The Metalloreductase Fre6p in Fe-Efflux from the Yeast Vacuole

Abstract

The yeast vacuole is the storage depot for cellular iron. In this report we quantify the import-export balance in the vacuole because of the import of iron by Ccc1p and to export by the combined activity of Smf3p and the ferroxidase, permease pair of proteins, Fet5p and Fth1p. Our data indicate that the two efflux pathways are equally efficient in trafficking iron out of the vacuole. A major focus of this work was to identify the ferrireductase(s) that supplies the FeII for efflux whether by Smf3p or the Fet5p-Fth1p complex. Using a combination of flameless atomic absorption spectrophotometry to quantify vacuolar and whole cell iron content and a reporter assay for cytoplasmic iron we demonstrate that Fre6p supplies FeII to both efflux systems, while Fre7p plays no role in Fe-efflux from the vacuole. Enzymatic assay shows the two fusions to have similar reductase activity, however. Confocal fluorescence microscopy demonstrates that Fre6:GFP localizes to the vacuolar membrane; in contrast, Fre7:GFP fusions exhibit a variable and diffuse cellular distribution. Demonstrating a role for a vacuolar metalloreductase in Fe-efflux supports the model that iron is stored in the vacuole in the ferric state. The yeast vacuole is the storage depot for cellular iron. In this report we quantify the import-export balance in the vacuole because of the import of iron by Ccc1p and to export by the combined activity of Smf3p and the ferroxidase, permease pair of proteins, Fet5p and Fth1p. Our data indicate that the two efflux pathways are equally efficient in trafficking iron out of the vacuole. A major focus of this work was to identify the ferrireductase(s) that supplies the FeII for efflux whether by Smf3p or the Fet5p-Fth1p complex. Using a combination of flameless atomic absorption spectrophotometry to quantify vacuolar and whole cell iron content and a reporter assay for cytoplasmic iron we demonstrate that Fre6p supplies FeII to both efflux systems, while Fre7p plays no role in Fe-efflux from the vacuole. Enzymatic assay shows the two fusions to have similar reductase activity, however. Confocal fluorescence microscopy demonstrates that Fre6:GFP localizes to the vacuolar membrane; in contrast, Fre7:GFP fusions exhibit a variable and diffuse cellular distribution. Demonstrating a role for a vacuolar metalloreductase in Fe-efflux supports the model that iron is stored in the vacuole in the ferric state. Across all superkingdoms, transition metal ion homeostasis is strongly dependent on the regulation of nutrient accumulation rather than on excess nutrient excretion (1Finney L.A. O'Halloran T.V. Science. 2003; 300: 931-936Crossref PubMed Scopus (940) Google Scholar, 2Aisen P. Enns C. Wessling-Resnick M. Int. J. Biochem. Cell Biol. 2001; 33: 940-959Crossref PubMed Scopus (611) Google Scholar, 3Curie C. Briat J.F. Annu. Rev. Plant Biol. 2003; 54: 183-206Crossref PubMed Scopus (448) Google Scholar, 4De Freitas J. Wintz H. Kim J.H. Poynton H. Fox T. Vulpe C. Biometals. 2003; 16: 185-197Crossref PubMed Scopus (129) Google Scholar, 5Luk E. Jensen L.T. Culotta V.C. J. Biol. Inorg. Chem. 2003; 8: 803-809Crossref PubMed Scopus (114) Google Scholar). Metal ion storage appears to be a complement of this regulatory pattern, storage that is illustrated by ferritin, the metallothioneins, and compartmentalization into cell organelles such as the mitochondria and vacuole. Although in some cases, this storage provides a detoxification mechanism (e.g. of heavy metals by the thioneins), it is as likely to play a dynamic role in cell and/or organismal metal ion homeostasis. This is certainly the case with ferritin in regards to vertebrate cell iron handling (2Aisen P. Enns C. Wessling-Resnick M. Int. J. Biochem. Cell Biol. 2001; 33: 940-959Crossref PubMed Scopus (611) Google Scholar), and is very likely to be the case with the vacuoles found in the cells of many diverse eukaryotic genera; the vacuole in fungi and plants is a dynamic organelle that plays a significant role in the overall nutritional status of the organism (3Curie C. Briat J.F. Annu. Rev. Plant Biol. 2003; 54: 183-206Crossref PubMed Scopus (448) Google Scholar, 4De Freitas J. Wintz H. Kim J.H. Poynton H. Fox T. Vulpe C. Biometals. 