Heteromeric Protein Complexes Mediate Zinc Transport into the Secretory Pathway of Eukaryotic Cells
2005; Elsevier BV; Volume: 280; Issue: 31 Linguagem: Inglês
10.1074/jbc.m505500200
ISSN1083-351X
AutoresCharissa D. Ellis, Colin W. MacDiarmid, David Eide,
Tópico(s)Plant Micronutrient Interactions and Effects
ResumoThe cation diffusion facilitator (CDF) family of metal ion transporters plays important roles in zinc transport at all phylogenetic levels. In this report, we describe a novel interaction between two members of the CDF family in Saccharomyces cerevisiae. One CDF member in yeast, Msc2p, was shown recently to be involved in zinc transport into the endoplasmic reticulum (ER) and required for ER function. We describe here a newly recognized CDF family member in yeast, Zrg17p. ZRG17 was previously identified as a zinc-regulated gene controlled by the zinc-responsive Zap1p transcription factor. A zrg17 mutant exhibits the same zinc-suppressible phenotypes as an msc2 mutant, including an induction of the unfolded protein response in low zinc. Moreover, a significant fraction of the total Zrg17p protein appears to localize to the ER. Their common phenotypes and localization suggested that these two proteins function together to mediate zinc transport into the ER. Consistent with this hypothesis, Msc2p and Zrg17p physically interact with each other, as determined by co-immunoprecipitation. Therefore, we propose that Msc2p and Zrg17p form a heteromeric zinc transport complex in the ER membrane. We also demonstrate that ZnT5 and ZnT6, mammalian homologues of Msc2p and Zrg17p, functionally interact as well. These results suggest that heteromeric complexes formed by different CDF members may be a common phenomenon for this ubiquitous family of metal ion transporters. The cation diffusion facilitator (CDF) family of metal ion transporters plays important roles in zinc transport at all phylogenetic levels. In this report, we describe a novel interaction between two members of the CDF family in Saccharomyces cerevisiae. One CDF member in yeast, Msc2p, was shown recently to be involved in zinc transport into the endoplasmic reticulum (ER) and required for ER function. We describe here a newly recognized CDF family member in yeast, Zrg17p. ZRG17 was previously identified as a zinc-regulated gene controlled by the zinc-responsive Zap1p transcription factor. A zrg17 mutant exhibits the same zinc-suppressible phenotypes as an msc2 mutant, including an induction of the unfolded protein response in low zinc. Moreover, a significant fraction of the total Zrg17p protein appears to localize to the ER. Their common phenotypes and localization suggested that these two proteins function together to mediate zinc transport into the ER. Consistent with this hypothesis, Msc2p and Zrg17p physically interact with each other, as determined by co-immunoprecipitation. Therefore, we propose that Msc2p and Zrg17p form a heteromeric zinc transport complex in the ER membrane. We also demonstrate that ZnT5 and ZnT6, mammalian homologues of Msc2p and Zrg17p, functionally interact as well. These results suggest that heteromeric complexes formed by different CDF members may be a common phenomenon for this ubiquitous family of metal ion transporters. Zinc is required by all organisms because of the essential roles this metal plays in cells. For example, zinc is important for the activity of many proteins that reside in or move through the secretory pathway. In pancreatic β cells, zinc is needed for the assembly of proinsulin into homohexamers in the endoplasmic reticulum (ER) 1The abbreviations used are: ER, endoplasmic reticulum; GPI, glycosylphosphatidylinositol; ZRE, zinc-responsive element; CDF, cation diffusion facilitator; UPR, unfolded protein response; TAP, tandem affinity purification. 1The abbreviations used are: ER, endoplasmic reticulum; GPI, glycosylphosphatidylinositol; ZRE, zinc-responsive element; CDF, cation diffusion facilitator; UPR, unfolded protein response; TAP, tandem affinity purification. and/or Golgi (1.Huang X.F. Arvan P. J. Biol. Chem. 1995; 270: 20417-20423Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 2.Dodson G. Steiner D. Curr. Opin. Struct. Biol. 1998; 8: 189-194Crossref PubMed Scopus (428) Google Scholar). The subsequent packaging of insulin into crystalline structures within secretory granules also requires zinc (1.Huang X.F. Arvan P. J. Biol. Chem. 1995; 270: 20417-20423Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Killer T cells present antigen peptides on their cell surface bound to major histocompatibility complex class I molecules. The processing of some of these peptides takes place in the ER by the ER-associated aminopeptidase, which utilizes zinc in its active site (3.Rock K.L. York I.A. Goldberg A.L. Nat. Immunol. 