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.

Transcription factors and transporters in zinc homeostasis: lessons learned from fungi.

Zinc is an essential nutrient for all organisms because this metal serves as a critical structural or catalytic cofactor for many proteins. These zinc-dependent proteins are abundant in the cytosol as well as within organelles of eukaryotic cells such as the nucleus, mitochondria, endoplasmic reticulum, Golgi, and storage compartments such as the fungal vacuole. Therefore, cells need zinc transporters so that they can efficiently take up the metal and move it around within cells. In addition, because zinc levels in the environment can vary drastically, the activity of many of these transporters and other components of zinc homeostasis is regulated at the level of transcription by zinc-responsive transcription factors. Mechanisms of post-transcriptional control are also important for zinc homeostasis. In this review, the focus will be on our current knowledge of zinc transporters and their regulation by zinc-responsive transcription factors and other mechanisms in fungi because these organisms have served as useful paradigms of zinc homeostasis in all organisms. With this foundation, extension to other organisms will be made where warranted.

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.

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.

Bacillithiol, a new role in buffering intracellular zinc.

Zinc is a catalytic or structural cofactor of numerous proteins but can also be toxic if cells accumulate too much of this essential metal. Therefore, mechanisms of zinc homeostasis are needed to maintain a low but adequate amount of free zinc so that newly translated zinc-dependent proteins can bind their cofactor without confounding issues of toxicity. These mechanisms include the regulation of uptake and efflux transporters and buffering of the free metal concentration by low-molecular-weight ligands in the cytosol. While many of the transporters involved in zinc homeostasis have been discovered in recent years, the molecules that buffer zinc have remained largely a mystery. In the new report highlighted by this commentary, Ma et al. (2014) provide convincing evidence that bacillithiol, the major low-molecular-weight thiol compound in Bacillus subtilis, serves as an important zinc buffer in those cells. Their discovery provides an important piece to the puzzle of how zinc buffering occurs in a large number of microbes and provides new clues about the role and relative importance of zinc buffering in all organisms.

Genome-wide functional profiling identifies genes and processes important for zinc-limited growth of Saccharomyces cerevisiae.

Zinc is an essential nutrient because it is a required cofactor for many enzymes and transcription factors. To discover genes and processes in yeast that are required for growth when zinc is limiting, we used genome-wide functional profiling. Mixed pools of ∼4,600 deletion mutants were inoculated into zinc-replete and zinc-limiting media. These cells were grown for several generations, and the prevalence of each mutant in the pool was then determined by microarray analysis. As a result, we identified more than 400 different genes required for optimal growth under zinc-limiting conditions. Among these were several targets of the Zap1 zinc-responsive transcription factor. Their importance is consistent with their up-regulation by Zap1 in low zinc. We also identified genes that implicate Zap1-independent processes as important. These include endoplasmic reticulum function, oxidative stress resistance, vesicular trafficking, peroxisome biogenesis, and chromatin modification. Our studies also indicated the critical role of macroautophagy in low zinc growth. Finally, as a result of our analysis, we discovered a previously unknown role for the ICE2 gene in maintaining ER zinc homeostasis. Thus, functional profiling has provided many new insights into genes and processes that are needed for cells to thrive under the stress of zinc deficiency.

Promotion of vesicular zinc efflux by ZIP13 and its implications for spondylocheiro dysplastic Ehlers-Danlos syndrome.

Zinc is essential but potentially toxic, so intracellular zinc levels are tightly controlled. A key strategy used by many organisms to buffer cytosolic zinc is to store it within vesicles and organelles.It is yet unknown whether vesicular or organellar sites perform this function in mammals. Human ZIP13, a member of the Zrt/Irt-like protein (ZIP) metal transporter family, might provide an answer to this question. Mutations in the ZIP13 gene, SLC39A13, previously were found to cause the spondylocheiro dysplastic form of Ehlers­Danlos syndrome (SCD-EDS), a heritable connective tissue disorder.Those previous studies suggested that ZIP13 transports excess zinc out of the early secretory pathway and that zinc overload in the endoplasmic reticulum (ER) occurs in SCD-EDS patients. In contrast,this study indicates that ZIP13's role is to release labile zinc from vesicular stores for use in the ER and other compartments. We propose that SCD-EDS is the result of vesicular zinc trapping and ER zinc deficiency rather than 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.