2003; 16: 185-197Crossref PubMed Scopus (129) Google Scholar, 5Luk E. Jensen L.T. Culotta V.C. J. Biol. Inorg. Chem. 2003; 8: 803-809Crossref PubMed Scopus (114) Google Scholar). Vacuoles provide a storage depot for newly arrived nutrients as well as being the site of macromolecular degradation and nutrient recycling (6Kucharczyk R. Rytka J. Acta Biochim. Pol. 2001; 48: 1025-1042Crossref PubMed Scopus (15) Google Scholar, 7Herman E.M. Larkins B.A. Plant Cell. 1999; 11: 601-614Crossref PubMed Scopus (321) Google Scholar). In regards to metal metabolism the vacuoles in plants and fungi have been associated with the handling of copper, iron, manganese, and zinc, in addition to magnesium and calcium; the yeast vacuole is essential for the detoxification of several first row transition metals (8Ramsay L.M. Gadd G.M. FEMS Microbiol. Lett. 1997; 152: 293-298Crossref PubMed Google Scholar, 9Szczypka M.S. Zhu Z. Silar P. Thiele D.J. Yeast. 1997; 13: 1423-1435Crossref PubMed Scopus (80) Google Scholar). Iron metabolism in the yeast Saccharomyces cerevisiae has served as a paradigm for iron handling in many other fungi, plants and even humans (1Finney L.A. O'Halloran T.V. Science. 2003; 300: 931-936Crossref PubMed Scopus (940) Google Scholar, 4De Freitas J. Wintz H. Kim J.H. Poynton H. Fox T. Vulpe C. Biometals. 2003; 16: 185-197Crossref PubMed Scopus (129) Google Scholar, 5Luk E. Jensen L.T. Culotta V.C. J. Biol. Inorg. Chem. 2003; 8: 803-809Crossref PubMed Scopus (114) Google Scholar, 10Kaplan J. Cell. 2002; 111: 603-606Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Under normal nutrient conditions ([Fe]environmental ≤ 1 μm) iron uptake is mediated by the high affinity iron uptake complex consisting of the multicopper ferroxidase Fet3p and iron permease Ftr1p. Homologous iron uptake complexes are likely to be found in most fungi and plants (10Kaplan J. Cell. 2002; 111: 603-606Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 11Kosman D.J. Mol. Microbiol. 2003; 47: 1185-1197Crossref PubMed Scopus (261) Google Scholar, 12Hell R. Stephan U.W. Planta. 2003; 216: 541-551Crossref PubMed Scopus (470) Google Scholar). Uptake by yeast of environmental FeIII is preceded by ferric iron reduction to FeII by plasma membrane reductase activity supplied principally by Fre1p; reduction of environmental FeIII (and CuII) is paradigmatic of the uptake of these metal ions by all aerobic organisms (3Curie C. Briat J.F. Annu. Rev. Plant Biol. 2003; 54: 183-206Crossref PubMed Scopus (448) Google Scholar, 11Kosman D.J. Mol. Microbiol. 2003; 47: 1185-1197Crossref PubMed Scopus (261) Google Scholar, 13Andrews N.C. Fleming M.D. Gunshin H. Nutr. Rev. 1999; 57: 114-123Crossref PubMed Scopus (74) Google Scholar, 14Schroder I. Johnson E. de Vries S. FEMS Microbiol. Rev. 2003; 27: 427-447Crossref PubMed Scopus (242) Google Scholar). Given the reduction potential of the cytoplasm, iron is undoubtedly present there as FeII; based on the GSH/GSSG ratio quantified in a wild-type yeast cell, this potential can be as low as -300 mV (15Muller E.G. Mol. Biol. Cell. 1996; 7: 1805-1813Crossref PubMed Scopus (166) Google Scholar, 16Grant C.M. Mol. Microbiol. 2001; 39: 533-541Crossref PubMed Scopus (339) Google Scholar, 17Schafer F.Q. Buettner G.R. Free Rad. Biol. Med. 2001; 30: 1191-1212Crossref PubMed Scopus (3676) Google Scholar). This is likely to be the case in the cytoplasm where a major fraction of this GSH/GSSG is localized; in contrast, because of the overall non-equilibrium distribution of GSH in the cell, the reduction potential for the endoplasmic reticulum has been estimated at around -200 mV (17Schafer F.Q. Buettner G.R. Free Rad. Biol. Med. 2001; 30: 1191-1212Crossref PubMed Scopus (3676) Google Scholar). Also, the GSH/GSSG reduction potential is strongly pH-dependent: ΔE/ΔpH is +60 mV for a decrease in pH = 1.0 (17Schafer F.Q. Buettner G.R. Free Rad. Biol. Med. 2001; 30: 1191-1212Crossref PubMed Scopus (3676) Google Scholar). Thus, relatively acidic compartments like endosomes and the vacuole will be oxidizing in comparison to the cytoplasm; the presence of iron in the yeast vacuole as a ferric (poly)phosphate is consistent with this electrochemical condition (18Raguzzi F. Lesuisee E. Crichton R.R. FEBS Lett. 1988; 231: 253-258Crossref PubMed Scopus (130) Google Scholar). One hallmark of iron metabolism in yeast is, therefore, redox cycling between FeIII (exocytoplasmic, vesicular) and FeII (substrate for uptake, cytoplasmic) (1Finney L.A. O'Halloran T.V. Science. 2003; 300: 931-936Crossref PubMed Scopus (940) Google Scholar, 6Kucharczyk R. Rytka J. Acta Biochim. Pol. 2001; 48: 1025-1042Crossref PubMed Scopus (15) Google Scholar, 11Kosman D.J. Mol. Microbiol. 2003; 47: 1185-1197Crossref PubMed Scopus (261) Google Scholar). As noted, Fre1p supports this cycling at the plasma membrane where exocytoplasmic ferric iron needs to be reduced prior to ferroxidation and uptake by the Fet3p, Ftr1p complex. The FeII/FeIII ratio within the cell is likely determined by the overall reduction potential which, as noted, is strongly dependent on the [glutathione]total and pH. In addition, the GSH/GSSG ratio will be sensitive to the level of dioxygen; indeed, this ratio is a measure of cellular oxidative stress (17Schafer F.Q. Buettner G.R. Free Rad. Biol. Med. 2001; 30: 1191-1212Crossref PubMed Scopus (3676) Google Scholar, 19Wheeler G.L. Grant C.M. Physiol. Plant. 2004; 120: 12-20Crossref PubMed Scopus (61) Google Scholar). The differential redox speciation of iron in the cytoplasm and vacuole provides the context for the mechanism of the cycling of iron between these two compartments. Some of the proteins involved in this cycling in S. cerevisiae have been identified. Ccc1p is localized to the vacuolar membrane and appears to function as a divalent metal ion transporter; Ccc1p has been associated with calcium, manganese, and iron homeostasis in yeast (20Lapinskas P.J. Lin S.J. Culotta V.C. Mol. Microbiol. 1996; 21: 519-528Crossref PubMed Scopus (87) Google Scholar, 21Li L. Chen O.S. McVey Ward D. Kaplan J. J. Biol. Chem. 2001; 276: 29515-29519Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 22Fu D. Beeler T. Dunn T. Yeast. 1994; 10: 515-521Crossref PubMed Scopus (24) Google Scholar). Smf3p is a member of the Nramp divalent metal ion transporter family; Smf3p also exhibits relatively little selectivity among first row transition ions (23Nelson N. EMBO J. 1999; 18: 4361-4371Crossref PubMed Scopus (255) Google Scholar, 24Portnoy M.E. Liu X.F. Culotta V.C. Mol. Cell. Biol. 2000; 20: 7893-7902Crossref PubMed Scopus (182) Google Scholar). Smf3p localizes to the vacuolar membrane (24Portnoy M.E. Liu X.F. Culotta V.C. Mol. Cell. Biol. 2000; 20: 7893-7902Crossref PubMed Scopus (182) Google Scholar). Both transporters are thought to support vectorial iron flux with Ccc1p supporting import (21Li L. Chen O.S. McVey Ward D. Kaplan J. J. Biol. Chem. 2001; 276: 29515-29519Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar) and Smf3p supporting export of iron from the vacuolar lumen (24Portnoy M.E. Liu X.F. Culotta V.C. Mol. Cell. Biol. 2000; 20: 7893-7902Crossref PubMed Scopus (182) Google Scholar) although neither activity has been directly demonstrated. A complex of Fet5p and Fth1p is likely to support export of vacuolar iron also; this complex is localized to the vacuolar membrane with an orientation that is identical to that of its homologues, Fet3p and Ftr1p (25Urbanowski J.L. Piper R.C. J. Biol. Chem. 1999; 274: 38061-38070Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). In other words, both complexes transport iron into the cytoplasmic compartment: Fet3p, Ftr1p from the exocytoplasmic space, and Fth1p from the vacuolar As is the case for Fet3p, Ftr1p the for Fth1p transport is most as the of vacuolar iron is likely In with plasma membrane iron import that with Fth1p export with the reduction the yeast that reductase proteins, is of the activity of the Jensen L.T. G.