Effects of three permeases on arginine utilization in Saccharomyces cerevisiae

Arginine plays an important role in cellular function and metabolism. Arginine uptake mainly occurs through three amino acid permeases, Alp1p, Gap1p and Can1p, which act as both transporters and receptors for amino acid utilization. In this study, seven mutants were constructed with different combinations of permease deficiencies that inhibit arginine utilization. Their effects on arginine metabolism were measured. The three amino acid permeases were also individually overexpressed in wild-type (WT), Δalp1Δgap1Δcan1 and Δnpr1 strains. The growth and arginine utilization of Δcan1, Δgap1Δcan1 and Δalp1Δgap1Δcan1 mutants were suppressed in YNB medium when arginine was the sole nitrogen source. Meanwhile, overexpression of Alp1p and Can1p enhanced growth and arginine utilization in WT, Δalp1Δgap1Δcan1 and Δnpr1. Besides, overexpression of Can1p caused a 26.7% increase in OD600 and 29.3% increase in arginine utilization compared to that of Alp1p in Δalp1Δgap1Δcan1. Transcription analysis showed that the effects of three amino acid permeases on the arginine utilization and the regulation of related genes, were tightly related to their individual characteristics. However, their overall effects were different for different combinations of mutants. The results presented here suggest some possible synergistic effects of different amino acid permeases on regulation of amino acid utilization and metabolism.

Scientific RepoRts | 6:20910 | DOI: 10.1038/srep20910 ubiquitin ligase Rsp5p [16][17][18] . In nitrogen-poor environments, the TORC1 pathway is blocked. Then some of the amino acid permeases are phosphorylated by Npr1p, activating the transporter protein function. When glutamine and other preferred nitrogen source are present, the TORC1 pathway is activated while the Npr1p phosphorylation is inhibited, arginine permeases could be polyubiquitined by ubiquitin ligase Rsp5p and its associated binding adaptor proteins. The permease is subsequently transported to the vacuole for degradation 19 .
The mechanisms of regulation for amino acid permeases have been well analyzed 3,19 . However, for amino acid permeases with the ability to transport the same amino acid, knowledge of the precise regulation scheme is essential for further investigation. In this study, different arginine-related amino acid permease disruption mutants were constructed to investigate the roles of the three arginine permeases in arginine utilization, metabolism and transcriptional regulation. The three amino acid permeases were overexpressed in wild type (WT), Δ alp1Δ gap1Δ can1 and Δ npr1 S. cerevisiae strains. Under culture conditions with arginine as the sole nitrogen source, the transcription of the three arginine permeases, arginine metabolism and amino acid permease regulation genes were analyzed. The results showed the effects of three amino acid permeases on the arginine utilization and the regulation of related genes, were tightly related to their individual characteristics. The results presented here suggested that arginine permeases disruption combinations might have a role in the precise regulation of arginine utilization and metabolism by changing arginine uptake patterns.

Results
Arginine utilization in different arginine permease disrupted strains. Seven mutants were used to investigate the influence of different arginine permease disruptions on arginine utilization. The results showed that the growth of these mutants with CAN1 disruption was more suppressed than the WT strain. The triple disruption strain AP-AGC (Δ alp1Δ gap1Δ can1) could still survive in YNB (yeast nitrogen base without amino acids and ammonia) medium with 2 mM arginine as the sole nitrogen source (Fig. 1A). Besides, arginine was present in the culture broth of individual permease disrupted mutants (Fig. 1B) and double/triple permease disrupted mutants ( Fig. 1C) at 80 min. The WT strain, AP-A (Δ alp1), AP-AG (Δ alp1Δ gap1) showed more than 15.2% consumption of external arginine, while AP-G (Δ gap1), AP-AC (Δ alp1Δ can1) showed a 10.0% to 12.0% reduction. Arginine utilization by AP-C (Δ can1), AP-GC (Δ gap1Δ can1) and AP-AGC were severely suppressed (only 7.2% to 8.0% consumption) at 80 min.
Effects of permease disruption on transcription of arginine metabolism-related genes. Arginine uptake is the first step in arginine metabolism. To address the transcriptional influence of the different arginine permeases disruption on arginine metabolism, arginine permeases and arginine metabolism related genes were assayed by qRT-PCR (Fig. 2). The expression level of the CAN1 was down-regulated in AP-A (1.9-fold), AP-G (1.4-fold) and AP-AG (6.5-fold) mutants. The expression level of GAP1 was up-regulated in the AP-A (1.5-fold), AP-C (3.5-fold) and AP-AC (1.6-fold) mutants. The expression level of ALP1 was down-regulated in the AP-G (2.7-fold), AP-AC (0.1-fold) and AP-GC (1.2-fold) mutants. The expression levels of ARG1 and VBA2 were down-regulated at least 2.6-fold in all mutants except for the ARG1 was up-regulated in AP-GC (2.36-fold). However, the CAR1 was up-regulated at least 1.5-fold in these mutants except for AP-GC (6.5-fold down-regulated).

