The arginine transporter Can1 negatively regulates biofilm formation in yeasts.

The arginine transporter Can1 is a multifunctional protein of the conventional yeast Saccharomyces cerevisiae. Apart from facilitating arginine uptake, Can1 plays a pivotal role in regulating proline metabolism and maintaining cellular redox balance. Here, we report a novel function of Can1 in the control of yeast biofilm formation. First, the S. cerevisiae CAN1 gene knockout strain displayed a significant growth delay compared to the wild-type strain. Our genetic screening revealed that the slow growth of the CAN1 knockout strain is rescued by a functional deficiency of the FLO8 gene, which encodes the master transcription factor associated with biofilm formation, indicating that Can1 is involved in biofilm formation. Intriguingly, the CAN1 knockout strain promoted the Flo11-dependent aggregation, leading to higher biofilm formation. Furthermore, the CAN1 knockout strain of the pathogenic yeast Candida glabrata exhibited slower growth and higher biofilm formation, similar to S. cerevisiae. More importantly, the C. glabrata CAN1 gene knockout strain showed severe toxicity to macrophage-like cells and nematodes. The present results could help to elucidate both the molecular mechanism underlying yeast biofilm formation and the role it plays. Future investigations may offer insights that contribute to development of antibiofilm agents.

Identification of an arginine transporter in Candida glabrata.

Arginine is a proteinogenic amino acid that organisms additionally exploit both for nitrogen storage and as a stress protectant. The location of arginine, whether intra- or extracellular, is important in maintaining physiological homeostasis. Here, we identified an arginine transporter ortholog of the emerging fungal pathogenic Candida glabrata. Blast searches revealed that the C. glabrata genome contains two potential orthologs of the Saccharomyces cerevisiae arginine transporter gene CAN1 (CAGL0J08162g and CAGL0J08184g). We then found that CAGL0J08162g is stably located on the plasma membrane and performs cellular uptake of arginine. Moreover, CAGL0J08162-disrupted cells of C. glabrata showed a partial resistance to canavanine, a toxic analog of arginine. Our data suggest that CAGL0J08162g is a key arginine transporter in the pathogenic C. glabrata (CgCan1).

Isolation of Yeast Strains with Higher Proline Uptake and Their Applications to Beer Fermentation.

Although proline is the most or second most abundant amino acid in wort and grape must, it is not fully consumed by the yeast Saccharomyces cerevisiae during alcoholic fermentation, unlike other amino acids. Our previous studies showed that arginine, the third most abundant amino acid in wort, inhibits the utilization of proline in most strains of S. cerevisiae. Furthermore, we found that some non-Saccharomyces yeasts utilized proline in a specific artificial medium with arginine and proline as the only nitrogen source, but these yeasts were not suitable for beer fermentation due to their low alcohol productivity. For yeasts to be useful for brewing, they need to utilize proline and produce alcohol during fermentation. In this study, 11 S. cerevisiae strains and 10 non-Saccharomyces yeast strains in the Phaff Yeast Culture Collection were identified that utilize proline effectively. Notably, two of these S. cerevisiae strains, UCDFST 40-144 and 68-44, utilize proline and produce sufficient alcohol in the beer fermentation model used. These strains have the potential to create distinctive beer products that are specifically alcoholic but with a reduction in proline in the finished beer.

PKA-Msn2/4-Shy1 cascade controls inhibition of proline utilization under wine fermentation models.

Proline, which is a predominant amino acid in grape musts, is involved in the taste and flavor of foods and beverages. The yeast Saccharomyces cerevisiae poorly utilizes proline in wine-making processes, leading to a nitrogen deficiency during fermentation and proline accumulation in wine. Previous studies have shown that the protein kinase A (PKA) pathway is involved in inhibitory mechanisms of proline utilization. In this study, we screened the PKA pathway-related genes that regulate proline utilization. Using a yeast culture collection of disrupted strains associated with the downstream of the PKA cascade, we revealed that the stress-responsive transcription factor genes MSN2/4 regulate proline utilization. Moreover, we found that Msn2/4 up-regulate the SHY1 gene during the cell growth of the wine fermentation model, which may cause the inhibition of proline utilization. The SHY1-deleted strain of the commercial wine yeast clearly showed proline consumption and average ethanol production under the wine fermentation model. The present data indicate that the PKA-Msn2/4-Shy1 cascade controls the inhibition of proline utilization under wine-making processes. Our study could hold promise for the development of wine yeast strains that can efficiently reduce proline during wine fermentation.