Roles of two activation domains in Zap1 in the response to zinc deficiency in Saccharomyces cerevisiae.

Previous studies suggested that the zinc-responsive Zap1 transcriptional activator directly regulates the expression of over 80 genes in Saccharomyces cerevisiae. Many of these genes play key roles to enhance the ability of yeast cells to grow under zinc-limiting conditions. Zap1 is unusual among transcriptional activators in that it contains two activation domains, designated AD1 and AD2, which are regulated independently by zinc. These two domains are evolutionarily conserved among Zap1 orthologs suggesting that they are both important for Zap1 function. In this study, we have examined the roles of AD1 and AD2 in low zinc growth and the regulation of Zap1 target gene expression. Using alleles that are specifically disrupted for either AD1 or AD2 function, we found that these domains are not redundant, and both are important for normal growth in low zinc. AD1 plays the primary role in zinc-responsive gene regulation, whereas AD2 is required for maximal expression of only a few target promoters. AD1 alone is capable of driving full expression of most Zap1 target genes and dictates the kinetics of Zap1 gene induction in response to zinc withdrawal. Surprisingly, we found that AD1 is less active in zinc-limited cells under heat stress and AD2 plays a more important role under those conditions. These results suggest that AD2 may contribute more to Zap1 function when zinc deficiency is combined with other environmental stresses. In the course of these studies, we also found that the heat shock response is induced under conditions of severe zinc deficiency.

Transcriptional regulation of the Zrg17 zinc transporter of the yeast secretory pathway.

The Msc2 and Zrg17 proteins of Saccharomyces cerevisiae are members of the cation diffusion facilitator family of zinc transporters. These proteins form heteromeric complexes that transport zinc into the ER (endoplasmic reticulum). Previous studies suggested that the ZRG17 gene is regulated in response to zinc status by the Zap1 transcription factor. Zap1 activates the expression of many genes in zinc-deficient cells. In the present study, we assessed whether ZRG17 is a direct Zap1 target gene. We showed that ZRG17 mRNA levels were elevated in zinc-limited cells in a Zap1-dependent manner and were also elevated in zinc-replete cells expressing a constitutively active allele of Zap1. Furthermore, Zrg17 protein levels correlated closely with mRNA levels. A candidate Zap1-binding site [ZRE (zinc-responsive element)] in the ZRG17 promoter was required for this induction. Using electrophoretic mobility-shift assays and chromatin immunoprecipitation, we demonstrated that Zap1 binds specifically to the ZRG17 ZRE both in vitro and in vivo. By using a chromosomal ZRG17 mutant with a non-functional ZRE, we found that Zap1 induction of ZRG17 is required for ER function as indicated by elevated ER stress under zinc-limited conditions. Together, these results establish that ZRG17 is a direct Zap1 target gene and its regulation has biological importance in maintaining ER function.

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.

Zinc status and vacuolar zinc transporters control alkaline phosphatase accumulation and activity in Saccharomyces cerevisiae.

Little is known about how metalloproteins in the secretory pathway obtain their metal ion cofactors. We used the Pho8 alkaline phosphatase of the yeast Saccharomyces cerevisiae to probe this process in vivo. We found that both Pho8 activity and protein accumulation are zinc-dependent and decrease in zinc-limited cells. Low Pho8 accumulation was the result of degradation by vacuolar proteases. Surprisingly, the protective effect of zinc on Pho8 stability was not solely due to Zn(2+) binding to the active-site ligands suggesting that the Pho8 protein is targeted for degradation in zinc-limited cells by another mechanism. Pho8 appears to be a rare example of a metalloprotein whose stability is regulated by its metal cofactor independently of active-site binding. We also assessed which zinc transporters are responsible for supplying zinc to Pho8. We found that the Zrc1 and Cot1 vacuolar zinc transporters play the major role while the Msc2/Zrg17 zinc transporter complex active in the endoplasmic reticulum is not involved. These results demonstrate that the vacuolar zinc transporters, previously implicated in metal detoxification, also deliver zinc to certain metalloproteins within intracellular compartments. These data suggest that Pho8 receives its metal cofactor in the vacuole rather than in earlier compartments of the secretory pathway.