Effects of permease overexpression on arginine utilization and cell growth.
To further determine the effects of the three amino acid permeases on arginine utilization and cell growth, they were overexpressed in WT, AP-AGC and AP-NPR1. The cell growth, concentrations of arginine and galactose, were measured after 48 h (Fig. 4). Compared to the control strain, overexpression of Alp1p and Can1p caused an increase in OD 600 in WT, AP-AGC and AP-NPR1. Overexpression of Can1p alone caused a 26.7% increase in OD 600 compared to Alp1p in AP-AGC at 48 h. Arginine utilization was more efficient by overexpression of Alp1p in WT and AP-AGC. However, overexpression of Can1p increase the arginine utilization by 29.3% compared to that of Alp1p in AP-AGC at 48 h.
Effects of permease overexpression on the transcription of arginine metabolism-related genes. In order to find out the reasons for the influence of overexpression of the three amino acid permeases on arginine utilization and cell growth, the expression levels of the three amino acid permeases, arginine metabolism and arginine permease regulation genes were measured in WT, AP-AGC and AP-NPR1 (Fig. 5). Compared to the control strains, overexpression of one arginine permease gene showed different influence on the other two arginine permeases. The expression level of ALP1 gene was down-regulated in WT + G (1.9-fold). The expression level of GAP1 gene was up-regulated in AP-NPR1 + A (2.4-fold). The expression level of CAN1 gene was down-regulated in WT + A (1.8-fold), while up-regulated in AP-NPR1 + A (2.6-fold) and AP-NPR1 + G (4.2-fold). Furthermore, the rest of arginine permease genes showed no significant expression level changes (all up-/down-regulations were below 1.5-fold).
Overexpression of three arginine permeases also showed different influence on arginine metabolism related genes. The expression level of ARG1 gene was up-regulated at least 1.6-fold while ALP1 and CAN1 genes were individually overexpressed in WT and AP-NPR1. However, the ARG1 gene was down-regulated at least 2.8-fold while three arginine permease genes were overexpressed in AP-AGC. The expression level of VBA2 gene was down-regulated while CAN1 was overexpressed in WT (1.7-fold) and AP-NPR1 (2.2-fold), and down-regulated at least 1.6-fold while three arginine permeases genes were overexpressed in AP-AGC. The expression level of CAR1 was up-regulated at least 1.6-fold and 30.4-fold while three arginine permeases were overexpressed in AP-NPR1 and AP-AGC, respectively. The rest of arginine metabolism genes showed no significant expression level changes (up/down-regulated by less than 1.5-fold).