Plasmid-free CRISPR/Cas9 genome editing in Saccharomyces cerevisiae.

The current CRISPR/Cas9 systems in the yeast Saccharomyces cerevisiae cannot be considered a non-genetic modification technology because it requires the introduction of Cas9 and sgRNA into yeast cells using plasmid expression systems. Our present study showed that the yeast genome can be edited without plasmid expression systems by using a commercially available protein transfection reagent and chemically modified sgRNAs.

Large-scale screening of yeast strains that can utilize proline.

Proline contributes to the taste and flavor of foods. The yeast Saccharomyces cerevisiae poorly assimilates proline during fermentation processes, resulting in the accumulation of proline in fermentative products. We performed here a screening of in total 1138 yeasts to obtain strains that better utilize proline. Our results suggest that proline utilization occurs in the genera of Zygoascus, Galactomyces, and Magnusiomyces.

The arginine transporter Can1 acts as a transceptor for regulation of proline utilization in the yeast Saccharomyces cerevisiae.

Proline is the most abundant amino acid in wine and beer, because the yeast Saccharomyces cerevisiae hardly assimilates proline during fermentation processes. Our previous studies showed that arginine induces endocytosis of the proline transporter Put4, resulting in inhibition of proline utilization. We here report a possible role of arginine sensing in the inhibition of proline utilization. We first found that two basic amino acids, ornithine, and lysine, inhibit proline utilization by inducing Put4 endocytosis in a manner similar to arginine, but citrulline does not. Our genetic screening revealed that the arginine transporter Can1 is involved in the inhibition of proline utilization by arginine. Intriguingly, the arginine uptake activity of Can1 was not required for the arginine-dependent inhibition of proline utilization, suggesting that Can1 has a function beyond its commonly known function of transporting arginine. More importantly, our biochemical analyses revealed that Can1 activates signaling cascades of protein kinase A in response to extracellular arginine. Hence, we proposed that Can1 regulates proline utilization by functioning as a transceptor possessing the activity of both a transporter and receptor of arginine.

The Cdc25/Ras/cAMP-dependent protein kinase A signaling pathway regulates proline utilization in wine yeast Saccharomyces cerevisiae under a wine fermentation model.

Proline is a predominant amino acid in grape must, but it is poorly utilized by the yeast Saccharomyces cerevisiae in wine-making processes. This sometimes leads to a nitrogen deficiency during fermentation and proline accumulation in wine. In this study, we clarified that a glucose response is involved in an inhibitory mechanism of proline utilization in yeast. Our genetic screen showed that strains with a loss-of-function mutation on the CDC25 gene can utilize proline even under fermentation conditions. Cdc25 is a regulator of the glucose response consisting of the Ras/cAMP-dependent protein kinase A (PKA) pathway. Moreover, we found that activation of the Ras/PKA pathway is necessary for the inhibitory mechanism of proline utilization. The present data revealed that crosstalk exists between the carbon and proline metabolisms. Our study could hold promise for the development of wine yeast strains that can efficiently assimilate proline during the fermentation processes.

A novel yeast-based screening system for potential compounds that can alleviate human α-synuclein toxicity.

AIMS: This study aimed to establish a yeast-based screening system for potential compounds that can alleviate the toxicity of α-synuclein (α-syn), a neuropathological hallmark of Parkinson's disease, either inhibition of α-syn aggregation or promotion of ubiquitin-mediated degradation of α-syn. METHODS AND RESULTS: A powerful yeast-based screening assay using the rsp5A401E -mutant strain, which is hypersensitive to α-syn aggregation, was established by two-step gene replacement and further overexpressed the GFP-fused α-syn in the drug-sensitive yeast strain with a galactose-inducible multicopy plasmid. The rsp5A401E -mutant strain treated with baicalein, a known α-syn aggregation inhibitor, showed better α-syn toxicity alleviation than the same background wild type strain as accessed by comparison on the reduction kinetics of viable dye resazurin fluorometrically (λex 540/λem 590 nm). The rsp5A401E -mutant yeast-based assay system showed high sensitivity as it could detect as low as 3.13 µmol l-1 baicalein, the concentration that lower than previously report detected by the in vitro assay. CONCLUSIONS: Our yeast-based system has been effective for screening potential compounds that can alleviate α-syn toxicity with high sensitivity and specificity. SIGNIFICANCE AND IMPACT OF THE STUDY: Yeast-based assay system can be used to discover novel neuroprotective drug candidates which may be either efficiently suppress-α-syn aggregation or enhance ubiquitin-dependent degradation.

