Restricted glycolysis is a primary cause of the reduced growth rate of zinc-deficient yeast cells.

Zinc is required for many critical processes, including intermediary metabolism. In Saccharomyces cerevisiae, the Zap1 activator regulates the transcription of ∼80 genes in response to Zn supply. Some Zap1-regulated genes are Zn transporters that maintain Zn homeostasis, while others mediate adaptive responses that enhance fitness. One adaptive response gene encodes the 2-cysteine peroxiredoxin Tsa1, which is critical to Zn-deficient (ZnD) growth. Depending on its redox state, Tsa1 can function as a peroxidase, a protein chaperone, or a regulatory redox sensor. In a screen for possible Tsa1 regulatory targets, we identified a mutation (cdc19S492A) that partially suppressed the tsa1Δ growth defect. The cdc19S492A mutation reduced activity of its protein product, pyruvate kinase isozyme 1 (Pyk1), implicating Tsa1 in adapting glycolysis to ZnD conditions. Glycolysis requires activity of the Zn-dependent enzyme fructose-bisphosphate aldolase 1, which was substantially decreased in ZnD cells. We hypothesized that in ZnD tsa1Δ cells, the loss of a compensatory Tsa1 regulatory function causes depletion of glycolytic intermediates and restricts dependent amino acid synthesis pathways, and that the decreased activity of Pyk1S492A counteracted this depletion by slowing the irreversible conversion of phosphoenolpyruvate to pyruvate. In support of this model, supplementing ZnD tsa1Δ cells with aromatic amino acids improved their growth. Phosphoenolpyruvate supplementation, in contrast, had a much greater effect on growth rate of WT and tsa1Δ ZnD cells, indicating that inefficient glycolysis is a major factor limiting yeast growth. Surprisingly however, this restriction was not primarily due to low fructose-bisphosphate aldolase 1 activity, but instead occurs earlier in glycolysis.

Changes in transcription start sites of Zap1-regulated genes during zinc deficiency: Implications for HNT1 gene regulation.

Changes in RNA are often poor predictors of protein accumulation. One factor disrupting this relationship are changes in transcription start sites (TSSs). Therefore, we explored how alterations in TSS affected expression of genes regulated by the Zap1 transcriptional activator of Saccharomyces cerevisiae. Zap1 controls their response to zinc deficiency. Among over 80 known Zap1-regulated genes, several produced long leader transcripts (LLTs) in one zinc status condition and short leader transcripts (SLTs) in the other. Fusing LLT and SLT transcript leaders to green fluorescent protein indicated that for five genes, the start site shift likely has little effect on protein synthesis. For four genes, however, the different transcript leaders greatly affected translation. We focused on the HNT1 gene. Zap1 caused a shift from SLT HNT1 RNA in zinc-replete cells to LLT HNT1 RNA in deficient cells. This shift correlated with decreased protein production despite increased RNA. The LLT RNA contains multiple upstream open reading frames that can inhibit translation. Expression of the LLT HNT1 RNA was dependent on Zap1. However, expression of the long transcript was not required to decrease SLT HNT1 mRNA. Our results suggest that the Zap1-activated LLT RNA is a "fail-safe" mechanism to ensure decreased Hnt1 protein in zinc deficiency.

Elevation of cellular Mg2+ levels by the Mg2+ transporter, Alr1, supports growth of polyamine-deficient Saccharomyces cerevisiae cells.