2004; 5: 670-677Crossref PubMed Scopus (206) Google Scholar, 4.Serwold T. Gonzalez F. Kim J. Jacob R. Shastri N. Nature. 2002; 419: 480-483Crossref PubMed Scopus (470) Google Scholar). Zinc metalloenzymes responsible for attaching phosphoethanolamine groups to glycosylphosphatidylinositol (GPI) anchors are resident ER proteins (5.Galperin M.Y. Jedrzejas M.J. Proteins. 2001; 45: 318-324Crossref PubMed Scopus (108) Google Scholar, 6.Mann K.J. Sevlever D. Biochemistry. 2001; 40: 1205-1213Crossref PubMed Scopus (28) Google Scholar). As a last example, the Scj1p protein chaperone in the ER of yeast is a DnaJ homologue needed for protein folding and ER-associated degradation (7.Shen Y. Meunier L. Hendershot L.M. J. Biol. Chem. 2002; 277: 15947-15956Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 8.Silberstein S. Schlenstedt G. Silver P.A. Gilmore R. J. Cell Biol. 1998; 143: 921-933Crossref PubMed Scopus (76) Google Scholar). Scj1p has two zinc finger motifs and probably requires zinc for its function (9.Linke K. Wolfram T. Bussemer J. Jakob U. J. Biol. Chem. 2003; 278: 44457-44466Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 10.Ellis C.D. Wang F. MacDiarmid C.W. Clark S. Lyons T. Eide D.J. J. Cell Biol. 2004; 166: 325-335Crossref PubMed Scopus (152) Google Scholar). Because of these and the many other needs for zinc, eukaryotic cells must utilize zinc transport proteins in their plasma membranes and intracellular organelles for the uptake and intracellular distribution of zinc. The activities of these transporters are often regulated to maintain consistent intracellular zinc levels in the face of changing zinc availability. The budding yeast Saccharomyces cerevisiae has been an excellent model system for studying zinc transport and regulation. The initial uptake of zinc into the cytoplasm of yeast is accomplished by three transporters on the plasma membrane: Zrt1p, Zrt2p, and Fet4p (11.Zhao H. Eide D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2454-2458Crossref PubMed Scopus (440) Google Scholar, 12.Zhao H. Eide D. J. Biol. Chem. 1996; 271: 23203-23210Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 13.Waters B.M. Eide D.J. J. Biol. Chem. 2002; 277: 33749-33757Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). In times of zinc deficiency, zinc stores in the vacuole are mobilized by the Zrt3p transporter protein to supply zinc to the cytoplasm and other organelles (14.MacDiarmid C.W. Gaither L.A. Eide D. EMBO J. 2000; 19: 2845-2855Crossref PubMed Scopus (298) Google Scholar). Alternatively, when zinc levels in the cytoplasm become replete or excessive, the Zrc1p protein is involved in transporting extra zinc into the vacuole to be stored (15.MacDiarmid C.W. Milanick M.A. Eide D.J. J. Biol. Chem. 2002; 277: 39187-39194Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). The genes for these zinc transporter proteins are regulated by the zinc-responsive transcription factor, Zap1p. Zap1p up-regulates genes in zinc-deficient conditions by binding to an 11-base pair consensus sequence, a zinc-responsive element (ZRE), found in one or more copies in the promoters of its target genes (16.Zhao H. Eide D.J. Mol. Cell. Biol. 1997; 17: 5044-5052Crossref PubMed Scopus (214) Google Scholar). Lyons et al. (17.Lyons T.J. Gasch A.P. Gaither L.A. Botstein D. Brown P.O. Eide D.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7957-7962Crossref PubMed Scopus (250) Google Scholar) performed microarray analysis on the yeast transcriptome and estimated that as many as 46 genes in the yeast genome are directly regulated by Zap1p. Whereas many of the yeast zinc transporters known to date have been shown to be Zap1p targets, the Msc2p protein is not. Msc2p is a member of the cation diffusion facilitator (CDF) family of metal ion transporters (18.Gaither L.A. Eide D.J. Biometals. 2001; 14: 251-270Crossref PubMed Scopus (420) Google Scholar, 19.Paulsen I.T. Saier M.H. J. Membr. Biol. 1997; 156: 99-103Crossref PubMed Scopus (293) Google Scholar). Many members of the CDF family transport metal ions such as zinc either out of the cell or into intracellular organelles (18.Gaither L.A. Eide D.J. Biometals. 