediated functions and TORC1 signaling events play an important role in cell growth, possibly by maintaining vacuole integrity.

Keywords: Atg8, Hfl1, rapamycin, TORC1, vacuole, yeast

Summary

  • atg8Δ mutant is sensitive to rapamycin.

  • Both lipidation‐dependent and ‐independent functions of Atg8 are necessary.

  • Atg8‐ and TORC1‐mediated functions may maintain vacuole function.

The atg8Δ mutant is sensitive to rapamycin, and both lipidation‐dependent and ‐independent functions of Atg8 are necessary for survival during impaired TORC1 activity. Atg8‐ and TORC1‐mediated functions may contribute to maintaining vacuolar functions.

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1. Introduction

In response to nutrient availability, cells regulate their growth by coordinating various metabolic processes, such as anabolism and catabolism. During this process, the target of rapamycin complex 1 (TORC1) protein kinase, an evolutionarily conserved Ser/Thr kinase, acts as a central controller of the signaling networks to regulate cell growth (Loewith and Hall 2011). In yeast, TORC1 consists of Tor1, Tor2, Kog1, Lst8, and the yeast‐specific subunit Tco89. Nutrients regulate TORC1 activity using many regulators. Rapamycin, an immunosuppressant and anticancer agent, binds selectively with and inhibits TORC1 (Loewith and Hall 2011). The loss of function of an activator of TORC1 or a downstream factor of TORC1 required for cell growth renders cells hypersensitive to rapamycin (Chan et al. 2000; Dubouloz et al. 2005).

TORC1 promotes cell growth by regulating various cellular events, including transcription, translation, and macromolecule synthesis (Loewith and Hall 2011; G. Y. Liu and Sabatini 2020). Under nutrient‐starvation conditions, TORC1 is inactivated, leading to the repression of both its activities and cell growth. For example, translation initiation is blocked after rapamycin treatment and it appears that phosphorylation of the translation initiation factor eIF2 interferes with mRNA translation (Yang et al. 2022). Under nutrient‐rich conditions, TORC1 inhibits macroautophagy (hereinafter autophagy), and its inactivation induces autophagy‐mediated macromolecular degradation (Reggiori and Klionsky 2013; Nakatogawa 2020).

Vacuoles correspond to the lysosome in other higher eukaryotes. They play an important role in cellular homeostasis, including the degradation of molecules, such as proteins, lipids, or nucleic acids as well as pH homeostasis and the storage of small molecules. TORC1 localizes to the vacuole and its regulation extends to the vacuole. TORC1 promotes the vacuolar fission process during hyperosmotic stress ER stress (Michaillat et al. 2012; Stauffer and Powers 2017). In addition, TORC1 phosphorylates the lipid kinase Fab1, which generates lipid PI(3,5)P2, and phosphorylated Fab1, in turn, regulates TORC1 activity (Chen et al. 2021). The increase in PI(3,5)P2 levels on the vacuolar membrane is an important step for vacuolar fission (Weisman 2006).

Autophagy‐related protein 8 (Atg8), a member of the ubiquitin‐like family of proteins, was originally identified as one of the core elements of autophagic events in yeast (Tsukada and Ohsumi 1993). During autophagy, Atg8 binds to autophagic receptors and recruits cargo proteins for their degradation in the growing autophagosome (Abdollahzadeh et al. 2017; Noda et al. 2010). Moreover, Atg8 binds to phospholipid phosphatidylethanolamine (PE) through a ubiquitin‐like conjugative reaction known as lipidation (Ichimura et al. 2000). PE‐conjugated Atg8 has membrane‐tethering and ‐hemifusion activities in vitro, which reflects its in vivo activity during autophagosome formation (Nakatogawa et al. 2007). In addition to its role in autophagy, Atg8 exerts autophagy‐independent functions, such as vesicular transport, resistance to oxidative stress, vacuolar fusion, lipid body formation, and the suppression of vacuolar membrane invagination following heat stress (Legesse‐Miller et al. 2000; Mikawa et al. 2010; Tamura et al. 2010; Maeda et al. 2017; Ishii et al. 2019). As these autophagy‐independent functions can be performed by lipidation‐defective Atg8, this specific modification of the protein is not always required for its activity. Therefore, Atg8 has lipidation‐dependent and ‐independent functions.

