Zinc homeostasis in the secretory pathway in yeast.

It is estimated that up to 10% of proteins in eukaryotes require zinc for their function. Although the majority of these proteins are located in the nucleus and cytosol, a small subset is secreted from cells or is located within an intracellular compartment. As many of these compartmentalized metalloproteins fold to their native state and bind their zinc cofactor inside an organelle, cells require mechanisms to maintain supply of zinc to these compartments even under conditions of zinc deficiency. At the same time, intracellular compartments can also be the site for storing zinc ions, which then can be mobilized when needed. In this review, we highlight insight that has been obtained from yeast models about how zinc homeostasis is maintained in the secretory pathway and vacuole.

The Loz1 transcription factor from Schizosaccharomyces pombe binds to Loz1 response elements and represses gene expression when zinc is in excess.

In Schizosaccharomyces pombe, the expression of the zrt1 zinc uptake gene is tightly regulated by zinc status. When intracellular zinc levels are low, zrt1 is highly expressed. However, when zinc levels are high, transcription of zrt1 is blocked in a manner that is dependent upon the transcription factor Loz1. To gain additional insight into the mechanism by which Loz1 inhibits gene expression in high zinc, we used RNA-seq to identify Loz1-regulated genes, and ChIP-seq to analyze the recruitment of Loz1 to target gene promoters. We find that Loz1 is recruited to the promoters of 27 genes that are also repressed in high zinc in a Loz1-dependent manner. We also find that the recruitment of Loz1 to the majority of target gene promoters is dependent upon zinc and the motif 5'-CGN(A/C)GATCNTY-3', which we have named the Loz1 response element (LRE). Using reporter assays, we show that LREs are both required and sufficient for Loz1-mediated gene repression, and that the level of gene repression is dependent upon the number and sequence of LREs. Our results elucidate the Loz1 regulon in fission yeast and provide new insight into how eukaryotic cells are able to respond to changes in zinc availability in the environment.

Zinc-dependent activation of the Pho8 alkaline phosphatase in Schizosaccharomyces pombe.

Genome-wide analyses have revealed that during metal ion starvation, many cells undergo programmed changes in their transcriptome or proteome that lower the levels of abundant metalloproteins, conserving metal ions for more critical functions. Here we investigated how changes in cellular zinc status affect the expression and activity of the zinc-requiring Pho8 alkaline phosphatase from fission yeast (Schizosaccharomyces pombe). In S. pombe, Pho8 is a membrane-tethered and processed glycoprotein that resides in the vacuole. Using alkaline phosphatase activity assays along with various biochemical analyses, we found that Pho8 is active when zinc is plentiful and inactive when zinc is limited. Although Pho8 activity depended on zinc, we also found that higher levels of pho8 mRNAs and Pho8 protein accumulate in zinc-deficient cells. To gain a better understanding of the inverse relationship between pho8 mRNA levels and Pho8 activity, we examined the effects of zinc on the stability and processing of the Pho8 protein. We show that Pho8 is processed regardless of zinc status and that mature Pho8 accumulates under all conditions. We also noted that alkaline phosphatase activity is rapidly restored when zinc is resupplied to cells, even in the presence of the protein synthesis inhibitor cycloheximide. Our results suggest that S. pombe cells maintain inactive pools of Pho8 proteins under low-zinc conditions and that these pools facilitate rapid restoration of Pho8 activity when zinc ions become available.

Zinc transporters belonging to the Cation Diffusion Facilitator (CDF) family have complementary roles in transporting zinc out of the cytosol.

