The Gene Encodes the Low Affinity Zinc Transporter in
1996; Elsevier BV; Volume: 271; Issue: 38 Linguagem: Inglês
10.1074/jbc.271.38.23203
ISSN1083-351X
Autores Tópico(s)Aluminum toxicity and tolerance in plants and animals
ResumoZinc accumulation in Saccharomyces cerevisiae occurs through either of two uptake systems. A high affinity system is active in zinc-limited cells, and the ZRT1 gene encodes the transporter protein of this system. In this study, we characterized the low affinity system that is active in zinc-replete cells. The low affinity system is time-, temperature-, and concentration-dependent and prefers zinc over other metals as its substrate. Our results suggest that the ZRT2 gene encodes the transporter of this system. The amino acid sequence of Zrt2p is remarkably similar to those of Zrt1p and Irt1p, an Fe2+ transporter from Arabidopsis thaliana. Overexpressing ZRT2 increased low affinity uptake, whereas disrupting this gene eliminated that activity, but had little effect on the high affinity system. Therefore, the high and low affinity systems are separate uptake pathways. Analysis of the zinc levels required for growth of zrt2 mutant strains as well as the effects of the zrt2 mutation on the regulation of the high affinity system demonstrated that the low affinity system is a biologically relevant mechanism of zinc accumulation. Finally, a zrt1zrt2 mutant was viable, indicating the existence of additional zinc uptake pathways. Zinc accumulation in Saccharomyces cerevisiae occurs through either of two uptake systems. A high affinity system is active in zinc-limited cells, and the ZRT1 gene encodes the transporter protein of this system. In this study, we characterized the low affinity system that is active in zinc-replete cells. The low affinity system is time-, temperature-, and concentration-dependent and prefers zinc over other metals as its substrate. Our results suggest that the ZRT2 gene encodes the transporter of this system. The amino acid sequence of Zrt2p is remarkably similar to those of Zrt1p and Irt1p, an Fe2+ transporter from Arabidopsis thaliana. Overexpressing ZRT2 increased low affinity uptake, whereas disrupting this gene eliminated that activity, but had little effect on the high affinity system. Therefore, the high and low affinity systems are separate uptake pathways. Analysis of the zinc levels required for growth of zrt2 mutant strains as well as the effects of the zrt2 mutation on the regulation of the high affinity system demonstrated that the low affinity system is a biologically relevant mechanism of zinc accumulation. Finally, a zrt1zrt2 mutant was viable, indicating the existence of additional zinc uptake pathways. INTRODUCTIONHow the cells of all organisms acquire metal ions from their extracellular environment is one of the central unresolved questions in the biochemistry of these important nutrients. Zinc is essential because it is an integral cofactor of many proteins and is a critical determinant of their catalytic activity and/or structural stability (1Vallee B.L. Auld D.S. Biochemistry. 1990; 9: 5647-5659Crossref Scopus (1512) Google Scholar). Moreover, zinc is an important component of many transcription factors, the zinc finger proteins, that regulate gene expression (2Rhodes D. Klug A. Sci. Am. 1993; 268: 56-62Crossref PubMed Scopus (85) Google Scholar). Biochemical assays of zinc uptake in yeast indicated that this process is transporter-mediated, i.e. zinc uptake is time-, temperature-, and concentration-dependent and requires metabolic energy (3Fuhrmann G.F. Rothstein A. Biochim. Biophys. Acta. 1968; 163: 325-330Crossref PubMed Scopus (118) Google Scholar, 4Mowll J.L. Gadd G.M. J. Gen. Microbiol. 1983; 129: 3421-3425Google Scholar, 5White C. Gadd G.M. J. Gen. Microbiol. 1987; 133: 727-737Google Scholar). Recent studies suggested the presence of two separate uptake systems (6Zhao H. Eide D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2454-2458Crossref PubMed Scopus (445) Google Scholar). One system has high affinity for zinc with an apparent Km of 0.5-1 μ and is required for zinc-limited growth. The ZRT1 (for zinc-regulated transporter) gene appears to encode the transporter protein of this system. ZRT1 is a member of a closely related family of transporter genes found in organisms as diverse as fungi, plants, nematodes, and humans. This family includes the IRT1 gene from Arabidopsis thaliana, which encodes an Fe2+ transporter expressed in plant roots (7Eide D. Broderius M. Fett J. Guerinot M.