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). from the and is also conditions of iron Freitas Kim J.H. Poynton H. T. Wintz H. Fox T. P. S. M. Vulpe C. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). Although the of the is of the S. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), the that all are of has been to as the iron or that be of the iron trafficking that cytoplasmic and vacuolar iron In this we that Fre6p supplies the reductase activity to FeII from vacuolar ferric (poly)phosphate The data support this yeast in this from and are in The was a from D. The was by the a from of yeast with this in of the from to was by The was by the a from to The was by the from the R. P. Yeast. 1994; 10: PubMed Scopus Google that from the and from the The was into was by for the a the and a in the The for of activity by and from with the by was at the in this in a of yeast on yeast The for in a yeast and of The the the atomic absorption with and for and a and for flameless atomic absorption spectrophotometry plasma membrane endoplasmic reticulum by the and this into and P. PubMed Google Scholar). The of and of from yeast while the and was from the a from D. into in the The was by from and it into the at the The was in the to for this This the in a to be in the In the case of the was at in and and prior to the to a from of cells in The cells by at for in of and and for at The cells with pH and with for at by at for and in of in pH was to the and the for on and for at The was to two and with of of and 1 of pH The at for The vacuolar fraction was from the this fraction to vacuoles with little with other or membrane The iron content of vacuoles was quantified by flameless atomic absorption spectrophotometry on a in on from two The in the from these combined and the reporter in cells in of pH 1 of and of The was for at and of was The was by addition of of 1 The was for at high at The of the was at and activity was in J.H. in Scholar). condition was in from for The reductase activity of fusions of or Fre7p in membrane was determined as by J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). proteins as in a in the presence of and iron to reductase A membrane fraction was differential The membrane was in the presence of and of was with and in 1 pH in a of The was at in a out in the of or the reductase quantified dependent on and in all Confocal and from the yeast cells in by with a of for was to the of the vacuoles with for and prior to the cells in and with of for 1 The cells and in with and the yeast cells in to of was to the cells and to to The cells and in with and for The by two in the yeast cells in to a of The from cells was with and in of The a to a a by at a Iron in the iron in the than (21Li L. Chen O.S. McVey Ward D. Kaplan J. J. Biol. Chem. 2001; 276: 29515-29519Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). In contrast, vacuoles the strongly iron from the cytoplasm (24Portnoy M.E. Liu X.F. Culotta V.C. Mol. Cell. Biol. 2000; 20: 7893-7902Crossref PubMed Scopus (182) Google Scholar). This of vacuolar iron content is illustrated by the first in In addition, vacuoles Fre6p even iron in the than vacuoles from the One of this is that Fre6p supplies FeII for both Smf3p and the vacuolar of the Fet5p ferroxidase and Fth1p iron permease (25Urbanowski J.L. Piper R.C. J. Biol. Chem. 1999; 274: 38061-38070Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). The between and export was in by of the of Ccc1p and Smf3p in and Using as the in the presence and of we the role of Fre6p in the balance between import and export of iron in the vacuole. of Ccc1p in wild-type cells in a in vacuolar iron accumulation of Smf3p reduced this iron accumulation by a of two with of Smf3p with Ccc1p reduced by the excess iron accumulation because of Ccc1p In all cases, vacuolar in the was the most in cells in Fre6p the of the accumulation because of The data indicate Fre6p is of Smf3p in the from the vacuole. As noted, the Fth1p pair a vacuolar Fe-efflux The role of Fre6p