Zinc is an essential trace element that is required for the function of a large number of proteins. As these zinc-binding proteins are found within the cytosol and organelles, all eukaryotes require mechanisms to ensure that zinc is delivered to organelles, even under conditions of zinc deficiency. Although many zinc transporters belonging to the Cation Diffusion Facilitator (CDF) families have well characterized roles in transporting zinc into the lumens of intracellular compartments, relatively little is known about the mechanisms that maintain organelle zinc homeostasis. The fission yeast Schizosaccharomyces pombe is a useful model system to study organelle zinc homeostasis as it expresses three CDF family members that transport zinc out of the cytosol into intracellular compartments: Zhf1, Cis4, and Zrg17. Zhf1 transports zinc into the endoplasmic reticulum, and Cis4 and Zrg17 form a heterodimeric complex that transports zinc into the cis-Golgi. Here we have used the high and low affinity ZapCY zinc-responsive FRET sensors to examine cytosolic zinc levels in yeast mutants that lack each of these CDF proteins. We find that deletion of cis4 or zrg17 leads to higher levels of zinc accumulating in the cytosol under conditions of zinc deficiency, whereas deletion of zhf1 results in zinc accumulating in the cytosol when zinc is not limiting. We also show that the expression of cis4, zrg17, and zhf1 is independent of cellular zinc status. Taken together our results suggest that the Cis4/Zrg17 complex is necessary for zinc transport out of the cytosol under conditions of zinc-deficiency, while Zhf1 plays the dominant role in removing zinc from the cytosol when labile zinc is present. We propose that the properties and/or activities of individual CDF family members are fine-tuned to enable cells to control the flux of zinc out of the cytosol over a broad range of environmental zinc stress.

The Zap1 transcriptional activator negatively regulates translation of the RTC4 mRNA through the use of alternative 5' transcript leaders.

The zinc-responsive transcription activator Zap1 plays a central role in zinc homeostasis in the budding yeast Saccharomyces cerevisiae. In zinc-deficient cells, Zap1 binds to zinc responsive elements in target gene promoters and activates gene expression. In most cases, Zap1-dependent gene activation results in increased levels of mRNAs and proteins. However, Zap1-dependent activation of RTC4 results in increased levels of the RTC4 mRNA and decreased levels of the Rtc4 protein. This atypical regulation results from Zap1-mediated changes in the transcriptional start site for RTC4 and the production of a RTC4 transcript with a longer 5' leader. This long RTC4 transcript contains small upstream open reading frames that prevent translation of the downstream RTC4 ORF. The new studies with Zap1 highlight how a transcriptional activator can facilitate decreased protein expression.

The gluconate shunt is an alternative route for directing glucose into the pentose phosphate pathway in fission yeast.

Glycolysis and the pentose phosphate pathway both play a central role in the degradation of glucose in all domains of life. Another metabolic route that can facilitate glucose breakdown is the gluconate shunt. In this shunt glucose dehydrogenase and gluconate kinase catalyze the two-step conversion of glucose into the pentose phosphate pathway intermediate 6-phosphogluconate. Despite the presence of these enzymes in many organisms, their only established role is in the production of 6-phosphogluconate for the Entner-Doudoroff pathway. In this report we performed metabolic profiling on a strain of Schizosaccharomyces pombe lacking the zinc-responsive transcriptional repressor Loz1 with the goal of identifying metabolic pathways that were altered by cellular zinc status. This profiling revealed that loz1Δ cells accumulate higher levels of gluconate. We show that the altered gluconate levels in loz1Δ cells result from increased expression of gcd1 By analyzing the activity of recombinant Gcd1 in vitro and by measuring gluconate levels in strains lacking enzymes of the gluconate shunt we demonstrate that Gcd1 encodes a novel NADP+-dependent glucose dehydrogenase that acts in a pathway with the Idn1 gluconate kinase. We also find that cells lacking gcd1 and zwf1, which encode the first enzyme in the pentose phosphate pathway, have a more severe growth phenotype than cells lacking zwf1 We propose that in S. pombe Gcd1 and Idn1 act together to shunt glucose into the pentose phosphate pathway, creating an alternative route for directing glucose into the pentose phosphate pathway that bypasses hexokinase and the rate-limiting enzyme glucose-6-phosphate dehydrogenase.