The polyamines putrescine, spermidine, and spermine are required for normal eukaryotic cellular functions. However, the minimum requirement for polyamines varies widely, ranging from very high concentrations (mm) in mammalian cells to extremely low in the yeast Saccharomyces cerevisiae Yeast strains deficient in polyamine biosynthesis (spe1Δ, lacking ornithine decarboxylase, and spe2Δ, lacking SAM decarboxylase) require externally supplied polyamines, but supplementation with as little as 10-8 m spermidine restores their growth. Here, we report that culturing a spe1Δ mutant or a spe2Δ mutant in a standard polyamine-free minimal medium (SDC) leads to marked increases in cellular Mg2+ content. To determine which yeast Mg2+ transporter mediated this increase, we generated mutant strains with a deletion of SPE1 or SPE2 combined with a deletion of one of the three Mg2+ transporter genes, ALR1, ALR2, and MNR2, known to maintain cytosolic Mg2+ concentration. Neither Alr2 nor Mnr2 was required for increased Mg2+ accumulation, as all four double mutants (spe1Δ alr2Δ, spe2Δ alr2Δ, spe1Δ mnr2Δ, and spe2Δ mnr2Δ) exhibited significant Mg2+ accumulation upon polyamine depletion. In contrast, a spe2Δ alr1Δ double mutant cultured in SDC exhibited little increase in Mg2+ content and displayed severe growth defects compared with single mutants alr1Δ and spe2Δ under polyamine-deficient conditions. These findings indicate that Alr1 is required for the up-regulation of the Mg2+ content in polyamine-depleted cells and suggest that elevated Mg2+ can support growth of polyamine-deficient S. cerevisiae mutants. Up-regulation of cellular polyamine content in a Mg2+-deficient alr1Δ mutant provided further evidence for a cross-talk between Mg2+ and polyamine metabolism.

The cellular economy of the Saccharomyces cerevisiae zinc proteome.

Zinc is an essential cofactor for many proteins. A key mechanism of zinc homeostasis during deficiency is "zinc sparing" in which specific zinc-binding proteins are repressed to reduce the cellular requirement. In this report, we evaluated zinc sparing across the zinc proteome of Saccharomyces cerevisiae. The yeast zinc proteome of 582 known or potential zinc-binding proteins was identified using a bioinformatics analysis that combined global domain searches with local motif searches. Protein abundance was determined by mass spectrometry. In zinc-replete cells, we detected over 2500 proteins among which 229 were zinc proteins. Based on copy number estimates and binding stoichiometries, a replete cell contains ∼9 million zinc-binding sites on proteins. During zinc deficiency, many zinc proteins decreased in abundance and the zinc-binding requirement decreased to ∼5 million zinc atoms per cell. Many of these effects were due at least in part to changes in mRNA levels rather than simply protein degradation. Measurements of cellular zinc content showed that the level of zinc atoms per cell dropped from over 20 million in replete cells to only 1.7 million in deficient cells. These results confirmed the ability of replete cells to store excess zinc and suggested that the majority of zinc-binding sites on proteins in deficient cells are either unmetalated or mismetalated. Our analysis of two abundant zinc proteins, Fba1 aldolase and Met6 methionine synthetase, supported that hypothesis. Thus, we have discovered widespread zinc sparing mechanisms and obtained evidence of a high accumulation of zinc proteins that lack their cofactor during deficiency.

The GIS2 Gene Is Repressed by a Zinc-Regulated Bicistronic RNA in Saccharomyces cerevisiae.

Zinc homeostasis is essential for all organisms. The Zap1 transcriptional activator regulates these processes in the yeast Saccharomyces cerevisiae. During zinc deficiency, Zap1 increases expression of zinc transporters and proteins involved in adapting to the stress of zinc deficiency. Transcriptional activation by Zap1 can also repress expression of some genes, e.g., RTC4. In zinc-replete cells, RTC4 mRNA is produced with a short transcript leader that is efficiently translated. During deficiency, Zap1-dependent expression of an RNA with a longer transcript leader represses the RTC4 promoter. This long leader transcript (LLT) is not translated due to the presence of small open reading frames upstream of the RTC4 coding region. In this study, we show that the RTC4 LLT RNA also plays a second function, i.e., repression of the adjacent GIS2 gene. In generating the LLT transcript, RNA polymerase II transcribes RTC4 through the GIS2 promoter. Production of the LLT RNA correlates with the decreased expression of GIS2 mRNA and mutations that prevent synthesis of the LLT RNA or terminate it before the GIS2 promoter renders GIS2 mRNA expression and Gis2 protein accumulation constitutive. Thus, we have discovered an unusual regulatory mechanism that uses a bicistronic RNA to control two genes simultaneously.

Zap1-dependent transcription from an alternative upstream promoter controls translation of RTC4 mRNA in zinc-deficient Saccharomyces cerevisiae.

