CCC1 Is a Transporter That Mediates Vacuolar Iron Storage in Yeast

The budding yeast Saccharomyces cerevisiae can grow for generations in the absence of exogenous iron, indicating a capacity to store intracellular iron. As cells can accumulate iron by endocytosis we studied iron metabolism in yeast that were defective in endocytosis. We demonstrated that endocytosis-defective yeast (Δend4) can store iron in the vacuole, indicating a transfer of iron from the cytosol to the vacuole. Using several different criteria we demonstrated that CCC1encodes a transporter that effects the accumulation of iron and Mn2+ in vacuoles. Overexpression of CCC1, which is localized to the vacuole, lowers cytosolic iron and increases vacuolar iron content. Conversely, deletion ofCCC1 results in decreased vacuolar iron content and decreased iron stores, which affect cytosolic iron levels and cell growth. Furthermore Δccc1 cells show increased sensitivity to external iron. The sensitivity to iron is exacerbated by ectopic expression of the iron transporter FET4. These results indicate that yeast can store iron in the vacuole and thatCCC1 is involved in the transfer of iron from the cytosol to the vacuole. The budding yeast Saccharomyces cerevisiae can grow for generations in the absence of exogenous iron, indicating a capacity to store intracellular iron. As cells can accumulate iron by endocytosis we studied iron metabolism in yeast that were defective in endocytosis. We demonstrated that endocytosis-defective yeast (Δend4) can store iron in the vacuole, indicating a transfer of iron from the cytosol to the vacuole. Using several different criteria we demonstrated that CCC1encodes a transporter that effects the accumulation of iron and Mn2+ in vacuoles. Overexpression of CCC1, which is localized to the vacuole, lowers cytosolic iron and increases vacuolar iron content. Conversely, deletion ofCCC1 results in decreased vacuolar iron content and decreased iron stores, which affect cytosolic iron levels and cell growth. Furthermore Δccc1 cells show increased sensitivity to external iron. The sensitivity to iron is exacerbated by ectopic expression of the iron transporter FET4. These results indicate that yeast can store iron in the vacuole and thatCCC1 is involved in the transfer of iron from the cytosol to the vacuole. bathophenanthroline disulfonate 5-(and-6)-carboxy-2′,7′-dichlorofluorescein diacetate inductively coupled atomic absorption spectroscopy N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino) phenyl) hexatrienyl)-pyridinium dibromide carboxypeptidase While iron is a required element for all eucaryotes, it is also potentially toxic. Organisms tightly regulate the concentration of cytosolic iron through regulation of iron uptake and storage. In the past few years the mechanisms that mediate plasma membrane iron transport in the budding yeast Saccharomyces cerevisiae have been described in molecular detail (1Radisky D. Kaplan J. J. Biol. Chem. 1999; 274: 4481-4484Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Many of the genes required for plasma membrane transport have been cloned. Much less is known, however, about iron storage. Yeast is distinguished from most eucaryotes in not having ferritin as an iron storage molecule. In this regard yeast are more analogous to plants than vertebrates. In plants, ferritin is restricted to the chloroplasts, as opposed to the cytosol in animals. Plants and yeast are thought to store iron in the vacuole, although there is little compelling proof of that supposition. Three lines of evidence have been used to support the view that the vacuole is an iron storage organelle: 1) vacuolar mutants show increased metal sensitivity (2Szczypka M.S. Zhu Z. Silar P. Thiele D.J. Yeast. 1997; 13: 1423-1435Crossref PubMed Scopus (79) Google Scholar, 3Bode H.P. Dumschat M. Garotti S. Fuhrmann G.F. Eur. J. Biochem. 