Zinc sensing and regulation in yeast model systems.

The Zap1 transcription factor of Saccharomyces cerevisiae and the Loz1 transcription factor of Schizosaccharomyces pombe both play a central role in zinc homeostasis by controlling the expression of genes necessary for zinc metabolism. Zap1 activates gene expression when cells are limited for zinc, while Loz1 is required for gene repression when zinc is in excess. In this review we highlight what is known about the underlying mechanisms by which these factors are regulated by zinc, and how transcriptional activation and repression in eukaryotic cells can be finely tuned according to intracellular zinc availability.

Cellular sensing and transport of metal ions: implications in micronutrient homeostasis.

Micronutrients include the transition metal ions zinc, copper and iron. These metals are essential for life as they serve as cofactors for many different proteins. On the other hand, they can also be toxic to cell growth when in excess. As a consequence, all organisms require mechanisms to tightly regulate the levels of these metal ions. In eukaryotes, one of the primary ways in which metal levels are regulated is through changes in expression of genes required for metal uptake, compartmentalization, storage and export. By tightly regulating the expression of these genes, each organism is able to balance metal levels despite fluctuations in the diet or extracellular environment. The goal of this review is to provide an overview of how gene expression can be controlled at a transcriptional, posttranscriptional and posttranslational level in response to metal ions in lower and higher eukaryotes. Specifically, I review what is known about how these metalloregulatory factors sense fluctuations in metal ion levels and how changes in gene expression maintain nutrient homeostasis.

The double zinc finger domain and adjacent accessory domain from the transcription factor loss of zinc sensing 1 (loz1) are necessary for DNA binding and zinc sensing.

The Loz1 transcription factor from Schizosaccharomyces pombe plays an essential role in zinc homeostasis by repressing target gene expression in zinc-replete cells. To determine how Loz1 function is regulated by zinc, we employed a genetic screen to isolate mutants with impaired zinc-dependent gene expression and analyzed Loz1 protein truncations to map a minimal zinc-responsive domain. In the screen, we isolated 36 new loz1 alleles. 27 of these alleles contained mutations resulting in the truncation of the Loz1 protein. The remaining nine alleles contained point mutations leading to an amino acid substitution within a C-terminal double zinc finger domain. Further analysis of two of these substitutions revealed that they disrupted Loz1 DNA activity in vitro. By analyzing Loz1 protein truncations, we found that the last 96 amino acids of Loz1 was the smallest region that was able to confer partial zinc-dependent repression in vivo. This 96-amino acid region contains the double zinc finger domain and an accessory domain that enhances DNA binding. These results were further supported by the findings that MtfA, a transcription factor from Aspergillus nidulans that contains a related double zinc finger, is unable to complement loz1Δ, whereas a chimera of MtfA containing the Loz1 accessory domain is able to complement loz1Δ. Together, our studies indicate that the double zinc finger domain and adjacent accessory domain preceding zinc finger 1 are necessary for DNA binding and zinc-dependent repression.

Zinc'ing sensibly: controlling zinc homeostasis at the transcriptional level.

Zinc-responsive transcription factors are found in all kingdoms of life and include the transcriptional activators ZntR, SczA, Zap1, bZip19, bZip23, and MTF-1, and transcriptional repressors Zur, AdcR, Loz1, and SmtB. These factors have two defining features; their activity is regulated by zinc and they all play a central role in zinc homeostasis by controlling the expression of genes that directly affect zinc levels or its availability. This review summarizes what is known about the mechanisms by which each of these factors sense changes in intracellular zinc levels and how they control zinc homeostasis through target gene regulation. Other factors that influence zinc ion sensing are also discussed.

Zinc finger protein Loz1 is required for zinc-responsive regulation of gene expression in fission yeast.