Recently, the vacuolar membrane protein Hfl1 was identified as an Atg8‐binding protein in the yeasts, Schizosaccharomyces pombe and Saccharomyces cerevisiae, and was suggested to mediate lipidation‐independent activities of Atg8 in S. pombe (X.‐M. Liu et al. 2018). In S. pombe, hfl1Δ mutants exhibited metal ion sensitivity, suggesting impaired vacuolar function. In heat‐stressed yeast, Hfl1 and Atg8 regulate vacuolar invaginations, which were increased by both mutants (Kimura et al. 2023; Ishii et al. 2019). Hfl1 is localized at the neck of vacuolar invaginations following heat stress, suggesting its direct involvement in suppressing their formation (Kimura et al. 2023).

After discovering that an atg8Δ mutant of S. cerevisiae was sensitive to rapamycin, we demonstrated that Atg8 may function in maintaining vacuole functions together with the TORC1‐mediated signaling events through its autophagy‐dependent and ‐independent functions.

2. Materials and Methods

2.1. Media, Yeast Strains, and Plasmids

The yeast strains were grown in YPD medium [1% yeast extract, 2% Bacto–Peptone or Hipolypepton (Nihon Seiyaku), 2% glucose, and 0.002% adenine], synthetic complete medium (0.67% yeast nitrogen base and 2% glucose supplemented with amino acids), or synthetic casamino acid medium (0.67% yeast nitrogen base, 2% glucose, and 0.5% casamino acids; tryptophan, uracil, or adenine was added as necessary). S. cerevisiae W303 was used as the parental strain (see Table S1 for a list of the yeast strains used in this study). AZC, [l‐Azetidine‐2‐carboxylic Acid, an analog of l‐Proline] (Tokyo Chemical Industry) was added from a 1 M stock solution. Zinc chloride (Wako) containing plates were prepared by adding a 0.5 M ZnCl2 stock solution. The plasmids, YEplac195‐HA‐NPR1 and pRS416‐KOG1‐3xGFP, were kindly provided by Dr. M. Hall and Y. Jin, respectively. For pH‐adjusted plates, pH was changed by adding 1 M NaOH to the YPD containing 50 mM TAPS buffer.

2.2. Rapamycin Treatment

For liquid culture, the yeast cells were grown to early log phase in YPD medium at 25°C and treated with 200 ng/mL rapamycin. For the plate assay, rapamycin was included in the YPD agar at 2, 4, 5, or 10 ng/mL.

2.3. Western Blot Analysis

Western blot analysis was used to assess the phosphorylation status of serine/threonine‐protein kinase SCH9 (Sch9) and protein kinase Npr1. The proteins were extracted from yeast cells using the post‐alkaline extraction method described previously (Mostofa et al. 2018). Briefly, 10 mL of the cell culture (OD600 = 0.4–0.8) was incubated with 200 µL of 0.1 M NaOH for 5 min, the pellet was collected by centrifugation, and resuspended in sample buffer [60 mM Tris‐HCl (pH 6.8), 5% glycerol, 2% sodium dodecyl sulfate (SDS), 4% 2‐mercaptoethanol, and 0.0025% bromophenol blue] at 95°C for 5 min. After centrifugation of the suspension, the supernatant was subject to western blot analysis using the following primary antibodies: anti‐phospho‐Sch9‐S758 rabbit or mouse polyclonal antibody (a gift from R. Loewith) (Karuppasamy et al. 2017), anti‐hemagglutinin (HA) mouse monoclonal antibody (#014021881, Wako Pure Chemical, Osaka, Japan), and anti‐phosphoglycerate kinase 1 (Pgk1) mouse monoclonal antibody (#A‐6457, Thermo Fisher Scientific). The chemiluminescence signals from the Western BLoT Quant horseradish peroxidase Substrate (#DS‐T7102; Takara) and the immunoreaction enhancer reagent IMMUNO SHOT (#IS‐012‐250; Cosmo Bio) were detected using an image analyzer (LAS4000mini; Fuji). The phosphorylation status of Npr1 was assessed by SDS‐polyacrylamide gel electrophoresis on a 7.5% polyacrylamide gel. All western blot experiments were performed at least three times independently to confirm the reproducibility of the results.

