Exogenous addition of histidine reduces copper availability in the yeast Saccharomyces cerevisiae

The basic amino acid histidine inhibited yeast cell growth more severely than lysine and arginine. Overexpression of CTR1, which encodes a high-affinity copper transporter on the plasma membrane, or addition of copper to the medium alleviated this cytotoxicity. However, the intracellular level of copper ions was not decreased in the presence of excess histidine. These results indicate that histidine cytotoxicity is associated with low copper availability inside cells, not with impaired copper uptake. Furthermore, histidine did not affect cell growth under limited respiration conditions, suggesting that histidine cytotoxicity is involved in deficiency of mitochondrial copper.


INTRODUCTION
Free amino acids play pivotal roles as building blocks of proteins, as intermediates in metabolism, and also as regulators of a wide variety of cellular functions. In our previous studies, several amino acids including proline and arginine show cryoprotective activity in the budding yeast Saccharomyces cerevisiae [1][2][3]. We recently showed that, under oxidative stress conditions, increased conversion of proline into arginine led to the flavoprotein Tah18-dependent synthesis of nitric oxide, which confers stress tolerance on S. cerevisiae cells [4][5][6]. Although other charged amino acids, such as lysine and glutamate, also effectively enhance the freeze tolerance for yeast cells [1], the mechanism underlying the freezing stress tolerance by these amino acids remained unclear. In contrast, intracellular excessive levels of basic amino acids (lysine, arginine, and histidine) in mammals were reported to induce toxicity leading to gastrointestinal diseases, such as hepatomegaly and acute pancreatitis [7][8][9]. Although pleiotropic disorders in lipid metabolism, protein synthesis, and mitochondrial functions have been observed in cells damaged by basic amino acids, their primary cellular effects are not fully understood.
Overexpression of ALP1 leads to specific uptake of arginine, although it is unclear whether this gene is expressed under physiological conditions [15]. While these permeases transport amino acids with substrate preferences, the general amino-acid permease Gap1 is a transporter for all of 20 L-forms and also D-forms of the common α-amino acids, as well as other related compounds, such as citrulline, ornithine, γ-aminobutyric acid (GABA), and polyamines [16][17][18][19]. Gap1 is most closely related to Hip1 in terms of amino acid sequence, although Hip1 seems to be a rather specific permease for histidine [15,20,21]. Recent comprehensive studies revealed that single overexpression of these permeases decreases the growth rate [22,23], suggesting that S. cerevisiae can be utilized as a model to analyze the cytotoxicity caused by excess basic amino acids.
Among basic amino acids, histidine is especially related to copper transport [24]. Since the discovery of copper(II)bis(L-histidinato) complex in human blood [25], extensive research has been performed to determine its physiological roles. Consequently, histidine was found to facilitate copper uptake in hepatic, placental, and brain cells [26][27][28] by removing copper from albumin, which physically inhibits incorporation of copper ions [29]. The copper(II)-bis(Lhistidinato) complex has thus been applied for the treatment of Menkes disease and hypertrophic cardioencephalomyopathy, both of which are closely associated with copper deficiency [30,31]. In S. cerevisiae, copper uptake is mediated by the high-affinity transporters Ctr1 and Ctr3 and a ferric/cupric reductase Fre1, which oxidizes copper(II) into usable copper(I) ions in advance of their uptake [32][33][34]. To maintain copper homeostasis, the CTR1, CTR3, and FRE1 genes are upregulated or downregulated under copper starvation or excess copper conditions, respectively, via the action of the copper-sensing transcription factor Mac1 [35,36]. Intriguingly, the mutations in the histidine biosynthetic genes of S. cerevisiae increase sensitivity to the excess amounts of copper, which is suppressed by addition of histidine [37]. This finding supports the idea that histidine might directly interact with copper ions in yeast cells to alleviate the copper toxicity.
To explore novel roles of free amino acids, we analyzed here the cytotoxicity caused by exogenous addition of excess basic amino acids in S. cerevisiae.