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5624-5628Crossref PubMed Scopus (1034) Google Scholar). Like the other members of this family, Zrt1p is predicted to be an integral membrane protein containing eight potential transmembrane domains.The level of ZRT1 expression correlated with activity of the high affinity system; overexpression of ZRT1 increased high affinity uptake, whereas a zrt1 mutation eliminated high affinity activity and resulted in poor growth of the mutant on zinc-limited media (6Zhao H. Eide D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2454-2458Crossref PubMed Scopus (445) Google Scholar). The high affinity system was induced in activity >100-fold in response to zinc-limiting growth conditions. When cells were grown in media containing different zinc concentrations, high affinity uptake and ZRT1 mRNA levels were closely correlated, as was the β-galactosidase activity generated by a reporter gene in which the ZRT1 promoter was fused to the Escherichia coli lacZ gene. The ZRT1-lacZ fusion gene showed a similar pattern of regulation in response to cell-associated zinc levels in both wild-type and zrt1 mutant cells despite the 75-fold higher extracellular zinc level required to down-regulate the promoter in the mutant. These results indicate that the activity of the high affinity system is controlled, at least in part, by transcriptional regulation of the ZRT1 gene in response to a regulatory pool of intracellular zinc.The second system for zinc uptake in yeast has a lower affinity for substrate (apparent Km = 10 μ), and it is active in zinc-replete cells. Low affinity uptake was unaffected by the zrt1 mutation, suggesting that this system is a separate uptake pathway for zinc. As described in this report, an initial characterization was conducted to determine some of the biochemical properties of the low affinity system. We also report the analysis of another member of the IRT/ZRT gene family, ZRT2. ZRT2 was identified in the sequence data bases because of the close sequence similarity of its product to Irt1p and Zrt1p (6Zhao H. Eide D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2454-2458Crossref PubMed Scopus (445) Google Scholar, 7Eide D. Broderius M. Fett J. Guerinot M.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5624-5628Crossref PubMed Scopus (1034) Google Scholar). Our analysis of ZRT2 suggests that this gene encodes the transporter protein of the low affinity system.DISCUSSIONOur previous studies (6Zhao H. Eide D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2454-2458Crossref PubMed Scopus (445) Google Scholar) suggested that at least two zinc uptake systems are present in Saccharomyces cerevisiae. The high affinity system has an apparent Km of 1 μ total zinc, which corresponds to a calculated free zinc concentration of ∼10 n. The low affinity system has an apparent Km of 10 μ total zinc, which corresponds to ∼100 n free zinc. Although other roles are also possible, we propose that ZRT2 encodes the transporter of the low affinity system. Consistent with this hypothesis, the ZRT2 gene was isolated as a multicopy suppressor of the zinc-limited growth defect of a zrt1 mutant. Furthermore, the level of ZRT2 expression correlated with low affinity uptake activity. ZRT2 overexpression increased the activity of a system biochemically indistinguishable from the low affinity system. Conversely, disruption of the ZRT2 gene eliminated low affinity uptake. Thus, ZRT2 expression is both necessary and sufficient for low affinity activity. The predicted amino acid sequence of Zrt2p also suggests that this protein plays a direct role in the transport of zinc. Zrt2p shares remarkable similarity with Zrt1p (6Zhao H. Eide D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2454-2458Crossref PubMed Scopus (445) Google Scholar) and Irt1p, an Fe2+ transporter from A. thaliana (7Eide D. Broderius M. Fett J. Guerinot M.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5624-5628Crossref PubMed Scopus (1034) Google Scholar). The distribution of hydrophobic amino acids suggests that all three gene products are integral membrane proteins with eight transmembrane domains. It is possible that Zrt2p is only one subunit of a heteromeric transporter complex, but this hypothesis seems unlikely given that overexpression of ZRT2 alone increases zinc uptake activity.ZRT2 is a member of a new and rapidly growing gene family of putative metal transporters. We have identified closely related genes in organisms as diverse as fungi, plants, nematodes, and humans (6Zhao H. Eide D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2454-2458Crossref PubMed Scopus (445) Google Scholar, 7Eide D. Broderius M. Fett J. Guerinot M.