2.4. Microscopy

Before treatment with rapamycin, FM 4‐64 staining of the cells was performed based on a previously described method (Vida and Emr 1995). Briefly, 1.5–2 mL of the cell culture was grown in YPD medium at 25°C, collected by centrifugation, and resuspended in 49 μL of YPD medium. Then, 1 μL of 2 mM FM 4‐64 (Molecular Probes) was added at a final concentration of 40 μM and the cells were incubated for 20 min at ambient temperature. After washing with YPD medium, the cells were resuspended in 2–3 mL of medium and further incubated. Thereafter, the cells were briefly centrifuged and imaged. Images were acquired using a fluorescence microscope (BX61; Olympus) equipped with a 100× oil objective lens (N/A 1.45), a multiband‐filter set (CHROMO), and a CMOS camera (ORCA‐spark or ORCA‐fusion, Hamamatsu Photonics). The brightness and contrast of the processed images were adjusted using Olympus cellSens software.

3. Results

3.1. Sensitivity of Atg8Δ and Hfl1Δatg7Δ Mutants to Impaired TORC1 Activity

During a study of Atg8 function, we found that an atg8Δ mutant was highly sensitive to rapamycin, which inhibits TORC1 activity, compared with mutants of other core Atg family members, such as the atg7Δ or atg1Δ mutants, which showed minor growth defects in the presence of the drug (Figure 1A).

Figure 1.

Figure 1

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Rapamycin sensitivity of the atg8Δ and atg7Δhfl1Δ mutants. (A) Spot assay. Serial 10‐fold dilutions of the indicated strains grown in log phase were spotted on YPD or YPD plates containing 10 ng/mL rapamycin. The cells were incubated at 25°C for 3 days for the YPD plates and 8 days for the rapamycin‐containing plates. (B) Insufficient suppression of rapamycin sensitivity in the lipidation‐defective Atg8 mutant. Serial 10‐fold dilutions of atg8Δ (pRS306), atg8Δ cells expressing Atg8 or Atg8‐F115, atg7Δ (pRS306), and hfl1Δ (pRS306) were plated on YPD or YPD plates containing 10 ng/mL rapamycin. The cells were incubated at 25°C for 2 days for the YPD plates and 6 days for the rapamycin‐containing plates. (C) AZC sensitivity. The indicated strains were spotted on YPD or YPD plates containing 6 mM AZC.

In addition to its well‐known function in autophagy, Atg8 has autophagy‐unrelated functions. Although the autophagy‐related functions of Atg8 require lipidation, its autophagy‐unrelated functions do not. To determine whether the expression of only the non‐lipidated form of Atg8 could suppress the rapamycin sensitivity of the cell, we introduced the ATG8 gene or the atg8(F115) gene. The latter encodes Atg8‐F115, which lacks the critical Gly residue for lipidation, into the atg8Δ mutant (Figure 1B) (Maeda et al. 2017). Compared with the wild‐type Atg8‐carrying strain, rapamycin sensitivity was partially, but not fully, suppressed in the lipidation‐defective mutant, suggesting that both the lipidation‐dependent and ‐independent functions of Atg8 are required to suppress drug sensitivity.

The lipidation of Atg8 occurs through its conjugation with PE through a ubiquitin‐like conjugative reaction to form an Atg8–PE complex (Ichimura et al. 2000). The cysteine protease Atg4 first hydrolyzes the C‐terminal residue of Atg8 to expose a Gly residue at the C‐terminus. Then, the E1‐like enzyme Atg7 activates Atg8 to form an adenylate Atg8 intermediate that is transferred to the E2‐like enzyme Atg3 for PE attachment. Thus, a loss of Atg7 results in the loss of the lipidated Atg8 function. Moreover, Hfl1 was reported to mediate the lipidation‐independent functions of Atg8, which means that a loss of Hfl1 also results in the loss of non‐lipidated Atg8 function (X.‐M. Liu et al. 2018). Therefore, we examined the rapamycin sensitivity of the atg7Δ, hfl1Δ, and atg7Δhfl1Δ mutants to determine whether the lipidation‐dependent or ‐independent functions or both were required for the suppression of drug sensitivity. We observed that while the atg7Δ and hfl1Δ mutants showed a minor growth defect against rapamycin, the atg7Δhfl1Δ mutant was highly sensitive to rapamycin, similar to that for the atg8Δ mutant (Figure 1A). These results indicated that both lipidated and non‐lipidated forms of Atg8 are required for the suppression of rapamycin sensitivity.