Role of Gln79 in Feedback Inhibition of the Yeast γ-Glutamyl Kinase by Proline.

Awamori, the traditional distilled alcoholic beverage of Okinawa, Japan, is brewed with the yeast Saccharomyces cerevisiae. During the distillation process after the fermentation, enormous quantities of distillation residues containing yeast cells must be disposed of, and this has recently been recognized as a major problem both environmentally and economically. Proline, a multifunctional amino acid, has the highest water retention capacity among amino acids. Therefore, distillation residues with large amounts of proline could be useful in cosmetics. Here, we isolated a yeast mutant with high levels of intracellular proline and found a missense mutation (Gln79His) on the PRO1 gene encoding the γ-glutamyl kinase Pro1, a limiting enzyme in proline biosynthesis. The amino acid change of Gln79 to His in Pro1 resulted in desensitization to the proline-mediated feedback inhibition of GK activity, leading to the accumulation of proline in cells. Biochemical and in silico analyses showed that the amino acid residue at position 79 is involved in the stabilization of the proline binding pocket in Pro1 via a hydrogen-bonding network, which plays an important role in feedback inhibition. Our current study, therefore, proposed a possible mechanism underlying the feedback inhibition of γ-glutamyl kinase activity. This mechanism can be applied to construct proline-accumulating yeast strains to effectively utilize distillation residues.

Longevity Regulation by Proline Oxidation in Yeast.

Proline is a pivotal and multifunctional amino acid that is used not only as a nitrogen source but also as a stress protectant and energy source. Therefore, proline metabolism is known to be important in maintaining cellular homeostasis. Here, we discovered that proline oxidation, catalyzed by the proline oxidase Put1, a mitochondrial flavin-dependent enzyme converting proline into ∆1-pyrroline-5-carboxylate, controls the chronological lifespan of the yeast Saccharomyces cerevisiae. Intriguingly, the yeast strain with PUT1 deletion showed a reduced chronological lifespan compared with the wild-type strain. The addition of proline to the culture medium significantly increased the longevity of wild-type cells but not that of PUT1-deleted cells. We next found that induction of the transcriptional factor Put3-dependent PUT1 and degradation of proline occur during the aging of yeast cells. Additionally, the lifespan of the PUT3-deleted strain, which is deficient in PUT1 induction, was shorter than that of the wild-type strain. More importantly, the oxidation of proline by Put1 helped maintain the mitochondrial membrane potential and ATP production through the aging period. These results indicate that mitochondrial energy metabolism is maintained through oxidative degradation of proline and that this process is important in regulating the longevity of yeast cells.

Downregulation of the broad-specificity amino acid permease Agp1 mediated by the ubiquitin ligase Rsp5 and the arrestin-like protein Bul1 in yeast.

Most of plasma membrane transporters are downregulated by ubiquitination-dependent endocytosis to avoid the excess uptake of their substrates. In Saccharomyces cerevisiae, ubiquitination of transporters is mediated by the HECT-type ubiquitin ligase Rsp5. We report here a mechanism underlying the substrate-induced endocytosis of the broad-specificity amino acid permease Agp1. First, we found that Agp1 underwent ubiquitination and endocytosis in response to the addition of excess asparagine, which is a substrate of Agp1. Moreover, the substrate-induced internalization of Agp1 was dependent on the ubiquitination activity of Rsp5. Since Rsp5 requires α-arrestin family proteins as adaptors to bind with substrates, we next developed a method of genetic screening to identify adaptor proteins for Agp1 endocytosis. This screening and biochemical analysis revealed that Bul1, but not its paralogue Bul2, was essential for the substrate-induced endocytosis of Agp1. Our results support that the substrate-induced endocytosis of Agp1 requires Rsp5 and Bul1.

The C2 domain of the ubiquitin ligase Rsp5 is required for ubiquitination of the endocytic protein Rvs167 upon change of nitrogen source.