in this was in a similar Although of Fet5p and Fth1p little on iron accumulation in wild-type cells of 1 and it reduced by the excess iron accumulation because of Ccc1p This reduction was in the that Fre6p was of export as well Iron as a of the Iron balance between and iron in yeast has been by of a reporter in the is of the (21Li L. Chen O.S. McVey Ward D. Kaplan J. J. Biol. Chem. 2001; 276: 29515-29519Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 24Portnoy M.E. Liu X.F. Culotta V.C. Mol. Cell. Biol. 2000; 20: 7893-7902Crossref PubMed Scopus (182) Google Scholar, J.L. Piper R.C. J. Biol. Chem. 1999; 274: 38061-38070Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). iron is to the where it of in the in high to the cytoplasm R. R. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). Using this the of vacuolar iron accumulation on the of Ccc1p and Smf3p in and be data are in In wild-type cells Ccc1p exhibit an in this is by the of Fre6p first two of and This the vacuolar iron data in of Smf3p by the in activity because of vacuolar Ccc1p consistent with the iron data in to the of the of on Fe-efflux in the of Smf3p to the to the by Ccc1p was in the A similar was between the reporter and vacuolar iron in regards to the of the Fth1p ferroxidase, permease This export complex the of cytoplasmic iron because of of Ccc1p in this was by in the two of data support the that Fre6p of both of the two in the yeast vacuolar Fre7p from Fre6p in that is by the Also, Fre7p exhibits the to the other yeast although it all of the involved in and Jensen L.T. G.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). we the of to that because of the assay to it role in vacuolar The of this are in in the reporter assay was in a of Fe-efflux Smf3p and the Fth1p ferroxidase, permease pathways of no on Smf3p or Fth1p all to in of and Iron that of the two of vacuolar membrane iron trafficking we vacuolar iron and the of by iron, the assay was by the to plasma membrane iron trafficking to the level of cell iron. an vacuolar because of of Ccc1p decrease cytoplasmic iron and in the in an into the cytoplasm the from reporter In a the from a Smf3p on the in vacuolar iron efflux as a decrease in reporter the in cytoplasmic iron decrease uptake of iron into the cell as these by whole cell iron in the in these The data in this of plasma membrane and cycling of iron out of and into the vacuole Thus, in comparison to wild-type cells by the cell iron was in a in a two and cell iron in the and was the This is consistent with a model in the reduced Fe-efflux into the cytoplasm in the in comparison to the in a of at the This was with of vacuolar or efflux or of data that support the that Fre6p Fre7p is from both pathways for vacuolar Fre6p and Fre7p to demonstrate Fre6p or Fre7p a reductase activity, we membrane from a yeast fusions of or from a high of the The cells in the presence of and iron to of reductase In these cells these conditions we that the major fraction of the reductase activity from of a reductase such it was to metalloreductase with FeII or as of we reductase activity as a for proteins, E. M. P. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). The of this are in A and demonstrate that in this an of reductase activity is of the reductase being Although the in that the fusions at in the cell this Fre7p is as an reductase the in comparison to Fre1p or Fre6p Fre7p to the Fre6p Fre7p has been localized in the yeast the of the fusions by fluorescence this Fre6:GFP localized to the vacuolar membrane This was by with a for the vacuolar The of fluorescence from the vacuolar lumen that little of the was being and within the vacuole that the was a of the was to this of C. C. Microbiol. 