RESULTS AND DISCUSSION
To understand the mechanism by which excess of basic amino acids mediate cytotoxicity, we examined cell growth of S. cerevisiae under culture conditions supplemented with an elevated concentration of lysine, arginine, or histidine. As shown in Figure 1A, 5 mM of histidine severely impaired the growth of yeast cells, although a higher concentration of lysine or arginine (25 mM) did not affect growth. The concentrations for three basic amino acids were selected based on the intracellular contents of these amino acids in L5487 cells ( Figure 1B). When the CAN1 gene, encoding basic amino acids permease on the plasma membrane [12,15,38], was overexpressed, there was little effect on growth on SCGal medium that contained no excess of basic amino acids. In contrast, the overexpression of CAN1 markedly inhibited growth under elevated levels of basic amino acids. In particular, the growth of yeast cells that overexpress CAN1 was relatively slow on the medium in the presence of 5 mM of histidine. We confirmed that the overexpression of CAN1 increased the intracellular levels of basic amino acids in SCGal medium (approximately 1.6-to 4.1-fold increase) ( Figure 1B). Thus, these results suggest that the excess amount of intracellular basic amino acids exerts toxic effects on yeast cells. Considering that Can1 preferentially transports lysine and arginine [15], overexpression of the high-affinity histidine transporter gene HIP1 [15,21] might enhance histidine uptake and effectively confer more severe toxicity to yeast cells in the presence of excess histidine. As histidine conferred more sensitivity to yeast cells than lysine and arginine despite having the lowest intracellular level, we further analyzed histidine cytotoxicity.
To identify multicopy suppressor genes that alleviate histidine toxicity, a yeast genomic library YEp51B [39] was introduced into L5487 cells overexpressing CAN1, and the transformants were screened for growth on SCGal medium containing 10 mM histidine. Sixteen independent genomic DNA fragments were isolated from the transformant colonies, and 25 full-length open reading frames were included in these fragments. After subcloning into pYES2, each gene was tested for its effect on the growth of L5487 cells in the presence of excess histidine. Consequently, the CTR1 gene, which encodes a high-affinity copper transporter that predominantly mediates copper uptake under low copper conditions [32], exhibited the most significant suppression of the histidine-caused growth defect ( Figure 1C). Although the overexpression of CTR1 reduced cell growth in the presence of excess lysine by unknown mechanism(s), it is suggested that Ctr1 functions in alleviating histidine toxicity. Another high-affinity copper transporter gene, CTR3 [33], and a cupric reductase gene, FRE1, the latter of which is required for conversion of copper(II) to copper(I) ions prior to uptake [34], also suppressed the growth defect under histidine-excess conditions when overexpressed. In addition, deletion of the CTR1 or FRE1 gene slightly increased sensitivity to excess histidine (data not shown). These results indicate that the histidine cytotoxicity in S. cerevisiae is alleviated by enhancement of copper uptake. In agreement with these results, the growth defect caused by 5 mM histidine became significantly more severe when the concentration of CuSO 4 in SD medium was decreased (2.5 and 25 nM), although higher concentrations of CuSO 4 (2.5 and 25 µM) suppressed the histidine toxicity ( Figure  1D). It has been well studied that histidine directly binds to copper(II) to form copper(II)-bis(L-histidinato) complex under physiological conditions as in human blood [24]. However, considering that the overexpression of CAN1 enhanced histidine toxicity ( Figure 1A), it seems unlikely that the elevated level of histidine simply chelates copper(II) ions outside of the cells to inhibit copper uptake. Instead, excess histidine might interact with copper(I) ions to reduce the availability of copper after incorporation into yeast cells. To verify this hypothesis, we quantified the intracellular level of copper ions. Although cell growth was delayed starting after 4-hour incubation with 5 or 10 mM histidine (data not shown), intracellular copper ions were slightly increased in the histidine-treated cells ( Figure 1E). Therefore, our data consistently demonstrate that an excess level of histidine enhances copper uptake but impairs copper availability in yeast cells. Regarding the CTR1overexpressing cells ( Figure 1C), sufficient copper ions might be incorporated into cells in bioavailable forms and thus contributed to relieving histidine toxicity. In a similar manner as clioquinol [40], histidine may act as both a chelator and an ionophore: histidine chelates copper(II) outside of the cell and is taken in as a complex, which may facilitate uptake of copper ions, though copper ions in this complex are not bioavailable. Additionally, it is possible that histidine-induced copper deficiency upregulates expression of copper transporters, which may increase copper uptake.
What functions of copper does excess histidine inhibit? Intracellular copper is distributed to distinct target proteins via specific cytosolic copper chaperons, such as Atx1, Ccs1, and Cox17. Atx1 assists in the transport of copper to the cell-surface iron uptake protein Fet3 through the function of Ccc2, which has copper-transporting ATPase activity, on the post-Golgi vesicle [41]. Ccs1 delivers copper specifically to the superoxide dismutase Sod1, which scavenges reactive oxygen species in the intermembrane space of mitochondria [42,43]. Another copper chaperon Cox17 transfers copper to the mitochondrial inner membrane proteins OPEN ACCESS | www.microbialcell.com 243 Microbial Cell | July 2014 | Vol. 1 No. 7 and pRS416) in the absence (0 mM His) and presence of excess histidine (5 or 10 mM His). The values are the means and standard deviations of three independent experiments. Asterisks indicate a significant increase in copper levels compared to the control sample (0 mM His) (p < 0.05). (F) Growth phenotypes of strain L5487 (complemented with pRS415 and pRS416) (shown as ρ + ) and its spontaneous ρmutant. After overnight cultivation in SD liquid medium, approximately 10 6 cells of each strain, and serial dilutions of 10 -1 to 10 -4 (from left to right) were spotted and incubated onto SD agar medium in the absence (Control) and presence of 5 mM histidine (+ His) under aerobic or anaerobic conditions. OPEN ACCESS | www.microbialcell.com Cox11 and Sco1, both of which are essential for assembly of cytochrome c oxidase, which is the last enzyme in the respiratory electron transport chain [44]. In this study, we tested whether histidine cytotoxicity might be mediated by defective mitochondrial functions due to reduced copper availability. As shown in Figure 1F, L5487 cells were clearly sensitive to 5 mM histidine on SD plates, although histidine cytotoxicity was completely abrogated by a spontaneous cytoplasmic petite (ρ -) mutation, which inactivates mitochondrial respiratory functions. It is also worth noting that yeast cells showed similar growth phenotypes in the absence or presence of excess histidine under anaerobic conditions ( Figure 1F). Thus, histidine cytotoxicity was observed only when mitochondrial aerobic respiration should be functional. We hypothesize that the deficiency of the Cox17-bound copper ions due to excess of intracellular histidine might cause the abnormal assembly of cytochrome c oxidase complex in aerobically growing cells, leading to the observed toxic effect. This might be supported by the fact that the disturbance of cytochrome c oxidase induces apoptosis-like cell death in S. cerevisiae [45]. However, we cannot rule out the possibility that defective mitochondrial respiration reduces incorporation of histidine by some unknown mechanism(s), and hence, the excess level of histidine did not elicit the toxicity in ρcells under aerobic conditions or in ρ + cells under anaerobic conditions.
In this study, we discovered the cytotoxicity of excess histidine in S. cerevisiae, which is tightly associated with the reduced availability of intracellular copper ions. Similarly, Pearce and Sherman [37] revealed that intracellular histidine synthesis is required for the detoxification of excess copper. Both studies commonly suggest that intracellular histidine has a novel and important role in copper homeostasis.