To examine the specificity of atg8Δ and atg7Δhfl1Δ mutations to rapamycin, we determined the effect of the proline analog AZC, l‐azetidine‐2‐carboxylic acid, which causes sensitive growth in several mutants whose functions are related to protein quality control (Joshi et al. 2011; Finley et al. 1987; Amerik et al. 2000). The DOA4 mutant, which encodes a deubiquitinating enzyme, was sensitive to AZC, whereas atg8Δ and atg7Δhfl1Δ were insensitive, suggesting that sensitivity to rapamycin was likely specific to the atg8Δ and atg7Δhfl1Δ mutants (Figure 1C).

Next, we determined whether synthetic growth defects were observed when a temperature‐sensitive mutant of Kog1, which is an essential subunit of the TORC1 complex, contained an atg8Δ mutation (Nakashima et al. 2008). As expected, the temperature‐sensitive phenotype of the kog1‐105 mutant was significantly disrupted by the introduction of the atg8Δ mutation (Figure 2A). The kog1‐105, atg7Δhfl1Δ mutant also showed growth defects at higher temperatures compared with the kog1‐105 mutant, although the extent of the defect was less severe compared with that of the kog1‐101, atg8Δ mutant. Similar tendency was observed for the sensitivity to rapamycin (Figure 2B). Growth defect of kog1‐105, atg7Δhfl1Δ mutant was milder than that of kog1‐101, atg8Δ in the presence of 4 ng/mL of rapamycin, suggesting a possibility that Atg8 has some other unknown functions that Atg7 and Hfl1 are not involved.

Figure 2.

Figure 2

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Synergistic growth defects of kog1‐105, atg8Δ and atg7Δhfl1Δ mutants. (A) Serial 10‐fold dilutions of the indicated strains grown in log phase were spotted on YPD plates and incubated at the indicated temperatures. (B) Cells were spotted on YPD plates containing the indicated concentrations of rapamycin.

Taken together, the results suggest that when the impaired activity of TORC1 is combined with the atg8Δ mutation or atg7Δhfl1Δ combined mutation, synthetic growth defects occur.

3.2. Relationship of Atg8 With TORC1

Based on the above results, Atg8‐related functions may be an upstream or downstream positive regulator of TORC1 signaling. Alternatively, Atg8 may function independently from TORC1, although the impaired functions of Atg8 and TORC1 synergistically affect cell growth (Chan et al. 2000; Dubouloz et al. 2005). If Atg8 is an upstream activator of TORC1, then its loss should reduce TORC1 activity. To test this hypothesis, we assessed the activity of TORC1 in atg8Δ cells by monitoring the phosphorylation status of Sch9 and Npr1. TORC1 downstream signaling is mediated by at least two major effector branches, including the AGC protein kinase Sch9 and a protein phosphatase complex that contains Tap42 (Loewith and Hall 2011). Sch9 is a direct target of TORC1, whereas Npr1 is a downstream protein kinase of the Tap42 branches. The phosphorylation of both proteins was nearly the same as those in the wild‐type and atg8Δ cells, indicating that TORC1 activity was not compromised in atg8Δ cells (Figure 3A,B). This suggests that Atg8 is not an upstream activator of TORC1.

Figure 3.

