YKE4 (YIL023C) Encodes a Bidirectional Zinc Transporter in the Endoplasmic Reticulum of Saccharomyces cerevisiae

YIL023C encodes a member of the SLC39A, or ZIP, family, which we refer to as yeast KE4 (YKE4) after its mouse ortholog. Yke4p was localized to the endoplasmic reticulum (ER) membrane using Yke4p-specific antiserum. YKE4 is not an essential gene; however, deletion of YKE4 resulted in a sensitivity to calcofluor white and poor growth at 36 °C on respiratory substrates containing high zinc. Overexpression of transition metal transporters Zrc1p and Cot1p or the mouse orthologue mKe4 in Δyke4 suppressed the poor growth at 36 °C on respiratory substrates. We found that the role of Yke4p depends on the zinc status of the cells. In a zinc-adequate environment, Yke4p transports zinc into the secretory pathway, and the deletion of YKE4 leads to a zinc-suppressible cell wall defect. In high zinc medium, transport of zinc into the secretory pathway through Yke4p is a way to eliminate zinc from the cytosol, and deletion of YKE4 leads to toxic zinc accumulation in the cytosol. Under low cytosolic zinc conditions, however, Yke4p removes zinc from the secretory pathway, and deletion of YKE4 partially compensates for the loss of Msc2p, an ER zinc importer, and therefore helps to alleviate ER stress. In our model, Yke4p balances zinc levels between the cytosol and the secretory pathway, whereas the previously described Msc2p-Zrg17p ER zinc importer complex functions mainly in zinc-depleted conditions to ensure a ready supply of zinc essential for ER functions, such as phospholipid biosynthesis and unfolded protein response. YIL023C encodes a member of the SLC39A, or ZIP, family, which we refer to as yeast KE4 (YKE4) after its mouse ortholog. Yke4p was localized to the endoplasmic reticulum (ER) membrane using Yke4p-specific antiserum. YKE4 is not an essential gene; however, deletion of YKE4 resulted in a sensitivity to calcofluor white and poor growth at 36 °C on respiratory substrates containing high zinc. Overexpression of transition metal transporters Zrc1p and Cot1p or the mouse orthologue mKe4 in Δyke4 suppressed the poor growth at 36 °C on respiratory substrates. We found that the role of Yke4p depends on the zinc status of the cells. In a zinc-adequate environment, Yke4p transports zinc into the secretory pathway, and the deletion of YKE4 leads to a zinc-suppressible cell wall defect. In high zinc medium, transport of zinc into the secretory pathway through Yke4p is a way to eliminate zinc from the cytosol, and deletion of YKE4 leads to toxic zinc accumulation in the cytosol. Under low cytosolic zinc conditions, however, Yke4p removes zinc from the secretory pathway, and deletion of YKE4 partially compensates for the loss of Msc2p, an ER zinc importer, and therefore helps to alleviate ER stress. In our model, Yke4p balances zinc levels between the cytosol and the secretory pathway, whereas the previously described Msc2p-Zrg17p ER zinc importer complex functions mainly in zinc-depleted conditions to ensure a ready supply of zinc essential for ER functions, such as phospholipid biosynthesis and unfolded protein response. Zinc is not redox-active; however, its ability to form multiple bonds has made it an important structural component in hundreds of different enzymes; about 3% of human genes are thought to encode zinc-binding proteins (1Eide D.J. J. Nutr. 2003; 133: 1532S-1535SCrossref PubMed Google Scholar). Eukaryotic zinc transporters are grouped into two families, the cation diffusion family (CDF or SLC30) and the ZRT, IRT-like protein (ZIP or SLC39) family (2Eide D. Curr. Opin. Cell Biol. 1997; 9: 573-577Crossref PubMed Scopus (83) Google Scholar, 3Kambe T. Yamaguchi-Iwai Y. Sasaki R. Nagao M. Cell Mol. Life Sci. 2004; 61: 49-68Crossref PubMed Scopus (338) Google Scholar). Members of the CDF family include the yeast Zrc1p, Cot1p, Msc2p, and Zrg17p proteins and the human and mouse ZNT1–8. These transporters are thought to be proton-metal antiporters. Based on sequence similarity, there are now hundreds of proteins in the current sequence