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5624-5628Crossref PubMed Scopus (1034) Google Scholar). Given that three members of this family, IRT1, ZRT1, and now ZRT2, have been implicated in metal transport, it seems likely that the other genes in this family play similar roles in metal metabolism. The structural similarity of these different gene products suggests that they may use a similar mechanism to transport their substrates. Zinc uptake in yeast requires metabolic energy (5White C. Gadd G.M. J. Gen. Microbiol. 1987; 133: 727-737Google Scholar). What then is the driving force for zinc uptake by Zrt2p? Like the other members of this family, Zrt2p does not contain ATP-binding domains, nor does the protein bear any significant similarity to the ubiquitous P-type ATPase family of transport proteins. This observation suggests that uptake may be driven by indirect coupling to energy metabolism, perhaps through the electrical potential generated across the plasma membrane by the plasma membrane ATPase. Alternatively, uptake may be driven by a transmembrane gradient of another ion. Uptake of zinc by the low affinity system was not inhibited by high extracellular K+ (100 m), 3H. Zhao and D. Eide, unpublished result. indicating that a zinc/K+ antiport mechanism, as has been previously proposed (3Fuhrmann G.F. Rothstein A. Biochim. Biophys. Acta. 1968; 163: 325-330Crossref PubMed Scopus (118) Google Scholar, 25Okorokov L.A. Andreeva N.A. Lichko L.P. Valiakhmetov A.Y. Biochem. Int. 1983; 6: 463-472PubMed Google Scholar), is unlikely.A cluster of histidines in Zrt2p is also found in Zrt1p, Irt1p, and the other members of this gene family. In Zrt2p and Zrt1p, these histidines are located in a region with a highly negative net charge due to the abundance of acidic amino acids. Imidazole ring nitrogens and carboxylate groups frequently serve as coordinating ligands for zinc (1Vallee B.L. Auld D.S. Biochemistry. 1990; 9: 5647-5659Crossref Scopus (1512) Google Scholar), so these amino acids may be responsible for binding the metal substrate. In all of these proteins, the histidines are found in a region between two transmembrane domains that is predicted to be exposed on the cytoplasmic face of the membrane. Given this location, these amino acids may act in a late step in the uptake process by binding the metal after it has been transported across the membrane. Alternatively, these histidines may serve as part of a feedback regulation system. High intracellular zinc levels could result in binding of zinc to Zrt2p and, by some mechanism, reduce the activity of the transporter. Whatever their role, the conservation of these histidine residues within the IRT/ZRT gene family suggests that they are critical to the function of these proteins. This conclusion is further supported by the observation that similar histidine-rich domains are found in the sequences of four transport proteins implicated in zinc detoxification, i.e. Zrc1p and Cot1p from yeast and the mammalian ZnT-1p and ZnT-2p proteins (26Conklin D.S. Culbertson M.R. Kung C. Mol. Gen. Genet. 1994; 244: 303-311Crossref PubMed Scopus (58) Google Scholar, 27Conklin D.S. McMaster J.A. Culbertson M.R. Kung C. Mol. Cell. Biol. 1992; 12: 3678-3688Crossref PubMed Scopus (178) Google Scholar, 28Kamizono A. Nishizawa M. Teranishi Y. Murata K. Kimura A. Mol. Gen. Genet. 1989; 219: 161-167Crossref PubMed Scopus (160) Google Scholar, 29Palmiter R.D. Findley S.D. EMBO J. 1995; 14: 639-649Crossref PubMed Scopus (636) Google Scholar, 30Palmiter R.D. Cole T.B. Findley S.D. EMBO J. 1996; 15: 1784-1791Crossref PubMed Scopus (393) Google Scholar). These proteins are apparently efflux transporters that transport metal ions from the cytoplasm either into an intracellular compartment or outside of the cell and, aside from the histidine-rich domain, share no significant similarity with the IRT/ZRT gene family. In each case, the histidine-rich domain is predicted to be cytoplasmically located. The functional importance of the conserved histidines in the IRT/ZRT gene family is currently under investigation. Furthermore, the interplay between zinc uptake transporters like Zrt1p and Zrt2p and efflux transporters like ZnT-1p and ZnT-2p will play an important role in cellular zinc homeostasis and merits further study.Our results demonstrate that the high and low affinity systems are genetically and biochemically separate uptake pathways. We have also shown that the low affinity system is a relevant source of zinc for growing yeast cells. First, metal