1995; 228: 337-342Crossref PubMed Scopus (49) Google Scholar); 2) iron can be found in vacuoles (4Raguzzi F. Lesuisse E. Crichton R.R. FEBS Lett. 1988; 231: 253-258Crossref PubMed Scopus (127) Google Scholar); and 3) there are transport systems capable of extracting iron from the vacuole (5Portnoy M.E. Liu X.F. Culotta V.C. Mol. Cell. Biol. 2000; 20: 7893-7902Crossref PubMed Scopus (179) Google Scholar,6Urbanowski J.L. Piper R.C. J. Biol. Chem. 1999; 274: 38061-38070Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). The increased metal sensitivity of vacuolar mutants, however, can result from increased metal uptake rather than decreased storage (7Li L. Kaplan J. J. Biol. Chem. 1998; 273: 22181-22187Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Uptake of extracellular fluid by endocytosis, a steady state process would lead to vacuolar iron accumulation, which could be extracted by vacuolar iron transporters. There are little data that demonstrate that iron accumulated in the cytosol can be stored in the vacuole.To determine whether S. cerevisiae store iron in the vacuole we studied vacuolar iron storage in yeast strains unable to endocytose. Our studies demonstrated that yeast do store iron in the vacuole. We further demonstrated that CCC1 is an iron/Mn2+transporter responsible for storing these two metals in the vacuole.DISCUSSIONIn S. cerevisiae, high affinity iron transport is regulated at the level of transcription by Aft1p, which activates transcription of the iron-regulon in low iron conditions (23Yamaguchi-Iwai Y. Dancis A. Klausner R.D. EMBO J. 1995; 14: 1231-1239Crossref PubMed Scopus (313) Google Scholar). Once transcribed, the activity of the high affinity iron transport system is not inhibited by high iron (24Eide D. Davis-Kaplan S. Jordan I. Sipe D. Kaplan J. J. Biol. Chem. 1992; 267: 20774-20781Abstract Full Text PDF PubMed Google Scholar). In high iron media the expression of Fet3p/Ftr1p slowly declines, and cells can continue to accumulate iron even though they are iron replete. The inability to reduce high affinity iron transport when exposed to high concentrations of metal distinguishes the iron transport system from transport systems for Cu2+, Mn2+, and Zn2+ (1Radisky D. Kaplan J. J. Biol. Chem. 1999; 274: 4481-4484Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The activity of these transport systems is regulated by the metal, which accelerates the degradation of the transporters. Because of continued iron uptake even in the face of high iron stores, yeast must find ways of accommodating iron. That yeast can store iron is demonstrated by the observation that cells can grow for generations in the absence of exogenous iron.In this communication we presented direct biochemical data that show that iron can be stored in the vacuole. A previous concern with other studies that demonstrated vacuolar iron was the possibility that iron entered the vacuole through endocytic activity (3Bode H.P. Dumschat M. Garotti S. Fuhrmann G.F. Eur. J. Biochem. 1995; 228: 337-342Crossref PubMed Scopus (49) Google Scholar, 4Raguzzi F. Lesuisse E. Crichton R.R. FEBS Lett. 1988; 231: 253-258Crossref PubMed Scopus (127) Google Scholar). Endocytosis is a constitutive process, and exposure of cells to iron-rich media would lead to the accumulation of iron in vacuoles. To determine whether iron could enter the vacuole independently of endocytosis, we took advantage of yeast strains with a deletion in the END4 gene. TheΔend4 strain, while unable to internalize, has normal vacuoles by all criteria (Fig. 1). In the absence of endocytosis, however, Δend4 cells can accumulate iron in the vacuole, which can then be mobilized for cellular growth. These observations provide compelling evidence that the vacuole can function as an iron storage organelle.Further evidence that supports the hypothesis that vacuoles can store iron is the observation that CCC1 can effect vacuolar iron accumulation. CCC1 was first identified as a suppressor of a mutant that was unable to grow in high Ca2+ (25Fu D. Beeler T. Dunn T. Yeast. 1994; 10: 515-521Crossref PubMed Scopus (24) Google Scholar). The mutant had a defect in the synthesis of a mannosyl-lipid. A subsequent study identified CCC1 as a suppressor of Mn2+hypersensitivity resulting from a deletion in pmr1. Analysis of the effect of CCC1 suggested that it was a transporter that mediated the accumulation of Mn2+ into a membranous compartment. Subcellular fractionation and immunofluorescence suggested that an overexpressed hemagglutinin-tagged CCC1 was localized to the Golgi, and it was concluded that CCC1 was a Golgi Mn2+ transporter (22Lapinskas P.J. Lin S.J. Culotta V.C. Mol. Microbiol. 