data bases belonging to the ZIP family. The ZIP family can be divided into four large families. One of these families, the LIV1/KE4 family, contains several transporters, such as the human and mouse ZIP4, the fruit fly FOI, and the zebrafish LIV1 (4Taylor K.M. Nicholson R.I. Biochim. Biophys. Acta. 2003; 1611: 16-30Crossref PubMed Scopus (215) Google Scholar). These transporters (ZIP4, FOI, and LIV1) form a subfamily, usually named after LIV1, which was the first gene of this group to be identified. Although the Ke4-like proteins share many features with the LIV1 group, they form a separate subfamily that is present in all investigated eukaryotes. The human, mouse, zebrafish, Drosophila melanogaster, Arabidopsis thaliana, and Saccharomyces cerevisiae KE4-like genes are orthologous. It is of interest that the mammalian KE4 gene is located in the major histocompatibility complex. It is generally thought that the CDF and ZIP families play opposing roles in eukaryotic zinc transport; CDF proteins lower the cytosolic zinc content by transporting zinc out of the cell or into intracellular organelles, whereas the ZIP proteins increase cytosolic zinc by transporting zinc into the cells or out of intracellular organelles. Consistent with this view, a recent study has suggested that the mouse Ke4 is a Golgi zinc exporter (5Huang L. Kirschke C.P. Zhang Y. Yu Y.Y. J. Biol. Chem. 2005; 280: 15456-15463Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). We have investigated the function of the yeast Ke4 protein. Here we provide genetic and biochemical evidence that shows that Yke4p is capable of transporting zinc in both directions. Since the mouse Ke4 gene can substitute for the yeast and A. thaliana genes, we suggest that bidirectional transport may be conserved in all eukaryotes. Yeast Strains, Growth Media, and Plasmids—Yeast strains used in this study are shown in Table 1. YKE4 and PSD1 (phosphatidylserine decarboxylase 1) were deleted in W303 wild type cells (DY150), utilizing the KanMX4 cassette. KanMX4 cassette-disrupted open reading frames were PCR-amplified from the genome-wide deletion collection (Invitrogen) using the following primers: YKE4 PR1032, 5′-AGT AGA ATT TCT GCG GAG AAC-3′ and PR1033 5′-TCC AGT TAT CGA ATT CTC CTG-3′; PSD1 PR1235, 5′-GG TAC GTA GCG CTT TAA GGA-3′; and PR1236, 5′-AAG GGG TAC ATG ACA TGG CT-3′. The PCR-amplified yke4::KanMX4 cassette was transformed into DY150 cells. psd1::KanMX4 cassette was transformed into both DY1457 (MATα) and DY150 (MATa) strains. DY1457Δpsd1 was used to mate the DY150-based strains to create double and triple mutant cells. The double and triple deletion strains were confirmed by PCR analysis.TABLE 1Cell lines used in this studyStrainGenotypeSourceDY150MATa, ura3-52, leu2-3, 112, trp1-1, his3-11, ade2-1, can1-100(oc)W303 wild typeDY1457MATα, ura3-52, leu2-3, 112, trp1-1, his3-11, ade6-1, can1-100(oc)W303 wild typeΔyke4DY150 yke4::KanMX4This studyΔmsc2DY150 msc2::HIS3Li and Kaplan (11Li L. Kaplan J. J. Biol. Chem. 2001; 276: 5036-5043Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar)Δmsc2Δyke4DY150 yke4::KanMX4 msc2::HIS3This studyΔpsd1DY150 psd1::KanMX4This studyDY1457Δpsd1DY1457 psd1::KanMX4This studyΔpsd1Δyke4DY150 psd1::KanMX4 yke4::KanMX4This studyΔpsd1Δmsc2DY150 psd1::KanMX4 msc2::HIS3This studyΔpsd1Δmsc2Δyke4DY150 psd1::KanMX4 msc2::HIS3 yke4::KanMX4This studyΔgas1BY4741 gas1::KanMX4Deletion collectionΔzrc1MATa, ura3-52, leu2-3, 112, trp1-1, his3-11, ade2-1, can1-100(oc) zrc1::HIS3Conklin et al. (6Conklin D.S. McMaster J.A. Culbertson M.R. Kung C. Mol. Cell. Biol. 1992; 12: 3678-3688Crossref PubMed Scopus (181) Google Scholar)Δcot1MATa, ura3-52, leu2-3, 112, trp1-1, his3-11, ade2-1, can1-100(oc) cot1::URA3Conklin et al. (6Conklin D.S. McMaster J.A. Culbertson M.R. Kung C. Mol. Cell. Biol. 1992; 12: 3678-3688Crossref PubMed Scopus (181) Google Scholar)Δzrc1Δcot1zrc1::HIS3 cot1::URA3This studyΔzrc1Δcot1Δyke4zrc1::HIS3 cot1::URA3 yke4::KanMX4This study Open table in a new tab YKE4 was cloned and expressed with its own promoter (defined as a 1000-bp-long 5′ region of the start