inhibition studies indicate that the low affinity system is very similar to the high affinity system in its specificity for zinc over other metals. While copper and Fe2+ were capable of inhibiting zinc uptake by both the low and high affinity systems, further experiments will be required to determine if these metals are actually transported substrates. Second, the low affinity system is the major pathway for zinc uptake in wild-type cells grown under zinc-replete conditions (e.g. cells grown in SD glucose medium); no high affinity activity is detectable in these cells. Third, a zrt2 mutant strain that lacks the low affinity system has increased high affinity activity. This increased activity is presumably to compensate for loss of low affinity activity. In addition, the zrt1zrt2 mutant requires >1000-fold more zinc in the medium to grow and to supply the regulatory pool of intracellular zinc and to down-regulate the zinc-responsive ZRT1 promoter than does the zrt1 single mutant. These results indicate that the low affinity system is a major contributor to zinc accumulation in the zrt1 strain, and we infer that it also contributes to wild-type zinc accumulation under the same growth conditions.Additional evidence that the low affinity system is a relevant source of zinc is provided by the observation that this system is regulated by zinc. Low affinity activity was diminished in cells grown in a medium containing extremely high levels of zinc (2 m). The high affinity system and ZRT1 mRNA levels are regulated by zinc, and this regulation is mediated at the transcriptional level in response to an intracellular zinc pool (6Zhao H. Eide D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2454-2458Crossref PubMed Scopus (445) Google Scholar). The analysis of the low affinity system described here does not distinguish between transcriptional and post-transcriptional mechanisms. One possible mechanism, as discussed above, is down-regulation of the low affinity system by feedback inhibition of transporter activity. What is clear is that the regulatory systems that control high and low affinity uptake are responsive to very different levels of cell-associated zinc. A decrease in ZRT1 expression and high affinity activity was apparent when cell-associated zinc levels rose to as little as 30 pmol/106 cells (6Zhao H. Eide D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2454-2458Crossref PubMed Scopus (445) Google Scholar). In that same analysis, we found that cells with a cell-associated zinc level of 120 pmol/106 cells still had maximum low affinity activity (Vmax = 2 pmol/min/106 cells). Therefore, down-regulation of the low affinity system requires much higher levels of cell-associated zinc than is needed to repress the high affinity system. These observations pose an interesting regulatory question as to how these two systems respond to different levels of presumably the same signal, intracellular zinc.We demonstrated previously that zrt1 mutant cells are not more resistant to higher levels of extracellular zinc than are wild-type cells (6Zhao H. Eide D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2454-2458Crossref PubMed Scopus (445) Google Scholar). Neither zrt2 nor zrt1zrt2 strains are more resistant to extracellular zinc than are the wild-type or zrt1 strains.3 This observation is consistent with the low level of both high and low affinity activity observed in cells treated with extremely high levels of zinc and demonstrates that neither of these two systems plays a major role in zinc toxicity. Toxicity may result from zinc accumulation by one or more additional uptake pathways. The existence of this pathway(s) is demonstrated by the observation that a strain lacking both the high and low affinity systems, the zrt1zrt2 mutant, is still viable. Undoubtedly, these cells are obtaining zinc, and this uptake may represent the activity of a third system for zinc accumulation. The identity of this third system is suggested by earlier studies in which zinc uptake in yeast was attributed to a "divalent cation uptake system" that was also capable of transporting magnesium, cobalt, manganese, and nickel (3Fuhrmann G.F. Rothstein A. Biochim. Biophys. Acta. 1968; 163: 325-330Crossref PubMed Scopus (118) Google Scholar). The apparent Km of zinc uptake by this system was estimated to be ∼500 μ total zinc, i.e. 50- and 500-fold higher than the ZRT2- and ZRT1-dependent systems, respectively. This apparent Km is consistent with the high concentration of zinc required to confer maximum growth to the zrt1zrt2 mutant. Whatever the mechanism, given the 105-fold greater zinc requirement of the zrt1zrt2 mutant strain compared with the wild-type strain, it is unlikely that this third pathway plays a significant role in zinc accumulation under any but the most zinc-rich conditions. INTRODUCTIONHow the cells of all organisms acquire metal ions from their extracellular environment is one of the central unresolved questions in the biochemistry of these important nutrients. Zinc is essential because it is an integral cofactor of many proteins and is a critical determinant of their catalytic activity and/or structural stability (1Vallee B.L. Auld D.S. Biochemistry. 