1996; 21: 519-528Crossref PubMed Scopus (82) Google Scholar). Overexpressed CCC1could not, however, suppress a glycosylation defect ofΔpmr1 and provide Mn2+ to Golgi enzymes.We identified CCC1 as a suppressor of a Δyfh1strain (12Chen O.S. Kaplan J. J. Biol. Chem. 2000; 275: 7626-7632Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Δyfh1 strains can accumulate toxic levels of mitochondrial iron resulting in the selection of respiratory incompetent strains. Reduction in cytosolic iron can maintain the respiratory competence of Δyfh1 strains and permit them to grow on glycerol-ethanol even in high iron (26Radisky D.C. Babcock M.C. Kaplan J. J. Biol. Chem. 1999; 274: 4497-4499Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). We have discovered that iron accumulates within vacuoles in cells overexpressingCCC1. Iron sequestration within vacuoles would result in a low cytosolic iron level that precludes mitochondrial iron accumulation. Overexpression of CCC1 results in increased vacuolar iron accumulation in Δend4 cells, indicating that iron can enter the vacuole independently of endocytic activity. We have localized a FLAG-tagged CCC1 to the vacuolar membrane. Thus, our data are consistent in identifying the vacuole as a site of iron storage and CCC1 as a vacuolar iron transporter. Our studies also indicate that CCC1 can lead to Mn2+accumulation in vacuoles. This result suggests that CCC1 may be a Fe2+/Mn2+ transporter consistent with the finding that most transporters that recognize Mn2+ also recognize Fe2+.While there is one high affinity cell surface iron transporter there are also a number of low affinity iron transporters (1Radisky D. Kaplan J. J. Biol. Chem. 1999; 274: 4481-4484Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). There appear to be at least two transporters that can extract iron from the vacuole (FET5/FTH1, SMF3) (5Portnoy M.E. Liu X.F. Culotta V.C. Mol. Cell. Biol. 2000; 20: 7893-7902Crossref PubMed Scopus (179) Google Scholar, 6Urbanowski J.L. Piper R.C. J. Biol. Chem. 1999; 274: 38061-38070Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Thus, each membrane system has redundant iron transporters. We do not know if there are other transporters capable of mediating Fe2+ or Mn2+ accumulation in vacuoles. An attempt to examine this possibility by measuring vacuolar iron content in aΔend4Δccc1 deletion strain has been unfruitful as the strain grows poorly. We have also not been able to identify mammalian homologues of CCC1. There are, however, homologues in bothArabidopsis thaliana and Oryza sativa, indicating that plants may also store iron within vacuoles. While iron is a required element for all eucaryotes, it is also potentially toxic. Organisms tightly regulate the concentration of cytosolic iron through regulation of iron uptake and storage. In the past few years the mechanisms that mediate plasma membrane iron transport in the budding yeast Saccharomyces cerevisiae have been described in molecular detail (1Radisky D. Kaplan J. J. Biol. Chem. 1999; 274: 4481-4484Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Many of the genes required for plasma membrane transport have been cloned. Much less is known, however, about iron storage. Yeast is distinguished from most eucaryotes in not having ferritin as an iron storage molecule. In this regard yeast are more analogous to plants than vertebrates. In plants, ferritin is restricted to the chloroplasts, as opposed to the cytosol in animals. Plants and yeast are thought to store iron in the vacuole, although there is little compelling proof of that supposition. Three lines of evidence have been used to support the view that the vacuole is an iron storage organelle: 1) vacuolar mutants show increased metal sensitivity (2Szczypka M.S. Zhu Z. Silar P. Thiele D.J. Yeast. 1997; 13: 1423-1435Crossref PubMed Scopus (79) Google Scholar, 3Bode H.P. Dumschat M. Garotti S. Fuhrmann G.F. Eur. J. Biochem. 