Maintaining zinc homeostasis is an important property of all organisms. In the yeast Saccharomyces cerevisiae, the Zap1 transcriptional activator is a central player in this process. In response to zinc deficiency, Zap1 activates transcription of many genes and consequently increases accumulation of their encoded proteins. In this report, we describe a new mechanism of Zap1-mediated regulation whereby increased transcription of certain target genes results in reduced protein expression. Transcription of the Zap1-responsive genes RTC4 and RAD27 increases markedly in zinc-deficient cells but, surprisingly, their protein levels decrease. We examined the underlying mechanism further for RTC4 and found that this unusual regulation results from altered transcription start site selection. In zinc-replete cells, RTC4 transcription begins near the protein-coding region and the resulting short transcript leader allows for efficient translation. In zinc-deficient cells, RTC4 RNA with longer transcript leaders are expressed that are not efficiently translated due to the presence of multiple small open reading frames upstream of the coding region. This regulation requires a potential Zap1 binding site located farther upstream of the promoter. Thus, we present evidence for a new mechanism of Zap1-mediated gene regulation and another way that this activator protein can repress protein expression.

Activation of the Yeast UBI4 Polyubiquitin Gene by Zap1 Transcription Factor via an Intragenic Promoter Is Critical for Zinc-deficient Growth.

Stability of many proteins requires zinc. Zinc deficiency disrupts their folding, and the ubiquitin-proteasome system may help manage this stress. In Saccharomyces cerevisiae, UBI4 encodes five tandem ubiquitin monomers and is essential for growth in zinc-deficient conditions. Although UBI4 is only one of four ubiquitin-encoding genes in the genome, a dramatic decrease in ubiquitin was observed in zinc-deficient ubi4Δ cells. The three other ubiquitin genes were strongly repressed under these conditions, contributing to the decline in ubiquitin. In a screen for ubi4Δ suppressors, a hypomorphic allele of the RPT2 proteasome regulatory subunit gene (rpt2(E301K)) suppressed the ubi4Δ growth defect. The rpt2(E301K) mutation also increased ubiquitin accumulation in zinc-deficient cells, and by using a ubiquitin-independent proteasome substrate we found that proteasome activity was reduced. These results suggested that increased ubiquitin supply in suppressed ubi4Δ cells was a consequence of more efficient ubiquitin release and recycling during proteasome degradation. Degradation of a ubiquitin-dependent substrate was restored by the rpt2(E301K) mutation, indicating that ubiquitination is rate-limiting in this process. The UBI4 gene was induced ∼5-fold in low zinc and is regulated by the zinc-responsive Zap1 transcription factor. Surprisingly, Zap1 controls UBI4 by inducing transcription from an intragenic promoter, and the resulting truncated mRNA encodes only two of the five ubiquitin repeats. Expression of a short transcript alone complemented the ubi4Δ mutation, indicating that it is efficiently translated. Loss of Zap1-dependent UBI4 expression caused a growth defect in zinc-deficient conditions. Thus, the intragenic UBI4 promoter is critical to preventing ubiquitin deficiency in zinc-deficient cells.

The antifungal plant defensin AhPDF1.1b is a beneficial factor involved in adaptive response to zinc overload when it is expressed in yeast cells.

Antimicrobial peptides represent an expanding family of peptides involved in innate immunity of many living organisms. They show an amazing diversity in their sequence, structure, and mechanism of action. Among them, plant defensins are renowned for their antifungal activity but various side activities have also been described. Usually, a new biological role is reported along with the discovery of a new defensin and it is thus not clear if this multifunctionality exists at the family level or at the peptide level. We previously showed that the plant defensin AhPDF1.1b exhibits an unexpected role by conferring zinc tolerance to yeast and plant cells. In this paper, we further explored this activity using different yeast genetic backgrounds: especially the zrc1 mutant and an UPRE-GFP reporter yeast strain. We showed that AhPDF1.1b interferes with adaptive cell response in the endoplasmic reticulum to confer cellular zinc tolerance. We thus highlighted that, depending on its cellular localization, AhPDF1.1b exerts quite separate activities: when it is applied exogenously, it is a toxin against fungal and also root cells, but when it is expressed in yeast cells, it is a peptide that modulates the cellular adaptive response to zinc overload.