codon) into high copy episomal (pTF63) and low copy centromeric (YCp33) plasmids using TGG and TCT AGA GCG GTA of YKE4 was by the open reading into with ATG GCG ATT and CTC GAG The mouse Ke4 was from of mouse using ATG and AGA TAA and cloned using the from the mouse Ke4 gene in the mouse promoter and sequence were with the yeast promoter and the mKe4 was with GAG and TCT CGA GGG and the yeast promoter and were using and These a they the of mouse Ke4 with that encode create a The were cloned into containing or were from D. (6Conklin D.S. McMaster J.A. Culbertson M.R. Kung C. Mol. Cell. Biol. 1992; 12: 3678-3688Crossref PubMed Scopus (181) Google Scholar). The and plasmids were from of was (11Li L. Kaplan J. J. Biol. Chem. 2001; 276: 5036-5043Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). was PCR-amplified and cloned into using GGG TCT TCT AGT and GGG AGT TAT was by of the from the were in yeast and or in yeast and or zinc were made from yeast by and and the of zinc. high copy of the high zinc sensitivity of Δyke4 a cloned into was transformed into were containing zinc. The were at 36 °C for were identified. of these were found to on high zinc The plasmids were and transformed into by The in the plasmids were using and GCG and to the genes for the and Yke4p and mouse we of of and of mKe4 that are in of Ke4 The were to using and the was into using The were using to an to the cells were for as previously described M.R. L. Kaplan J. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). The was used at a by a of cells were with a of mouse by a of was used at a by a of or were on an mouse were on in with and for as described previously D. Kaplan J. 2003; PubMed Scopus Google Scholar). The was used at a by an were and and were by on and the were by were with to a Golgi mouse an ER used endoplasmic unfolded protein unfolded protein yeast yeast zinc and mouse a mouse and a membrane mouse by or Life was used for used gene to the ER and zinc The unfolded protein containing the from the promoter of the gene or T. D.J. J. Cell Biol. 2004; PubMed Scopus Google and the zinc containing the from or C. M. D.J. J. PubMed Scopus Google were used to the of the gene in The was as previously described (11Li L. Kaplan J. J. Biol. Chem. 2001; 276: 5036-5043Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). of Ke4 to the the of we both mouse and yeast Ke4 and were used to the The of was on from wild and Δyke4 cells in which YKE4 was on a from Δyke4 cells transformed with a containing YKE4 expressed the of its promoter a at that was not in from Δyke4 cells The of the to the the of the Yke4p The of the on the of is The of Yke4p was by were and and were by on and the were by using the were by The Golgi was in cells transformed with a which was with an The ER was by and using The Yke4p that Yke4p is present in in ER The of Yke4p was confirmed using type on a were with the by an or mouse to by The a of the ER in cerevisiae for Yke4p and a for the however, of Yke4p and the ER protein These suggest that Yke4p is localized to the The of Yke4p was investigated both high and low zinc conditions and was by zinc The mouse Ke4 protein was localized by with a a of the Golgi In to the Golgi large of secretory for et al. (5Huang L. Kirschke C.P. Zhang Y. Yu Y.Y. J. Biol. Chem. 2005; 280: 15456-15463Abstract Full Text Full Text PDF PubMed Scopus (164) Google using an a in cell such as and cells. in mouse cells was in zinc or zinc conditions not We that the Ke4 protein is mainly localized to the Golgi in mouse whereas it mainly to the ER in Δyke4 to YKE4 gene was in a W303 using a KanMX4 deletion and the gene deletion was confirmed by The Δyke4 was for such as growth on as as to cell and high and low transition We found that Δyke4 cells not at on high zinc on containing the respiratory substrates and as The high zinc sensitivity of Δyke4 cells was suppressed by the YKE4 gene on a low or high copy using its own promoter or the promoter We that of mouse Ke4 in Δyke4 cells the high of these that the function of yeast and mouse proteins is conserved of the of Δyke4 of