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.

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.

Evolution, and functional analysis of Natural Resistance-Associated Macrophage Proteins (NRAMPs) from Theobroma cacao and their role in cadmium accumulation.

The presence of the toxic metal cadmium (Cd2+) in certain foodstuffs is recognised as a global problem, and there is increasing legislative pressure to reduce the content of Cd in food. The present study was conducted on cacao (Theobroma cacao), the source of chocolate, and one of the crops known to accumulate Cd in certain conditions. There are a range of possible genetic and agronomic methods being tested as a route to such reduction. As part of a gene-based approach, we focused on the Natural Resistance-Associated Macrophage Proteins (NRAMPS), a family of proton/metal transporter proteins that are evolutionarily conserved across all species from bacteria to humans. The plant NRAMP gene family are of particular importance as they are responsible for uptake of the nutritionally vital divalent cations Fe2+, Mn2+, Zn2+, as well as Cd2+. We identified the five NRAMP genes in cacao, sequenced these genes and studied their expression in various organs. We then confirmed the expression patterns in response to variation in nutrient cation availability and addition of Cd2+. Functional analysis by expression in yeast provided evidence that NRAMP5 encoded a protein capable of Cd2+ transport, and suggested this gene as a target for genetic selection/modification.

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.

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.

An Autophagy-Independent Role for ATG41 in Sulfur Metabolism During Zinc Deficiency.

The Zap1 transcription factor of Saccharomyces cerevisiae is a key regulator in the genomic responses to zinc deficiency. Among the genes regulated by Zap1 during zinc deficiency is the autophagy-related gene ATG41 Here, we report that Atg41 is required for growth in zinc-deficient conditions, but not when zinc is abundant or when other metals are limiting. Consistent with a role for Atg41 in macroautophagy, we show that nutritional zinc deficiency induces autophagy and that mutation of ATG41 diminishes that response. Several experiments indicated that the importance of ATG41 function to growth during zinc deficiency is not because of its role in macroautophagy, but rather is due to one or more autophagy-independent functions. For example, rapamycin treatment fully induced autophagy in zinc-deficient atg41Δ mutants but failed to improve growth. In addition, atg41Δ mutants showed a far more severe growth defect than any of several other autophagy mutants tested, and atg41Δ mutants showed increased Heat Shock Factor 1 activity, an indicator of protein homeostasis stress, while other autophagy mutants did not. An autophagy-independent function for ATG41 in sulfur metabolism during zinc deficiency was suggested by analyzing the transcriptome of atg41Δ mutants during the transition from zinc-replete to -deficient conditions. Analysis of sulfur metabolites confirmed that Atg41 is needed for the normal accumulation of methionine, homocysteine, and cysteine in zinc-deficient cells. Therefore, we conclude that Atg41 plays roles in both macroautophagy and sulfur metabolism during zinc deficiency.

Zinc transporters and the cellular trafficking of zinc.

Zinc is an essential nutrient for all organisms because this metal serves as a catalytic or structural cofactor for many different proteins. Zinc-dependent proteins are found in the cytoplasm and within many organelles of the eukaryotic cell including the nucleus, the endoplasmic reticulum, Golgi, secretory vesicles, and mitochondria. Thus, cells require zinc transport mechanisms to allow cells to efficiently accumulate the metal ion and distribute it within the cell. Our current knowledge of these transport systems in eukaryotes is the focus of this review.