Peroxiredoxin chaperone activity is critical for protein homeostasis in zinc-deficient yeast.

Zinc is required for the folding and function of many proteins. In Saccharomyces cerevisiae, homeostatic and adaptive responses to zinc deficiency are regulated by the Zap1 transcription factor. One Zap1 target gene encodes the Tsa1 peroxiredoxin, a protein with both peroxidase and protein chaperone activities. Consistent with its regulation, Tsa1 is critical for growth under low zinc conditions. We previously showed that Tsa1's peroxidase function decreases the oxidative stress that occurs in zinc deficiency. In this report, we show that Tsa1 chaperone, and not peroxidase, activity is the more critical function in zinc-deficient cells. Mutations restoring growth to zinc-deficient tsa1 cells inactivated TRR1, encoding thioredoxin reductase. Because Trr1 is required for oxidative stress tolerance, this result implicated the Tsa1 chaperone function in tolerance to zinc deficiency. Consistent with this hypothesis, the tsa1Δ zinc requirement was complemented by a Tsa1 mutant allele that retained only chaperone function. Additionally, growth of tsa1Δ was also restored by overexpression of holdase chaperones Hsp26 and Hsp42, which lack peroxidase activity, and the Tsa1 paralog Tsa2 contributed to suppression by trr1Δ, even though trr1Δ inactivates Tsa2 peroxidase activity. The essentiality of the Tsa1 chaperone suggested that zinc-deficient cells experience a crisis of disrupted protein folding. Consistent with this model, assays of protein homeostasis suggested that zinc-limited tsa1Δ mutants accumulated unfolded proteins and induced a corresponding stress response. These observations demonstrate a clear physiological role for a peroxiredoxin chaperone and reveal a novel and unexpected role for protein homeostasis in tolerating metal deficiency.

Regulation of Alr1 Mg transporter activity by intracellular magnesium.

Mg homeostasis is critical to eukaryotic cells, but the contribution of Mg transporter activity to homeostasis is not fully understood. In yeast, Mg uptake is primarily mediated by the Alr1 transporter, which also allows low affinity uptake of other divalent cations such as Ni(2+), Mn(2+), Zn(2+) and Co(2+). Using Ni(2+) uptake to assay Alr1 activity, we observed approximately nine-fold more activity under Mg-deficient conditions. The mnr2 mutation, which is thought to block release of vacuolar Mg stores, was associated with increased Alr1 activity, suggesting Alr1 was regulated by intracellular Mg supply. Consistent with a previous report of the regulation of Alr1 expression by Mg supply, Mg deficiency and the mnr2 mutation both increased the accumulation of a carboxy-terminal epitope-tagged version of the Alr1 protein (Alr1-HA). However, Mg supply had little effect on ALR1 promoter activity or mRNA levels. In addition, while Mg deficiency caused a seven-fold increase in Alr1-HA accumulation, the N-terminally tagged and untagged Alr1 proteins increased less than two-fold. These observations argue that the Mg-dependent accumulation of the C-terminal epitope-tagged protein was primarily an artifact of its modification. Plasma membrane localization of YFP-tagged Alr1 was also unaffected by Mg supply, indicating that a change in Alr1 location did not explain the increased activity we observed. We conclude that variation in Alr1 protein accumulation or location does not make a substantial contribution to its regulation by Mg supply, suggesting Alr1 activity is directly regulated via as yet unknown mechanisms.

Zebrafish (Danio rerio) endomembrane antiporter similar to a yeast cation/H(+) transporter is required for neural crest development.