the of Δyke4 cells on at 36 °C to genes that that yeast cloned into a high copy was used to Δyke4 cells in that were to high zinc on at 36 plasmids and encodes a cell gene that is expressed in the J. PubMed Scopus Google encodes a protein that is of the large J. Biol. Chem. Full Text PDF PubMed Google and encodes a zinc that the of zinc from cytosol D. J. PubMed Scopus Google Scholar). Zrc1p is a zinc Overexpression of Zrc1p cytosolic zinc D. J. PubMed Scopus Google that the of the high zinc sensitivity of the Δyke4 cell is cytosolic zinc. is by a zinc importer, Cot1p D. J. PubMed Scopus Google in Δyke4 cells. on a high copy suppressed the zinc sensitivity of the deletion We the ability of and to the zinc sensitivity of cells and found that the high copy of the growth of cells on high zinc zinc in the of not not Overexpression of and the Cell by is a cell that to The families are by sequence and include of which are not by the cell in the of D. J. Mol. Cell. Biol. PubMed Scopus Google Scholar). that high levels of can cell the of the to high of the of cells in leads to of the cell at the The can be by the of a mutant J. Yamaguchi-Iwai Y. A. J. PubMed Scopus Google Scholar). is to zinc we transformed the high copy into cells. is a importer, and cells are to high they are to cytosolic in L. D. Kaplan J. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). Overexpression of was to the growth of as shown on that is not to zinc cells from high cytosolic The between zinc and is not is a component of the large and to the and proteins J. Biol. Chem. Full Text PDF PubMed Google Scholar). proteins is not thought to be of it contains zinc In to growth to high cytosolic we of the high growth of the The of of was not to was to the growth of cells Although the is both and can the cell by high cytosolic metal that the of cell growth of Δyke4 cells on high zinc is by zinc accumulation in the cytosol. of YKE4 with of an ER Zinc that Yke4p can lower cytosolic zinc by transporting zinc into the the of Yke4p to the secretory pathway zinc we cells with in both and is an ER zinc importer, and cells with a deletion in an ER that can be suppressed by growth in high zinc T. D.J. J. Cell Biol. 2004; PubMed Scopus Google Scholar). with a deletion in or Δyke4 or with a deletion in both genes have growth in at °C Δyke4 cells wild type there is growth for The double deletion not at that there is a both YKE4 and are The of zinc the growth of the however, the of zinc to the growth defect. The from deletion of YKE4 and these conditions that these transporters are zinc into the this we the of of the Msc2p-Zrg17p complex on the growth of Δyke4 cells. We that of the ER complex Msc2p-Zrg17p the growth of Δyke4 cells on high zinc Yke4p is an ER zinc type and Δyke4 cells were transformed with a containing its own promoter D.J. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google and a containing a promoter (11Li L. Kaplan J. J. Biol. Chem. 2001; 276: 5036-5043Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). The cells were at °C on to the of both and Overexpression of or resulted in in the growth of the wild type and Δyke4 strains Overexpression of and however, to growth of wild type cells at and this growth was not by high zinc. Overexpression of and the growth of Δyke4 cells on high zinc the that Yke4p may function as an ER zinc Yke4p for Cell found that Δyke4 are cells are to calcofluor a that to cell wall cell wall and cell growth Although it is a cell wall can the of calcofluor white A. R. PubMed Scopus Google Scholar). The calcofluor white is by of YKE4 or mouse Ke4 not We the sensitivity of Δyke4 to calcofluor white can be by the of zinc. We used cells as a to that zinc not with the of calcofluor is an which is for cell wall and deletion of leads to calcofluor white sensitivity PubMed Scopus Google Scholar). The be by zinc. the calcofluor white sensitivity of Δyke4 can be suppressed by zinc not the calcofluor white sensitivity of cells. We that zinc is for cell wall in the secretory pathway, cells have a cell wall defect. We found that cells are to calcofluor white Δyke4 cells. for Δyke4 the calcofluor white sensitivity was suppressed by zinc in the medium, the zinc was Zinc in the are proteins or in the yeast secretory et al. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google that low zinc conditions, the of the phospholipid are by the of decarboxylase which is a the for phospholipid biosynthesis are in the secretory pathway We a of mutant strains in which PSD1 is PSD1 encodes about of the decarboxylase and the is by the cells are on such as We that cells are to low zinc We this in two by cells on low zinc and by zinc transporters localized to the secretory We found that cells not on low zinc at 36 growth can be by zinc in the the of from and growth of the can be by in the growth can be through the pathway J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google zinc is for phospholipid biosynthesis in the secretory pathway, the growth of cells be by The growth of cells is the and can partially the growth of cells The for this be that the in the phospholipid pathway in to may be the of Yke4p on phospholipid we the of the deletion of YKE4 in cells as as in cells zinc transporters were We found that the cells the cells is a zinc importer and deletion of YKE4 cell Yke4p may be transporting zinc out of the secretory We that cells be to calcofluor are for cell wall shown in cells are to calcofluor and zinc in the can cell with a deletion in both PSD1 and YKE4 are to calcofluor white of the of YKE4 in the resulted in growth on calcofluor white from these is that the of zinc transport depends on the zinc status of the cytosol and Under low zinc conditions, Yke4p transports zinc out of the secretory deletion of YKE4 helps the growth of and cells on low zinc calcofluor white however, deletion of YKE4 the calcofluor white sensitivity of cells of YKE4 in however, helps growth on calcofluor that ER zinc by the deletion of in ER zinc levels T. D.J. J. Cell Biol. 2004; PubMed Scopus Google that the of zinc transport Under this Yke4p transports zinc out of the secretory We confirmed this using genes to zinc levels in the cytosol and the secretory way to the zinc levels is to that cytosolic zinc or ER zinc In zinc-depleted conditions, the is and to the zinc promoter (2Eide D. Curr. Opin. Cell Biol. 1997; 9: 573-577Crossref PubMed Scopus (83) Google Scholar). We a that contains the from the promoter of the high membrane zinc a of the cytosolic and zinc et al. T. D.J. J. Cell Biol. 2004; PubMed Scopus Google that zinc is in the the ER unfolded protein is Zinc in the ER and of the can be by or which encodes its protein D.J. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). We a to the levels of zinc in the We the and in wild and cells. a increase in and cells and in Δyke4 cells as the increase in Δyke4 cells and in and cells. These suggest that Yke4p to transport zinc out of the secretory The between these strains were we strains that in zinc between the cytosol and the secretory We a of mutant in which the zinc transporters and were cells zinc in the and cells are to the of zinc in the medium, zinc in the cytosol. Zrc1p and Cot1p to the zinc supply in the and deletion of these genes the of an deletion T. D.J. J. Cell Biol. 2004; PubMed Scopus Google Scholar). Growth of cells is by zinc on D.J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). °C on cells zinc sensitivity as on at °C however, cells are to high zinc cells 36 cells on and not at all on not the deletion of YKE4 in cells leads to growth on We transformed and plasmids into these deletion strains. The is expressed cytosolic zinc is The of zinc to wild type cells the of the by about In zinc was to the of these cells have cytosolic zinc was in cells with that Yke4p transports zinc into the cytosol, its deletion the cytosolic zinc The is in low zinc medium, as by the gene of YKE4 in cells the of in low zinc medium, that this Yke4p is an ER zinc exporter these suggest that Yke4p transports zinc to the zinc levels in the cytosol and The protein of the yeast open reading is a member of the ZIP, or family and is suggested to be a