Culture Media
The media used for growth of S. cerevisiae were a synthetic complete medium, SC (2% glucose, 0.67% yeast nitrogen base without amino acids (Difco), supplemented with synthetic drop-out amino acid and nucleotide mixture as required), and a synthetic defined medium, SD (2% glucose, 0.67% yeast nitrogen base without amino acids (Difco)). For overexpression of CAN1, CTR1, CTR3, and FRE1, SC with galactose as a carbon source (SCGal) (2% galactose, 0.67% yeast nitrogen base without amino acids (Difco), supplemented with synthetic dropout amino acid and nucleotide mixture as required) was used.
To evaluate the effect of copper sulfate (CuSO 4 ) addition, SD-Cu media (2% glucose, 0.67% yeast nitrogen base without amino acids and copper (ForMedium)) containing different concentrations of CuSO 4 were used. All experiments were performed at 30°C, and all growth media were adjusted to pH 6.5 with HEPES buffer (pH 7.0) and sodium hydroxide. When necessary, 2% agar was added to solidify the medium. For anaerobic cultivation, the inoculated plates were incubated with O 2 -absorber/CO 2 -generator AnaeroPouch-Anaero (Mitsubishi Gas Chemical Company) and O 2 indicators. The E. coli recombinant strains were grown in Luria-Bertani complete medium containing 50 µg/ml ampicillin or M9 minimal medium plus 2% Casamino acids containing 50 µg/ml ampicillin. If necessary, 2% agar was added to solidify the medium.

Measurements of Intracellular Amino Acids Levels
According to a method described previously [3], intracellular amino acids were extracted by boiling from log-phase cells cultivated in SCGal-Leu-Ura liquid medium, and were subsequently quantified with an amino acid analyzer AminoTac JLC-500/V (JEOL).

Determination of Intracellular Copper Ions
Cell lysates were prepared from log-phase cells cultivated in SC-Leu-Ura-Lys-Arg-His liquid medium, and copper ions concentrations were determined by the CUPRAC-BCS assay [25].