Figure 3

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TORC1 activity and localization are not altered in atg8Δ cells. (A) Phosphorylation status of Sch9 in atg8Δ cells. Exponentially growing wild‐type (WT) and atg8Δ cells harboring the plasmid pSCU2202 (pRS413‐3HA‐SCH9) were incubated at 30°C. Whole‐cell extracts were subjected to western blot analysis using an anti‐phospho‐Sch9 (S758) antibody. The total amount of Sch9 was determined using an anti‐(hemagglutinin) HA antibody. (B) Phosphorylation status of Npr1 in atg8Δ cells. WT and atg8Δ cells harboring the plasmid pSCU302 (YEplac195‐HA‐NPR1) (Schmidt et al. 1998) were incubated at 30°C. Whole‐cell extracts were subjected to western blot analysis using an anti‐HA antibody. Cells treated with rapamycin for 15 min were used as the control. Pgk1 was used as a loading control (using an anti‐Pgk1 antibody). (C) Localization of Kog1‐3xGFP following rapamycin treatment. Localization of Kog1‐3xGFP expressed in wild‐type (WT) and atg8Δ cells. Cells harboring pRS416‐KOG1‐3xGFP were grown to early log phase at 25°C and treated with FM 4‐64 immediately before treatment with 200 ng/mL rapamycin. The cells were observed at the indicated times. Cells not treated with FM 4‐64 are shown in the bottom panels (w/o FM 4‐64).

Next, we examined the localization of the TORC1 complex, which is normally localized to the vacuolar membrane. It has been reported that during sublethal conditions of severe heat stress, TORC1 molecules dissociate from the vacuolar membrane and are sequestered into stress granules, a process that negatively regulates their activities (Takahara and Maeda 2012). This suggests that TORC1 has a potential to move away from the vacuole. Therefore, we tested a possibility whether the localization of exogenously expressed 3xGFP‐tagged Kog1, an essential component of TORC1, changes after rapamycin treatment in the atg8Δ mutant. We observed that Kog1‐3xGFP remained associated with the vacuolar membrane following rapamycin treatment in the atg8Δ mutant and the wild‐type strains (Figure 3C), indicating that TORC1 was properly localized to the vacuolar membrane in the atg8Δ cells.

3.3. Zinc Sensitivity in Low‐Level Rapamycin‐Treated Atg8Δ and Atg7Δhfl1Δ Cells

We hypothesized that the rapamycin sensitivity of the atg8Δ mutant is caused by defects in vacuole function. Cells with impaired vacuole function are often sensitive to treatment with metal ions, such as Zn, Mn, Co, or Ni (Ortiz et al. 1992; Ramsay and Gadd 1997) because vacuoles function in metal homeostasis. Moreover, in S. pombe, hfl1Δ mutants exhibited metal ion sensitivity (X.‐M. Liu et al. 2018). Therefore, we determined whether simultaneous treatment with a low dose of rapamycin and metal ions causes severe defects. We incubated atg8Δ, atg7Δ, hfl1Δ, and atg7Δhfl1Δ mutants in the presence of a low concentration of rapamycin and ZnCl2. Although 5 or 10 ng/mL of rapamycin inhibited the growth of atg8Δ and atg7Δhfl1Δ mutants, a low dose of rapamycin (2 ng/mL) did not inhibit their growth (Figure 4). ZnCl2 at this concentration of rapamycin slowed the growth of the cells; however, combined treatment with low levels of rapamycin (2 ng/mL) and ZnCl2 caused a synthetic growth defect on the atg8Δ and atg7Δhfl1Δ mutants. The results suggest a possibility that Atg8 and TORC1 support cell growth through the maintenance of vacuolar function.

Figure 4.

Figure 4

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Sensitivity to low rapamycin concentrations in the presence of ZnCl2 and on the high pH. (A) Serial 10‐fold dilutions of the indicated strains grown in log phase were spotted on YPD plates with or without 2 ng/mL rapamycin or/and 7 mM ZnCl2. The cells were incubated on the plate with or without ZnCl2 at 25°C for 3 and 9 days, respectively. (B) Cells were spotted on YPD plates of the indicated pH with or without 2 ng/mL rapamycin, and incubated at 25°C.

We examined the effect of another metal ion Mn2+, however, we could not evaluate the result because all the cells acquired resistance to high concentration (8 mM) of MnCl2 to some extent in the presence of low concentration of rapamycin (data not shown). This result might be consistent with previous result that cytoplasmic Mn2+ is likely to cause rapamycin resistance (Nicastro et al. 2022).