CAtion/H(+) eXchangers (CAXs) are integral membrane proteins that transport Ca(2+) or other cations by exchange with protons. While several yeast and plant CAX proteins have been characterized, no functional analysis of a vertebrate CAX homologue has yet been reported. In this study, we further characterize a CAX from yeast, VNX1, and initiate characterization of a zebrafish CAX (Cax1). Localization studies indicated that both Vnx1 and Cax1 proteins are found in endomembrane compartments. Biochemical characterization of endomembrane fractions from vnx1 mutant cells and zebrafish Cax1-expressing yeast cells suggested that both yeast and fish CAXs have Ca(2+)/H(+) antiport activities. Additionally, the vnx1 mutation was associated with heightened pH-sensitivity. In zebrafish embryos, cax1 was specifically expressed in neural crest cells. Morpholino knockdown of cax1 caused defects in neural crest development, including alterations in pigmentation, defects in jaw development, and reduction in expression of the neural crest marker, Pax7. Collectively, our findings provide insights into Vnx1 function and support an unexpected role of CAX transporters in animal growth and development.

MNR2 regulates intracellular magnesium storage in Saccharomyces cerevisiae.

Magnesium (Mg) is an essential enzyme cofactor and a key structural component of biological molecules, but relatively little is known about the molecular components required for Mg homeostasis in eukaryotic cells. The yeast genome encodes four characterized members of the CorA Mg transporter superfamily located in the plasma membrane (Alr1 and Alr2) or the mitochondrial inner membrane (Mrs2 and Lpe10). We describe a fifth yeast CorA homolog (Mnr2) required for Mg homeostasis. MNR2 gene inactivation was associated with an increase in both the Mg requirement and the Mg content of yeast cells. In Mg-replete conditions, wild-type cells accumulated an intracellular store of Mg that supported growth under deficient conditions. An mnr2 mutant was unable to access this store, suggesting that Mg was trapped in an intracellular compartment. Mnr2 was localized to the vacuole membrane, implicating this organelle in Mg storage. The mnr2 mutant growth and Mg-content phenotypes were dependent on vacuolar proton-ATPase activity, but were unaffected by the loss of mitochondrial Mg uptake, indicating a specific dependence on vacuole function. Overexpression of Mnr2 suppressed the growth defect of an alr1 alr2 mutant, indicating that Mnr2 could function independently of the ALR genes. Together, our results implicate a novel eukaryotic CorA homolog in the regulation of intracellular Mg storage.

Heteromeric protein complexes mediate zinc transport into the secretory pathway of eukaryotic cells.

The cation diffusion facilitator (CDF) family of metal ion transporters plays important roles in zinc transport at all phylogenetic levels. In this report, we describe a novel interaction between two members of the CDF family in Saccharomyces cerevisiae. One CDF member in yeast, Msc2p, was shown recently to be involved in zinc transport into the endoplasmic reticulum (ER) and required for ER function. We describe here a newly recognized CDF family member in yeast, Zrg17p. ZRG17 was previously identified as a zinc-regulated gene controlled by the zinc-responsive Zap1p transcription factor. A zrg17 mutant exhibits the same zinc-suppressible phenotypes as an msc2 mutant, including an induction of the unfolded protein response in low zinc. Moreover, a significant fraction of the total Zrg17p protein appears to localize to the ER. Their common phenotypes and localization suggested that these two proteins function together to mediate zinc transport into the ER. Consistent with this hypothesis, Msc2p and Zrg17p physically interact with each other, as determined by co-immunoprecipitation. Therefore, we propose that Msc2p and Zrg17p form a heteromeric zinc transport complex in the ER membrane. We also demonstrate that ZnT5 and ZnT6, mammalian homologues of Msc2p and Zrg17p, functionally interact as well. These results suggest that heteromeric complexes formed by different CDF members may be a common phenomenon for this ubiquitous family of metal ion transporters.

Characterization of a Glomus intraradices gene encoding a putative Zn transporter of the cation diffusion facilitator family.

A full-length cDNA (GintZnT1) encoding a putative Zn transporter was isolated from the extraradical mycelium of Glomus intraradices. Based on its sequence analysis, GintZnT1 was classified as a member of the cation diffusion facilitator (CDF) family of heavy metal transporters. Functional analysis of GintZnT1 was performed by heterologous expression in yeast mutants defective in different CDFs. Although Zn sensitivity of the mutants was not reverted, an effect of GintZnT1 on the labile regulatory Zn pool was detected by using a Zn-regulated beta-galactosidase reporter gene. GintZnT1 expression was studied in the extraradical mycelium obtained from a symbiotic root organ culture. Gin +/- ZnT1 was up-regulated in the extraradical mycelium of G. intraradices upon short-time exposure to Zn and when the mycelia were developed in 75 microM Zn supplemented plates. These data suggest a role of GintZnT1 in Zn compartmentalization and in the protection of G. intraradices against Zn stress.