In Schizosaccharomyces pombe, alcohol dehydrogenase 1 (Adh1) is an abundant zinc-requiring enzyme that catalyses the conversion of acetaldehyde to ethanol during fermentation. In a zinc-replete cell, adh1 is highly expressed. However, in zinc-limited cells, adh1 gene expression is repressed, and cells induce the expression of an alternative alcohol dehydrogenase encoded by the adh4 gene. In our studies examining this zinc-dependent switch in alcohol dehydrogenase gene expression, we isolated an adh1Δ strain containing a partial loss of function mutation that resulted in higher levels of adh4 transcripts in zinc-replete cells. This mutation also led to the aberrant expression of other genes that are typically regulated by zinc. Using linkage analysis, we have mapped the position of this mutation to a single gene called Loss Of Zinc sensing 1 (loz1). Loz1 is a 55-kDa protein that contains a double C2H2-type zinc finger domain. The mapped mutation that disrupts Loz1 function leads to an arginine to glycine substitution in the second zinc finger domain, suggesting that the double zinc finger domain is important for Loz1 function. We show that loz1Δ cells hyperaccumulate zinc and that Loz1 is required for gene repression in zinc-replete cells. We also have found that Loz1 negatively autoregulates its own expression. We propose that Loz1 is a unique metalloregulatory factor that plays a central role in zinc homeostasis in S. pombe.

Zinc-dependent regulation of the Adh1 antisense transcript in fission yeast.

In yeast, Adh1 (alcohol dehydrogenase 1) is an abundant zinc-binding protein that is required for the conversion of acetaldehyde to ethanol. Through transcriptome profiling of the Schizosaccharomyces pombe genome, we identified a natural antisense transcript at the adh1 locus that is induced in response to zinc limitation. This antisense transcript (adh1AS) shows a reciprocal expression pattern to that of the adh1 mRNA partner. In this study, we show that increased expression of the adh1AS transcript in zinc-limited cells is necessary for the repression of adh1 gene expression and that the increased level of the adh1AS transcript in zinc-limited cells is a result of two mechanisms. At the transcriptional level, the adh1AS transcript is expressed at a high level in zinc-limited cells. In addition to this transcriptional control, adh1AS transcripts preferentially accumulate in zinc-limited cells when the adh1AS transcript is expressed from a constitutive promoter. This secondary mechanism requires the simultaneous expression of adh1. Our studies reveal how multiple mechanisms can synergistically control the ratio of sense to antisense transcripts and highlight a novel mechanism by which adh1 gene expression can be controlled by cellular zinc availability.

Hammering out details: regulating metal levels in eukaryotes.

The transition metals zinc, iron and copper are common constituents in a wide range of proteins. Although these metals are all essential for life, when present in excess, they are frequently toxic to cell growth and viability. Therefore, all organisms rely on sophisticated mechanisms to maintain optimal levels of each metal. Genes that encode metal transport or storage proteins are often regulated at the transcriptional level in response to changes in metal status. In this review, we focus on what is known about the transcription factors that mediate these metal-dependent changes. Specifically, we highlight recent advances in our understanding of the mechanisms by which these factors sense metal ions.

Zinc-regulated DNA binding of the yeast Zap1 zinc-responsive activator.

The Zap1 transcription factor of Saccharomyces cerevisiae plays a central role in zinc homeostasis by controlling the expression of genes involved in zinc metabolism. Zap1 is active in zinc-limited cells and repressed in replete cells. At the transcriptional level, Zap1 controls its own expression via positive autoregulation. In addition, Zap1's two activation domains are regulated independently of each other by zinc binding directly to those regions and repressing activation function. In this report, we show that Zap1 DNA binding is also inhibited by zinc. DMS footprinting showed that Zap1 target gene promoter occupancy is regulated with or without transcriptional autoregulation. These results were confirmed using chromatin immunoprecipitation. Zinc regulation of DNA binding activity mapped to the DNA binding domain indicating other parts of Zap1 are unnecessary for this control. Overexpression of Zap1 overrode DNA binding regulation and resulted in constitutive promoter occupancy. Under these conditions of constitutive binding, both the zinc dose response of Zap1 activity and cellular zinc accumulation were altered suggesting the importance of DNA binding control to zinc homeostasis. Thus, our results indicated that zinc regulates Zap1 activity post-translationally via three independent mechanisms, all of which contribute to the overall zinc responsiveness of Zap1.