2002; PubMed Scopus Google Scholar). In to the and of the to the vacuolar of the Fre7:GFP fusions and a fluorescence from these was diffuse and cytoplasmic with little accumulation in cellular the vacuolar or plasma all Fre7p the at to the Fre6:GFP this we that it was to the of the all of these fusions in the cell by Fre6p and Fre7p fusions of low and by iron in yeast is of iron are from vacuoles in yeast from to as from to (18Raguzzi F. Lesuisee E. Crichton R.R. FEBS Lett. 1988; 231: 253-258Crossref PubMed Scopus (130) Google Scholar). a vacuolar iron import or export complex wild-type in this to a (25Urbanowski J.L. Piper R.C. J. Biol. Chem. 1999; 274: 38061-38070Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). are two in the yeast vacuolar membrane to be involved in vacuolar iron the Fet5p-Fth1p complex (25Urbanowski J.L. Piper R.C. J. Biol. Chem. 1999; 274: 38061-38070Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar) and of the yeast of the Nramp divalent metal iron transporters (24Portnoy M.E. Liu X.F. Culotta V.C. Mol. Cell. Biol. 2000; 20: 7893-7902Crossref PubMed Scopus (182) Google Scholar). the redox of iron present in the cell is strongly dependent the in is strongly (17Schafer F.Q. Buettner G.R. Free Rad. Biol. Med. 2001; 30: 1191-1212Crossref PubMed Scopus (3676) Google Scholar), the relatively acidic vacuole is likely to be oxidizing in comparison to the cytoplasm; vacuolar iron is likely stored as a ferric and/or (18Raguzzi F. Lesuisee E. Crichton R.R. FEBS Lett. 1988; 231: 253-258Crossref PubMed Scopus (130) Google Scholar). In as as FeII is the for the Fet5p-Fth1p complex and for is for the of iron from the vacuole. the that reductase proteins in is by iron with and S. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). Our data indicate that Fre6p supplies the reductase activity to support the of FeII from the vacuolar ferric iron and the export of iron from the vacuole Smf3p and the Fet5p-Fth1p complex. have localized a of Fre6p to the vacuolar In the we for Fre7:GFP to the at the level of of the because of no cellular be for this the other at this level of Fre6p and Fre7p similar reductase activity, that was to that for the plasma membrane In a and Thiele E.M. Thiele D.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) a plasma membrane for a Fre7:GFP in an a that is involved in membrane proteins for is a sensitive that is at J. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar). In this plasma membrane of Fre7:GFP was at the This a metalloreductase activity conditions like that by this from of in the and the trafficking of the to the or to a of the in the an Fre6p and Fre7p in regulation with the of the of and the of the the of proteins that and Fth1p be to a the of ferric iron from the vacuole. In this that being Fre7p play a role in the redox of vacuolar copper, to as the plasma membrane Fre1p and in support of cell the S. Thiele D.J. Chem. Biol. 2002; PubMed Scopus Google Scholar). is the vacuolar to out of the vacuole M.E. P.J. Culotta V.C. Mol. 2001; PubMed Scopus Google Scholar, E.M. J. Thiele D.J. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). The work by and Thiele E.M. Thiele D.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), that from the vacuole is, like dependent on Thus, Fre6p is similar to Fre1p in that both proteins function from the iron and transport in the vacuolar and plasma of The two in regulation and is of both the and S. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, Fre1p activity is iron or is Fre6p activity is cytoplasmic iron is This regulatory that from the vacuole is an in is This can be in that the from the vacuole can be to the Fet3p ferroxidase in support of at the plasma membrane D.J. Mol. Microbiol. 2003; 47: 1185-1197Crossref PubMed Scopus (261) Google Scholar). we indicate the role of Fre7p as a metalloreductase will be found in some other than membrane One is that as the reductase in is for efficient of iron from Chen J. Fleming M.D. PubMed Scopus Google Scholar, Fleming M.D. PubMed Scopus Google Scholar), Fre7p be involved in the of iron, as from an ferric iron In as as S. cerevisiae can environmental iron from M. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar), it is a that these be the for this reductase that as of a for in the of fluorescence microscopy in the of in yeast and to and Thiele for work on metal ion from the vacuole. with