1990; 9: 5647-5659Crossref Scopus (1512) Google Scholar). Moreover, zinc is an important component of many transcription factors, the zinc finger proteins, that regulate gene expression (2Rhodes D. Klug A. Sci. Am. 1993; 268: 56-62Crossref PubMed Scopus (85) Google Scholar). Biochemical assays of zinc uptake in yeast indicated that this process is transporter-mediated, i.e. zinc uptake is time-, temperature-, and concentration-dependent and requires metabolic energy (3Fuhrmann G.F. Rothstein A. Biochim. Biophys. Acta. 1968; 163: 325-330Crossref PubMed Scopus (118) Google Scholar, 4Mowll J.L. Gadd G.M. J. Gen. Microbiol. 1983; 129: 3421-3425Google Scholar, 5White C. Gadd G.M. J. Gen. Microbiol. 1987; 133: 727-737Google Scholar). Recent studies suggested the presence of two separate uptake systems (6Zhao H. Eide D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2454-2458Crossref PubMed Scopus (445) Google Scholar). One system has high affinity for zinc with an apparent Km of 0.5-1 μ and is required for zinc-limited growth. The ZRT1 (for zinc-regulated transporter) gene appears to encode the transporter protein of this system. ZRT1 is a member of a closely related family of transporter genes found in organisms as diverse as fungi, plants, nematodes, and humans. This family includes the IRT1 gene from Arabidopsis thaliana, which encodes an Fe2+ transporter expressed in plant roots (7Eide D. Broderius M. Fett J. Guerinot M.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5624-5628Crossref PubMed Scopus (1034) Google Scholar). Like the other members of this family, Zrt1p is predicted to be an integral membrane protein containing eight potential transmembrane domains.The level of ZRT1 expression correlated with activity of the high affinity system; overexpression of ZRT1 increased high affinity uptake, whereas a zrt1 mutation eliminated high affinity activity and resulted in poor growth of the mutant on zinc-limited media (6Zhao H. Eide D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2454-2458Crossref PubMed Scopus (445) Google Scholar). The high affinity system was induced in activity >100-fold in response to zinc-limiting growth conditions. When cells were grown in media containing different zinc concentrations, high affinity uptake and ZRT1 mRNA levels were closely correlated, as was the β-galactosidase activity generated by a reporter gene in which the ZRT1 promoter was fused to the Escherichia coli lacZ gene. The ZRT1-lacZ fusion gene showed a similar pattern of regulation in response to cell-associated zinc levels in both wild-type and zrt1 mutant cells despite the 75-fold higher extracellular zinc level required to down-regulate the promoter in the mutant. These results indicate that the activity of the high affinity system is controlled, at least in part, by transcriptional regulation of the ZRT1 gene in response to a regulatory pool of intracellular zinc.The second system for zinc uptake in yeast has a lower affinity for substrate (apparent Km = 10 μ), and it is active in zinc-replete cells. Low affinity uptake was unaffected by the zrt1 mutation, suggesting that this system is a separate uptake pathway for zinc. As described in this report, an initial characterization was conducted to determine some of the biochemical properties of the low affinity system. We also report the analysis of another member of the IRT/ZRT gene family, ZRT2. ZRT2 was identified in the sequence data bases because of the close sequence similarity of its product to Irt1p and Zrt1p (6Zhao H. Eide D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2454-2458Crossref PubMed Scopus (445) Google Scholar, 7Eide D. Broderius M. Fett J. Guerinot M.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5624-5628Crossref PubMed Scopus (1034) Google Scholar). Our analysis of ZRT2 suggests that this gene encodes the transporter protein of the low affinity system.
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