Repression of sulfate assimilation is an adaptive response of yeast to the oxidative stress of zinc deficiency.

The Zap1 transcription factor is a central player in the response of yeast to changes in zinc status. Previous studies identified over 80 genes activated by Zap1 in zinc-limited cells. In this report, we identified 36 genes repressed in a zinc- and Zap1-responsive manner. As a result, we have identified a new mechanism of Zap1-mediated gene repression whereby transcription of the MET3, MET14, and MET16 genes is repressed in zinc-limited cells. These genes encode the first three enzymes of the sulfate assimilation pathway. We found that MET30, encoding a component of the SCF(Met30) ubiquitin ligase, is a direct Zap1 target gene. MET30 expression is increased in zinc-limited cells, and this leads to degradation of Met4, a transcription factor responsible for MET3, MET14, and MET16 expression. Thus, Zap1 is responsible for a decrease in sulfate assimilation in zinc-limited cells. We further show that cells that are unable to down-regulate sulfate assimilation under zinc deficiency experience increased oxidative stress. This increased oxidative stress is associated with an increase in the NADP(+)/NADPH ratio and may result from a decrease in NADPH-dependent antioxidant activities. These studies have led to new insights into how cells adapt to nutrient-limiting growth conditions.

Differential control of Zap1-regulated genes in response to zinc deficiency in Saccharomyces cerevisiae.

BACKGROUND: The Zap1 transcription factor is a central player in the response of yeast to changes in zinc status. We previously used transcriptome profiling with DNA microarrays to identify 46 potential Zap1 target genes in the yeast genome. In this new study, we used complementary methods to identify additional Zap1 target genes. RESULTS: With alternative growth conditions for the microarray experiments and a more sensitive motif identification algorithm, we identified 31 new potential targets of Zap1 activation. Moreover, an analysis of the response of Zap1 target genes to a range of zinc concentrations and to zinc withdrawal over time demonstrated that these genes respond differently to zinc deficiency. Some genes are induced under mild zinc deficiency and act as a first line of defense against this stress. First-line defense genes serve to maintain zinc homeostasis by increasing zinc uptake, and by mobilizing and conserving intracellular zinc pools. Other genes respond only to severe zinc limitation and act as a second line of defense. These second-line defense genes allow cells to adapt to conditions of zinc deficiency and include genes involved in maintaining secretory pathway and cell wall function, and stress responses. CONCLUSION: We have identified several new targets of Zap1-mediated regulation. Furthermore, our results indicate that through the differential regulation of its target genes, Zap1 prioritizes mechanisms of zinc homeostasis and adaptive responses to zinc deficiency.

Metallosensors, the ups and downs of gene regulation.

In fungal cells, transcriptional regulatory mechanisms play a central role in both the homeostatic regulation of the essential metals iron, copper and zinc and in the detoxification of heavy metal ions such as cadmium. Fungi detect changes in metal ion levels using unique metallo-regulatory factors whose activity is responsive to the cellular metal ion status. New studies have revealed that these factors not only regulate the expression of genes required for metal ion acquisition, storage or detoxification but also globally remodel metabolism to conserve metal ions or protect against metal toxicity. This review focuses on the mechanisms metallo-regulators use to up- and down-regulate gene expression.

Regulation of the yeast TSA1 peroxiredoxin by ZAP1 is an adaptive response to the oxidative stress of zinc deficiency.