Discussion
Amino acids are transported via different amino acid permeases 3 . However, permeases that transport the same amino acid may display different specific roles in amino acid transport 20 . The results present here demonstrated that different combinatorial disruption and/or overexpression of three arginine-related permeases showed different roles in promoting cell growth, arginine utilization and the transcription of arginine permeases and arginine metabolic genes. The results revealed that the characteristics and combined effects of three amino acid permeases on the regulation of arginine metabolism. It also provided useful clues for the regulation of amino acids metabolism through rational combinational engineering of different amino acid permeases.
In S. cerevisiae, arginine is transported into cells mainly through three different amino acid permeases 3 . However, how those permeases contribute synergistically to arginine uptake and their combinatorial effects remained unclear. Previous works on NH 4 + and L-phenylalanine permeases with different combinations of permease disruptions provided a comprehensive knowledge of the combined and individual roles of these permeases 20,21 . It was also reported that genotype Δ gap1Δ can1 could substantially eliminate arginine uptake 9 . Here, a systematic investigation on the three arginine permeases revealed combinatorial effects in both arginine utilization and its transcriptional regulation (Figs 1,2). The combinatorial effects of three arginine permeases could be dependent on the individual characteristics of three amino acid permeases and their corresponding upstream regulation processes.
These three amino acid permeases are not only used as transporters for amino acids, but also as receptors. Gap1p is reported to be an amino acid transport receptor 22 . Can1p is known as a GAAC-and NCR-regulated permease 3,23 . Since Alp1p and Can1p are considered to be transporters of cationic amino acids 24 , the fact that the expression level of GAP1 was up-regulated in the AP-AC mutant and down-regulated in the WT + A and WT + C strains may indicate an antagonistic effect while ALP1 and CAN1 were disrupted or overexpressed. It also showed that disruption of ALP1 or CAN1 accompanied with GAP1 disruption showed a synergetic effect on each other. Furthermore, overexpression of Alp1p supported adequate arginine uptake ability as Can1p (Fig. 4D-F),  The mutants were cultured in YNB + ammonium sulfate medium to log phase. Cells were then collected and transferred to YNB + 10 mM arginine medium for 2 h. The expression levels of three amino acid permease genes were measured in AP-VBA2, AP-ARG1 and AP-CAR1. The WT strain was used as a control. Data were normalized to the ACT1 gene. Error bars represent standard deviations (n = 3).
Scientific RepoRts | 6:20910 | DOI: 10.1038/srep20910 suggested the limited effect of ALP1 disruption on arginine utilization might be the result of lower promoter activity 9 .
TORC1 regulates ubiquitin-mediated endocytosis via Npr1p-mediated phosphoinhibition of a ubiquitin ligase adaptor 16 . Since Gap1p and Can1p are two of the most important Npr1-regulated amino acid permeases 16,25 . Disruption of NPR1 affects the expression level of arginine metabolic genes, while the expression level of these three arginine metabolism related genes were consistent in AP-AGC. This means that Npr1p influences the arginine transport function and regulation via arginine permeases. Previous reports showed that Alp1p was an effective transporter for arginine uptake 9 . Here, we further demonstrated that Alp1p are highly efficient for arginine utilization. However, Can1p showed higher capacity for arginine transport. Although the Alp1p was highly homologous to Can1p 26 , their characteristics and regulation mechanisms might be different, thus lead to different regulation patterns of arginine transport and metabolism.
In summary, this study demonstrated the combinatorial disruption and overexpression effects of the three arginine-related amino acid permeases on arginine utilization and metabolism. Although the amounts of amino acid permeases have been identified and characterized 3 , clearer differences between amino acid permeases that can transport the same amino acid still need to be characterized. This study provided systematic insight on fine regulation of arginine metabolism by the characteristics, combinatorial effects and upstream regulation processes of three arginine permeases. The results also showed that all of these amino acids permeases were well orchestrated and tightly linked between each other in the precise regulation of arginine uptake and metabolism process.

Materials and Methods
Strains, media and culture conditions. S. cerevisiae strains used in this study are listed in Table 1. S. cerevisiae BY4741 was used as the initial strain 27 . Strains were first cultured in YNB + ammonium sulfate medium (1.6 g/L yeast nitrogen base without amino acids or ammonium, 20 g/L glucose and 5 g/L ammonium sulfate); appropriate supplements (25 mg/L leucine, 25 mg/L histidine, 25 mg/L methionine and 25 mg/L uracil) were added when required. For analysis of arginine utilization, cells were cultured in YNB + arginine media in which arginine was the sole nitrogen source. YPD + G418 plates (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose, 20 g/L agar, 200 mg/L G418 sulfate) were used for selection of G418 resistance transformants.
For arginine utilization analysis, strains were cultured in YNB + ammonium sulfate medium at 30 °C until OD 600 = 0.6 ~ 1.0. Cells were then harvested, washed twice with YNB medium without ammonium or amino acids, and the solutions were adjusted to OD 600 = 1.0 and transferred to a YNB + 2 mM arginine medium 19 . Arginine was monitored for 80 min. For overexpression of the three amino acid permeases, cells were first cultured in YNB + ammonium sulfate medium to the log phase, then they were diluted to OD 600 = 0.3~0.5 (to reduce the lag phase) in YNB + 10 mM arginine with 30 g/L galactose as the sole carbon source, The concentration of arginine and OD 600 were monitored for 48 h.

Oligonucleotides for genes disruption verification
RNA preparation and DNA synthesis. Yeast cells were cultured in the YNB + ammonium sulfate medium at 30 °C to log phase, transferred to YNB medium with 10 mM arginine and 30 g/L galactose or 20 g/L glucose as the sole carbon source for 2 h. Cells were washed twice with double-distilled water and stored at − 80°C until RNA preparation. The procedures for RNA extraction and cDNA synthesis have been described previously 33 .

Real-time quantitative PCR (qRT-PCR) assay. Primers used for qRT-PCR were designed by Beacon
Designer 7.0 (  Table 2. Oligonucleotides used for quantitative RT-PCR and gene disruption. *Underlined letters represent the overlap of loxP plasmids and primers.