In addition, because cells with impaired vacuole function are often sensitive to high pH, we investigated pH effects on the growth of cells in the presence of a low concentration of rapamycin (Figure 4B). We observed the growth defects at pH8.4 in the presence of rapamycin in atg8Δ, hfl1Δ and atg7Δhfl1Δ as compared with wild‐type. Because the growth defect was observed in the hfl1Δ mutant, it is difficult to determine whether an additive growth defect occurred in the atg7Δhfl1Δ mutant.

4. Discussion

Because the TORC1 complex is localized on the vacuole membrane, it is reasonable to expect that the vacuole activities are related to TORC1‐regulated events. Indeed, for transmitting the TORC1 signal, the integrity of vacuolar functions and endolysosomal trafficking are required (Kingsbury et al. 2014; Takeda et al. 2018). Moreover, vacuoles undergo fission in response to TORC1 signaling (Stauffer and Powers 2017). In this study, we showed that Atg8 and TORC1 signaling plays an important role in cell growth. Based on the result that the atg8Δ as well as the atg7Δhfl1Δ mutant showed ZnCl2 sensitivity in the presence of a low dose of rapamycin, we suggest that TORC1 signaling events and Atg8‐mediated activities might be regulated to maintain vacuole integrity.

Although we found that both lipidated and non‐lipidated forms of Atg8 are involved in this process, the exact function of Atg8 in this process is currently unclear. The function of the non‐lipidated form of Atg8 is likely mediated by Hfl1 because the atg7Δhfl1Δ double‐mutant exhibited a similar rapamycin‐sensitive phenotype to that of the atg8Δ mutant, though they may not be the same from the result of kog1‐105 strains (Figure 2). In addition, because the simultaneous treatment of ZnCl2 and a low level of rapamycin caused a severe defect in atg8Δ and atg7Δhfl1Δ, vacuole integrity might be protected by Atg8‐mediated activities and TORC1‐mediated pathways. As for the lipidation‐dependent functions of Atg8, recent mammalian studies have indicated that membrane “atg8ylation,” that is, membranes covalently modified by Atg8, occurs at various sites, such as in autophagosomes, conventional phagosomes containing pathogens or microbial products, stressed or signaling endosomal compartments, lipid droplets, exocytic vesicles, and lysosomes, with some unlikely to be related to autophagy (Deretic et al. 2024). These events may occur in the vacuoles of yeast to protect vacuole function; however, further studies of the causes of rapamycin sensitivity in this mutant are needed to reveal additional functions of Atg8.

Author Contributions

Yumiko Oba and Miyuki Higuchi performed the growth assays. Haruko Katsuta and Naoki Koike performed the TORC1 activity experiments. Naoka Takahashi conducted the microscopic analyses. Miyuki Higuchi made the yeast strains. Yoko Kimura and Takashi Ushimaru acquired funding and supervised the experiments. Yoko Kimura designed the experiments. Yoko Kimura and Takashi Ushimaru wrote the main manuscript text.

Ethics Statement

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Sup Table S1 Strains.

YEA-42-161-s001.xls (39KB, xls)

Acknowledgments

We thank Dr. Robbie Loewith for the anti‐Sch9‐pS758 antibody, Drs. Michael Hall and Yui Jin for the plasmids, and Dr. Y. Kamada for the strains. We also thank Ms. S. Yoshizaki for her initial observations. This study was supported by the Collaborative Project of the Departments of Science and Agriculture at Shizuoka University (to Yoko Kimura and Takashi Ushimaru), the Takeda Foundation (to Yoko Kimura), the Japan Society for the Promotion of Science (JSPS) (Grants‐in‐Aid for Scientific Research; grant No. 20K06620 and 23K05145 to Yoko Kimura, and 18K06212, 20H05317, and 21H02475 to Takashi Ushimaru), and the Institute for Fermentation, Osaka (grant No. G‐2020‐2‐055 to Takashi Ushimaru).

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Sup Table S1 Strains.

YEA-42-161-s001.xls (39KB, xls)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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