1995; 228: 337-342Crossref PubMed Scopus (49) Google Scholar); 2) iron can be found in vacuoles (4Raguzzi F. Lesuisse E. Crichton R.R. FEBS Lett. 1988; 231: 253-258Crossref PubMed Scopus (127) Google Scholar); and 3) there are transport systems capable of extracting iron from the vacuole (5Portnoy M.E. Liu X.F. Culotta V.C. Mol. Cell. Biol. 2000; 20: 7893-7902Crossref PubMed Scopus (179) Google Scholar,6Urbanowski J.L. Piper R.C. J. Biol. Chem. 1999; 274: 38061-38070Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). The increased metal sensitivity of vacuolar mutants, however, can result from increased metal uptake rather than decreased storage (7Li L. Kaplan J. J. Biol. Chem. 1998; 273: 22181-22187Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Uptake of extracellular fluid by endocytosis, a steady state process would lead to vacuolar iron accumulation, which could be extracted by vacuolar iron transporters. There are little data that demonstrate that iron accumulated in the cytosol can be stored in the vacuole. To determine whether S. cerevisiae store iron in the vacuole we studied vacuolar iron storage in yeast strains unable to endocytose. Our studies demonstrated that yeast do store iron in the vacuole. We further demonstrated that CCC1 is an iron/Mn2+transporter responsible for storing these two metals in the vacuole. DISCUSSIONIn S. cerevisiae, high affinity iron transport is regulated at the level of transcription by Aft1p, which activates transcription of the iron-regulon in low iron conditions (23Yamaguchi-Iwai Y. Dancis A. Klausner R.D. EMBO J. 1995; 14: 1231-1239Crossref PubMed Scopus (313) Google Scholar). Once transcribed, the activity of the high affinity iron transport system is not inhibited by high iron (24Eide D. Davis-Kaplan S. Jordan I. Sipe D. Kaplan J. J. Biol. Chem. 1992; 267: 20774-20781Abstract Full Text PDF PubMed Google Scholar). In high iron media the expression of Fet3p/Ftr1p slowly declines, and cells can continue to accumulate iron even though they are iron replete. The inability to reduce high affinity iron transport when exposed to high concentrations of metal distinguishes the iron transport system from transport systems for Cu2+, Mn2+, and Zn2+ (1Radisky D. Kaplan J. J. Biol. Chem. 1999; 274: 4481-4484Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The activity of these transport systems is regulated by the metal, which accelerates the degradation of the transporters. Because of continued iron uptake even in the face of high iron stores, yeast must find ways of accommodating iron. That yeast can store iron is demonstrated by the observation that cells can grow for generations in the absence of exogenous iron.In this communication we presented direct biochemical data that show that iron can be stored in the vacuole. A previous concern with other studies that demonstrated vacuolar iron was the possibility that iron entered the vacuole through endocytic activity (3Bode H.P. Dumschat M. Garotti S. Fuhrmann G.F. Eur. J. Biochem. 1995; 228: 337-342Crossref PubMed Scopus (49) Google Scholar, 4Raguzzi F. Lesuisse E. Crichton R.R. FEBS Lett. 1988; 231: 253-258Crossref PubMed Scopus (127) Google Scholar). Endocytosis is a constitutive process, and exposure of cells to iron-rich media would lead to the accumulation of iron in vacuoles. To determine whether iron could enter the vacuole independently of endocytosis, we took advantage of yeast strains with a deletion in the END4 gene. TheΔend4 strain, while unable to internalize, has normal vacuoles by all criteria (Fig. 1). In the absence of endocytosis, however, Δend4 cells can accumulate iron in the vacuole, which can then be mobilized for cellular growth. These observations provide compelling evidence that the vacuole can function as an iron storage organelle.Further evidence that supports the hypothesis that vacuoles can store iron is the observation that CCC1 can effect vacuolar iron accumulation. CCC1 was first identified as a suppressor of a mutant that was unable to grow in high Ca2+ (25Fu D. Beeler T. Dunn T. Yeast. 1994; 10: 515-521Crossref PubMed Scopus (24) Google Scholar). The mutant had a defect in the synthesis of a mannosyl-lipid. A subsequent study identified CCC1 as a suppressor of Mn2+hypersensitivity resulting from a deletion in pmr1. Analysis of the effect of CCC1 suggested that it was a transporter that mediated the accumulation of Mn2+ into a membranous compartment. Subcellular fractionation and immunofluorescence suggested that an overexpressed hemagglutinin-tagged CCC1 was localized to the Golgi, and it was concluded that CCC1 was a Golgi Mn2+ transporter (22Lapinskas P.J. Lin S.J. Culotta V.C. Mol. Microbiol. 