transition metal found in all eukaryotes. suggest that all KE4-like genes are as the mouse Ke4 can to both yeast and Arabidopsis KE4 J. C. Cell. 12: PubMed Scopus Google Scholar). genetic that the genes are the proteins to in between yeast and Yke4p was localized to the ER in as in this and by et al. (5Huang L. Kirschke C.P. Zhang Y. Yu Y.Y. J. Biol. Chem. 2005; 280: 15456-15463Abstract Full Text Full Text PDF PubMed Scopus (164) Google the expressed mouse Ke4 is localized to the Golgi is a for yeast and mammalian different Msc2p, a zinc that is a member of the cation diffusion family, is found in the ER in yeast T. D.J. J. Cell Biol. 2004; PubMed Scopus Google Scholar, L. Kaplan J. J. Biol. Chem. 2001; 276: 5036-5043Abstract Full Text Full Text PDF PubMed Scopus (80) Google its mammalian orthologue is found in the Golgi T. Nagao M. Yamaguchi-Iwai Y. T. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). The of both the mouse (5Huang L. Kirschke C.P. Zhang Y. Yu Y.Y. J. Biol. Chem. 2005; 280: 15456-15463Abstract Full Text Full Text PDF PubMed Scopus (164) Google and the yeast Ke4 proteins was not by zinc has to be a transition metal on genetic for Ke4 have in D. and A. of Ke4 in named or have PubMed Google Scholar). It was that protein In KE4 were through a to to the of is a for many In is usually to or and are to the from the These usually or for 1) is a mutant of the and was found to the of J. C. Cell. 12: PubMed Scopus Google Scholar). It was suggested that transport for the or may that was thought not to be a not the sensitivity of a in the yeast Golgi mutant J. C. Cell. 12: PubMed Scopus Google Scholar). in both yeast and mammalian cells suggest that is a zinc et al. (5Huang L. Kirschke C.P. Zhang Y. Yu Y.Y. J. Biol. Chem. 2005; 280: 15456-15463Abstract Full Text Full Text PDF PubMed Scopus (164) Google used a to that mammalian cells with a have with cells a Golgi zinc The expressed the mouse Ke4 gene a in yeast cells in which the gene was is thought to zinc from the and deletion of leads to cytosolic zinc. of the mouse Ke4 protein the cytosolic zinc as by the an mouse Ke4 gene the zinc content in cells to zinc in the et al. (5Huang L. Kirschke C.P. Zhang Y. Yu Y.Y. J. Biol. Chem. 2005; 280: 15456-15463Abstract Full Text Full Text PDF PubMed Scopus (164) Google from these that mKe4 transports zinc from the Golgi into the is with of the 1) deletion of YKE4 helps the growth of cells on low zinc and deletion of YKE4 helps the growth of cells on calcofluor is to ER zinc the ER zinc importer in cells resulted in a growth growth was by zinc or The calcofluor white sensitivity of cells be suppressed by zinc in the or by deletion of YKE4 the of the ER These are with the that Yke4p is a secretory pathway zinc is however, that conditions Yke4p and mouse Ke4 expressed in yeast can transport zinc into the secretory this the 1) of YKE4 in a sensitivity to zinc at high in cells on respiratory substrates. sensitivity is by cytosolic zinc as suggested by the that of zinc transporters and this the of the two of the are our that they can growth from high cytosolic The is that they growth from high of cytosolic transition of YKE4 in a calcofluor white which is suppressed by high zinc. Since calcofluor white sensitivity to deletion of is not by we that deletion of YKE4 calcofluor white sensitivity by zinc. this is the that deletion of in a zinc-suppressible calcofluor white deletion of and YKE4 leads to a sensitivity to high of the and of and can the growth of Δyke4 cells on high respiratory at is that can function transporting zinc both into and out of the the is that Yke4p to the zinc between cytosol and as a zinc are One that we are is that the of transport not on the zinc is a of a response. of data shows of YKE4 in to conditions that to of M.R. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). of through a leads to a of YKE4 R. C. C. R. J. C. J. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). Based on these we that the of transport may be not by the zinc by of which be by Yke4p We for to start this and and the of the Kaplan for and