Zinc and the Msc2 zinc transporter protein are required for endoplasmic reticulum function.

In this report, we show that zinc is required for endoplasmic reticulum function in Saccharomyces cerevisiae. Zinc deficiency in this yeast induces the unfolded protein response (UPR), a system normally activated by unfolded ER proteins. Msc2, a member of the cation diffusion facilitator (CDF) family of metal ion transporters, was previously implicated in zinc homeostasis. Our results indicate that Msc2 is one route of zinc entry into the ER. Msc2 localizes to the ER when expressed at normal levels. UPR induction in low zinc is exacerbated in an msc2 mutant. Genetic and biochemical evidence indicates that this UPR induction is due to genuine ER dysfunction. Notably, we found that ER-associated protein degradation is defective in zinc-limited msc2 mutants. We also show that the vacuolar CDF proteins Zrc1 and Cot1 are other pathways of ER zinc acquisition. Finally, zinc deficiency up-regulates the mammalian ER stress response indicating a conserved requirement for zinc in ER function among eukaryotes.

Induction of the ZRC1 metal tolerance gene in zinc-limited yeast confers resistance to zinc shock.

Zinc is an essential nutrient but toxic to cells with overaccumulation. For this reason, intracellular zinc levels are tightly controlled. In the yeast Saccharomyces cerevisiae, the Zrc1 and Cot1 proteins have been implicated in the storage and detoxification of excess zinc in the vacuole. Surprisingly, transcription of ZRC1 is induced in zinc-limited cells by the zinc-responsive transcription factor Zap1. We show here that this increase in ZRC1 expression is a novel mechanism of zinc homeostasis and stress tolerance. Zinc-limited cells also express high levels of the plasma membrane zinc uptake transporters. As a consequence, when zinc-limited cells are resupplied with small amounts of zinc, large quantities quickly accumulate in the cell, a condition we refer to as "zinc shock." We show here that ZRC1 and its induction in zinc-limited cells are required for resistance to this zinc shock. Experiments using the zinc-responsive fluorophore FuraZin-1 as an indicator of vacuolar zinc levels indicated that Zrc1 is required for the rapid transport of zinc into the vacuole during zinc shock. We also present evidence that cytosolic zinc rises to higher levels in cells unable to sequester this excess zinc. Thus, the increase in ZRC1 expression occurs prior to the zinc shock stress for which this induction is important. We propose that this "proactive" strategy of homeostatic regulation, such as we document here for ZRC1, may represent a common but largely unrecognized phenomenon.

Biochemical properties of vacuolar zinc transport systems of Saccharomyces cerevisiae.

The yeast vacuole plays an important role in zinc homeostasis by storing zinc for later use under deficient conditions, sequestering excess zinc for its detoxification, and buffering rapid changes in intracellular zinc levels. The mechanisms involved in vacuolar zinc sequestration are only poorly characterized. Here we describe the properties of zinc transport systems in yeast vacuolar membrane vesicles. The major zinc transport activities in these vesicles were ATP-dependent, requiring a H+ gradient generated by the V-ATPase for function. One system we identified was dependent on the ZRC1 gene, which encodes a member of the cation diffusion facilitator family of metal transporters. These data are consistent with the proposed role of Zrc1 as a vacuolar zinc transporter. Zrc1-independent activity was also observed that was not dependent on the closely related vacuolar Cot1 protein. Both Zrc1-dependent and independent activities showed a high specificity for Zn(2+) over other physiologically relevant substrates such as Ca2+, Fe2+, and Mn2+. Moreover, these systems had high affinities for zinc with apparent K(m) values in the 100-200 nm range. These results provide biochemical insight into the important role of Zrc1 and related proteins in eukaryotic zinc homeostasis.