Zinc deficiency is a potential risk factor for disease in humans because it leads to increased oxidative stress and DNA damage. We show here that the yeast Saccharomyces cerevisiae also experiences oxidative stress when zinc-deficient, and we have identified one mechanism yeast cells use to defend themselves against this stress. The Zap1p transcription factor is a central player in the response of yeast to zinc deficiency. To identify genes important for growth in low zinc, DNA microarrays were used to identify genes directly regulated by Zap1p. We found that the TSA1 gene is one such Zap1p target whose expression is increased under zinc deficiency. TSA1 encodes a cytosolic thioredoxin-dependent peroxidase responsible for degrading hydrogen peroxide and organic hydroperoxides. Consistent with its regulation by Zap1p, we showed that tsa1delta mutants have a growth defect in low zinc that can be suppressed by zinc but not by other metals. Anaerobic conditions also suppressed the tsa1delta low zinc growth defect indicating that oxidative stress is the likely cause of the poor growth. Consistent with this hypothesis, we demonstrated that zinc deficiency causes increased reactive oxygen species in wild type cells and that this increase is further exacerbated in tsa1delta mutants. The role of this regulation by Zap1p in limiting oxidative stress in low zinc was confirmed when the Zap1p-binding site was specifically mutated in the chromosomal TSA1 promoter. Thus, we conclude that TSA1 induction by Zap1p is an adaptive response to deal with the increased oxidative stress caused by zinc deficiency.

Repression of ADH1 and ADH3 during zinc deficiency by Zap1-induced intergenic RNA transcripts.

The transcriptional activator Zap1 induces target gene expression in response to zinc deficiency. We demonstrate that during zinc starvation, Zap1 is required for the repression of ADH1 expression. ADH1 encodes the major zinc-dependent alcohol dehydrogenase that is utilized during fermentation. During zinc starvation, Zap1 binds upstream of the activator Rap1 and induces an intergenic RNA transcript, ZRR1. ZRR1 expression leads to the transient displacement of Rap1 from the ADH1 promoter resulting in ADH1 repression. Using a microarray-based approach, we screened for additional genes repressed by Zap1 intergenic transcripts. We found that ADH3, the major mitochondrial alcohol dehydrogenase, is regulated in a manner similar to ADH1. Thus, during zinc deficiency, Zap1 mediates the repression of two of the most abundant zinc-requiring enzymes.

Zinc metalloregulation of the zinc finger pair domain.

The yeast transcriptional activator Zap1 contains two uncommon structural motifs designated zinc finger pair domains. The hallmark of this domain is the packing of two zinc finger motifs in one globular unit. One finger pair domain in Zap1 contains the AD2 transactivation domain. Zn(II) binding to this domain (ZF1/2) is kinetically labile yielding a zinc-regulated transactivator. The second finger pair domain (ZF3/4) lies within the DNA-binding domain, and it stably binds Zn(II). The goal of this study was to map the determinant conferring lability in Zn(II) binding by using finger pair chimeras. Whereas ZF2 contains the transactivation function, zinc regulation is dependent on the presence of ZF1. ZF3 can functionally replace ZF1, and a ZF3/2 finger pair retains limited zinc regulation. Replacement of ZF3 by ZF1 creating a ZF1/4 chimera was found to stably bind Zn(II), suggesting that the presence of a stable motif (ZF4) can impart binding stability on a labile motif (ZF1). Zn(II) binding in finger pair domains is dependent on the presence of both motifs. Mutations in one finger motif markedly attenuate Zn(II) binding to the second motif. Kinetic lability in Zn(II) binding was mapped to the alpha-helix of ZF2. A ZF1/ZFbeta2alpha4 chimera resembles ZF3/4 in Zn(II) binding stability in incubation studies with the Zn(II) chelators. The present results demonstrate that zinc regulation of AD activity of ZF2 is dependent on determinants in ZF1 as well as the alpha-helix segment of ZF2.