1996; 21: 519-528Crossref PubMed Scopus (82) Google Scholar). Overexpressed CCC1could not, however, suppress a glycosylation defect ofΔpmr1 and provide Mn2+ to Golgi enzymes.We identified CCC1 as a suppressor of a Δyfh1strain (12Chen O.S. Kaplan J. J. Biol. Chem. 2000; 275: 7626-7632Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Δyfh1 strains can accumulate toxic levels of mitochondrial iron resulting in the selection of respiratory incompetent strains. Reduction in cytosolic iron can maintain the respiratory competence of Δyfh1 strains and permit them to grow on glycerol-ethanol even in high iron (26Radisky D.C. Babcock M.C. Kaplan J. J. Biol. Chem. 1999; 274: 4497-4499Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). We have discovered that iron accumulates within vacuoles in cells overexpressingCCC1. Iron sequestration within vacuoles would result in a low cytosolic iron level that precludes mitochondrial iron accumulation. Overexpression of CCC1 results in increased vacuolar iron accumulation in Δend4 cells, indicating that iron can enter the vacuole independently of endocytic activity. We have localized a FLAG-tagged CCC1 to the vacuolar membrane. Thus, our data are consistent in identifying the vacuole as a site of iron storage and CCC1 as a vacuolar iron transporter. Our studies also indicate that CCC1 can lead to Mn2+accumulation in vacuoles. This result suggests that CCC1 may be a Fe2+/Mn2+ transporter consistent with the finding that most transporters that recognize Mn2+ also recognize Fe2+.While there is one high affinity cell surface iron transporter there are also a number of low affinity iron transporters (1Radisky D. Kaplan J. J. Biol. Chem. 1999; 274: 4481-4484Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). There appear to be at least two transporters that can extract iron from the vacuole (FET5/FTH1, SMF3) (5Portnoy M.E. Liu X.F. Culotta V.C. Mol. Cell. Biol. 2000; 20: 7893-7902Crossref PubMed Scopus (179) Google Scholar, 6Urbanowski J.L. Piper R.C. J. Biol. Chem. 1999; 274: 38061-38070Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Thus, each membrane system has redundant iron transporters. We do not know if there are other transporters capable of mediating Fe2+ or Mn2+ accumulation in vacuoles. An attempt to examine this possibility by measuring vacuolar iron content in aΔend4Δccc1 deletion strain has been unfruitful as the strain grows poorly. We have also not been able to identify mammalian homologues of CCC1. There are, however, homologues in bothArabidopsis thaliana and Oryza sativa, indicating that plants may also store iron within vacuoles. In S. cerevisiae, high affinity iron transport is regulated at the level of transcription by Aft1p, which activates transcription of the iron-regulon in low iron conditions (23Yamaguchi-Iwai Y. Dancis A. Klausner R.D. EMBO J. 1995; 14: 1231-1239Crossref PubMed Scopus (313) Google Scholar). Once transcribed, the activity of the high affinity iron transport system is not inhibited by high iron (24Eide D. Davis-Kaplan S. Jordan I. Sipe D. Kaplan J. J. Biol. Chem. 1992; 267: 20774-20781Abstract Full Text PDF PubMed Google Scholar). In high iron media the expression of Fet3p/Ftr1p slowly declines, and cells can continue to accumulate iron even though they are iron replete. The inability to reduce high affinity iron transport when exposed to high concentrations of metal distinguishes the iron transport system from transport systems for Cu2+, Mn2+, and Zn2+ (1Radisky D. Kaplan J. J. Biol. Chem. 1999; 274: 4481-4484Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The activity of these transport systems is regulated by the metal, which accelerates the degradation of the transporters. Because of continued iron uptake even in the face of high iron stores, yeast must find ways of accommodating iron. That yeast can store iron is demonstrated by the observation that cells can grow for generations in the absence of exogenous iron. In this communication we presented direct biochemical data that show that iron can be stored in the vacuole. A previous concern with other studies that demonstrated vacuolar iron was the possibility that iron entered the vacuole through endocytic activity (3Bode H.P. Dumschat M. Garotti S. Fuhrmann G.F. Eur. J. Biochem. 