Ubiquitination is a key signal for endocytosis of proteins on the plasma membrane. The ubiquitin ligase Rsp5 of Saccharomyces cerevisiae, which contains an amino-terminal membrane-binding C2 domain, three substrate-recognizing tryptophan-tryptophan (WW) domains and a carboxyl-terminal catalytic homologous to the E6-AP carboxyl terminus (HECT) domain, can ubiquitinate plasma membrane proteins directing them for endocytosis. Here, we examined the roles of the C2 domain in endocytosis for the downregulation of the general amino acid permease Gap1, which is one of nitrogen-regulated permeases in S. cerevisiae. First, we constructed several rsp5 mutants producing Rsp5 variants without the C2 domain or with amino acid changes of membrane-binding lysine residues. These mutants showed defects in endocytosis of Gap1 in response to a preferred nitrogen source. Intriguingly, we found that ubiquitination of Gap1 in these mutant cells was highly similar to that in wild-type cells during endocytosis. These results indicate that the C2 domain is essential for endocytosis but not for ubiquitination of substrates such as Gap1. Moreover, genetic and biochemical analyses showed that the endocytic protein Rvs167 was ubiquitinated via Rsp5 and the C2 domain was required for efficient ubiquitination in response to a preferred nitrogen source. Here, we propose a mechanism for the C2 domain-mediated endocytosis of plasma membrane permeases.

The yeast α-arrestin Art3 is a key regulator for arginine-induced endocytosis of the high-affinity proline transporter Put4.

Proline is one of the abundant amino acids in grape must, but in winemaking processes it is poorly assimilated by the yeast Saccharomyces cerevisiae. This often causes a nitrogen deficiency during fermentation and proline accumulation in wine. Our previous study showed that arginine inhibits proline utilization by specifically inducing the endocytosis of the high-affinity proline transporter Put4. However, the detailed mechanisms underlying this induction are still unclear. Here, we propose a possible mechanism mediated by the ubiquitin ligase Rsp5 and its adaptor protein, Art3. First, we found that the ubiquitination activity of Rsp5 was essential for the arginine-induced endocytosis of Put4. Because Put4 contains no Rsp5-binding motif, we next screened an adaptor protein that plays a role in the arginine-induced endocytosis of Put4. Our genetic and biochemical analyses clearly revealed that the ART3 gene-disrupted cells were defective in Put4 endocytosis, indicating that Art3 is a key regulator for Put4 endocytosis. More importantly, we discovered that deletion of ART3 remarkably canceled the inhibitory effects of arginine on proline utilization. The present results could hold promise for the development of wine yeast strains that can efficiently assimilate the abundant proline in grape must during the fermentation processes.

Cooperative and selective roles of the WW domains of the yeast Nedd4-like ubiquitin ligase Rsp5 in the recognition of the arrestin-like adaptors Bul1 and Bul2.

The ubiquitin ligase Rsp5, which is the only yeast Saccharomyces cerevisiae member of the Nedd4-family, recognizes and ubiquitinates various substrate proteins through the functions of three conserved WW domains. To elucidate the role of each WW domain in endocytosis of the general amino acid permease Gap1 via interaction with the arrestin-like adaptor proteins Bul1 and Bul2 (Bul1/2), we investigated the effects of the double mutations that abrogate the recognition of PY motifs on target proteins (rsp5(W257F/P260A), rsp5(W359F/P362A), and rsp5(W415F/P418A)) and the alanine substitutions of the conserved threonine residues that are regarded as putative phosphorylation sites (rsp5(T255A), RSP5(T357A), and rsp5(T413A)), both of which are located within each WW domain. The rsp5(W257F/P260A), rsp5(W359F/P362A), and rsp5(W415F/P418A) mutations increased sensitivity to the proline analog azetidine-2-carboxylate (AZC), defective endocytosis of Gap1, and impaired interactions with Bul1. These results demonstrate that molecular recognition by each WW domain is responsible for the cooperative interaction with Bul1. Intriguingly, the RSP5(T357A) mutation enhanced AZC tolerance and endocytosis of Gap1, although rsp5(T255A) and rsp5(T413A) decreased both of them. While rsp5(T255A), RSP5(T357A), and rsp5(T413A) impaired the interaction of Rsp5 with Bul1, the RSP5(T357A) mutation specifically augmented the interaction with Bul2. The AZC tolerance enhanced by RSP5(T357A) was fully abolished by combining with each of the rsp5(W257F/P260A), rsp5(W359F/P362A), or rsp5(W415F/P418A) mutations. It was thus suggested that Thr357 in the WW2 domain has a unique role in preventing from the constitutive activation of Bul1/2-mediated endocytosis of Gap1. Taken together, our results highlight the cooperative and specific roles of WW domains in the regulation of Bul1/2-mediated cellular events.