1995; 228: 337-342Crossref PubMed Scopus (49) Google Scholar, 4Raguzzi F. Lesuisse E. Crichton R.R. FEBS Lett. 1988; 231: 253-258Crossref PubMed Scopus (127) Google Scholar). Endocytosis is a constitutive process, and exposure of cells to iron-rich media would lead to the accumulation of iron in vacuoles. To determine whether iron could enter the vacuole independently of endocytosis, we took advantage of yeast strains with a deletion in the END4 gene. TheΔend4 strain, while unable to internalize, has normal vacuoles by all criteria (Fig. 1). In the absence of endocytosis, however, Δend4 cells can accumulate iron in the vacuole, which can then be mobilized for cellular growth. These observations provide compelling evidence that the vacuole can function as an iron storage organelle. Further evidence that supports the hypothesis that vacuoles can store iron is the observation that CCC1 can effect vacuolar iron accumulation. CCC1 was first identified as a suppressor of a mutant that was unable to grow in high Ca2+ (25Fu D. Beeler T. Dunn T. Yeast. 1994; 10: 515-521Crossref PubMed Scopus (24) Google Scholar). The mutant had a defect in the synthesis of a mannosyl-lipid. A subsequent study identified CCC1 as a suppressor of Mn2+hypersensitivity resulting from a deletion in pmr1. Analysis of the effect of CCC1 suggested that it was a transporter that mediated the accumulation of Mn2+ into a membranous compartment. Subcellular fractionation and immunofluorescence suggested that an overexpressed hemagglutinin-tagged CCC1 was localized to the Golgi, and it was concluded that CCC1 was a Golgi Mn2+ transporter (22Lapinskas P.J. Lin S.J. Culotta V.C. Mol. Microbiol. 1996; 21: 519-528Crossref PubMed Scopus (82) Google Scholar). Overexpressed CCC1could not, however, suppress a glycosylation defect ofΔpmr1 and provide Mn2+ to Golgi enzymes. We identified CCC1 as a suppressor of a Δyfh1strain (12Chen O.S. Kaplan J. J. Biol. Chem. 2000; 275: 7626-7632Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Δyfh1 strains can accumulate toxic levels of mitochondrial iron resulting in the selection of respiratory incompetent strains. Reduction in cytosolic iron can maintain the respiratory competence of Δyfh1 strains and permit them to grow on glycerol-ethanol even in high iron (26Radisky D.C. Babcock M.C. Kaplan J. J. Biol. Chem. 1999; 274: 4497-4499Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). We have discovered that iron accumulates within vacuoles in cells overexpressingCCC1. Iron sequestration within vacuoles would result in a low cytosolic iron level that precludes mitochondrial iron accumulation. Overexpression of CCC1 results in increased vacuolar iron accumulation in Δend4 cells, indicating that iron can enter the vacuole independently of endocytic activity. We have localized a FLAG-tagged CCC1 to the vacuolar membrane. Thus, our data are consistent in identifying the vacuole as a site of iron storage and CCC1 as a vacuolar iron transporter. Our studies also indicate that CCC1 can lead to Mn2+accumulation in vacuoles. This result suggests that CCC1 may be a Fe2+/Mn2+ transporter consistent with the finding that most transporters that recognize Mn2+ also recognize Fe2+. While there is one high affinity cell surface iron transporter there are also a number of low affinity iron transporters (1Radisky D. Kaplan J. J. Biol. Chem. 1999; 274: 4481-4484Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). There appear to be at least two transporters that can extract iron from the vacuole (FET5/FTH1, SMF3) (5Portnoy M.E. Liu X.F. Culotta V.C. Mol. Cell. Biol. 2000; 20: 7893-7902Crossref PubMed Scopus (179) Google Scholar, 6Urbanowski J.L. Piper R.C. J. Biol. Chem. 1999; 274: 38061-38070Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Thus, each membrane system has redundant iron transporters. We do not know if there are other transporters capable of mediating Fe2+ or Mn2+ accumulation in vacuoles. An attempt to examine this possibility by measuring vacuolar iron content in aΔend4Δccc1 deletion strain has been unfruitful as the strain grows poorly. We have also not been able to identify mammalian homologues of CCC1. There are, however, homologues in bothArabidopsis thaliana and Oryza sativa, indicating that plants may also store iron within vacuoles. We thank our colleagues in the Utah metal group for their help in editing this manuscript.