2001; 14: 251-270Crossref PubMed Scopus (420) Google Scholar, 20.Liuzzi J.P. Cousins R.J. Annu. Rev. Nutr. 2004; 24: 151-172Crossref PubMed Scopus (459) Google Scholar). Msc2p was recently shown to be localized to the ER and involved with zinc transport into this compartment (10.Ellis C.D. Wang F. MacDiarmid C.W. Clark S. Lyons T. Eide D.J. J. Cell Biol. 2004; 166: 325-335Crossref PubMed Scopus (152) Google Scholar, 21.Li L. Kaplan J. J. Biol. Chem. 2000; 276: 5036-5043Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Under low zinc conditions, an msc2 mutant exhibits an up-regulation of the unfolded protein response (UPR) and has defects in ER-associated protein degradation, suggesting that Msc2p and zinc are needed for proper ER function (10.Ellis C.D. Wang F. MacDiarmid C.W. Clark S. Lyons T. Eide D.J. J. Cell Biol. 2004; 166: 325-335Crossref PubMed Scopus (152) Google Scholar). The UPR and ER-associated degradation defects as well as other phenotypes of the msc2 mutant are all suppressible by elevated zinc. These observations suggested that there are other zinc transporters active in the ER/secretory pathway. Two vacuolar zinc transporters, Zrc1p and Cot1p, were found to potentially contribute to ER zinc levels; an msc2 zrc1 cot1 triple mutant caused an up-regulation of the UPR in low zinc even higher than that seen in an msc2 mutant alone. However, even this triple mutant was suppressed by elevated zinc (10.Ellis C.D. Wang F. MacDiarmid C.W. Clark S. Lyons T. Eide D.J. J. Cell Biol. 2004; 166: 325-335Crossref PubMed Scopus (152) Google Scholar). These observations led us to look for more potential zinc transporters in the secretory pathway. One candidate for such a protein was Zrg17p. Previous studies of ZRG17 determined it to be a Zap1p target and regulated by zinc at the mRNA level (i.e. ZRG17 is up-regulated in low zinc and down-regulated in high zinc) (17.Lyons T.J. Gasch A.P. Gaither L.A. Botstein D. Brown P.O. Eide D.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7957-7962Crossref PubMed Scopus (250) Google Scholar, 22.Yuan D.S. Genetics. 2000; 156: 45-58Crossref PubMed Google Scholar). Zrg17p is a probable membrane protein with multiple transmembrane domains. It was previously proposed that Zrg17p was involved in zinc uptake, perhaps under environmental conditions where Zrt1p was less effective (22.Yuan D.S. Genetics. 2000; 156: 45-58Crossref PubMed Google Scholar). However, zrg17 mutants have an abnormal large cell morphology, which is suppressed by the addition of higher amounts of zinc in the medium (22.Yuan D.S. Genetics. 2000; 156: 45-58Crossref PubMed Google Scholar). An msc2 mutant also exhibits this zinc-suppressible large cell phenotype (21.Li L. Kaplan J. J. Biol. Chem. 2000; 276: 5036-5043Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). This suggested that Zrg17p may be involved in ER zinc transport. In this report, we determined that this is the case and that Zrg17p and Msc2p physically interact to form a complex that transports zinc into the ER. Last, we suggest that Zrg17p is a distant member of the CDF family and present evidence that other CDF family members interact to form heteromeric complexes for metal ion transport. Yeast Strains and Growth Conditions—Media used were YPD, SD + 2% glucose or galactose, YPGE, and LZM, as described previously (23.Gitan R.S. Luo H. Rodgers J. Broderius M. Eide D. J. Biol. Chem. 1998; 273: 28617-28624Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). We also used rich YP medium supplemented with 2% glycerol, 2% ethanol, and 2% galactose (YPGEgal) to induce expression of the GAL1 promoter in cells growing on poorly fermentable and nonfermentable carbon sources. Yeast strains DY150 (MATa ade2 can1 his3 leu2 trp1 ura3) and DY150 msc2 (DY150 msc2::HIS3) (21.Li L. Kaplan J. J. Biol. Chem. 2000; 276: 5036-5043Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), BY4741 (MATa his3 leu2 met15 ura3) (Research Genetics), ZRG17-TAP (BY4741 ZRG17::TAP), and MSC2-TAP (BY4741 MSC2::TAP) (Open Biosystems) have been described previously. To generate mutant strains CEY9 (DY150 zrg17::KanMX) and CEY11 (DY150 msc2::HIS3 zrg17::KanMX), the KanMX cassette with 500 bp flanking the ZRG17 open reading frame was amplified by PCR from the zrg17 yeast deletion mutant (Research Genetics). The PCR fragment was then transformed into DY150 or DY150 msc2 to generate CEY9 and CEY11, respectively. Plasmids—Plasmids used in this study are described in Table I. pZnT5 was a gift from Taiho Kambe (Kyoto University) (24.Kambe T. Narita H. Yamaguchi-Iwai Y. Hirose J. Amano T. Sugiura N. Sasaki R. Mori K. Iwanaga T. Nagao M. J. Biol. Chem. 2002; 277: 19049-19055Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar), and pZnT6 was a gift from Liping Huang (University of California, Davis, CA) (25.Huang L. Kirschke C.P. Gitschier J. J. Biol. Chem. 2002; 277: 26389-26395Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). pRS316GAL1LEU2, pYES2L, and pZnT5L were generated by swapping the URA3 gene on pRS316GAL1, pYES2, and pZnT5, respectively, with the LEU2 gene using the marker swap plasmid pUL9 (26.Cross F.R. Yeast. 1997; 13: 647-653Crossref PubMed Scopus (140) Google Scholar). pZRG17 and pZRG17HA were constructed the same way as pMSC2 and pMSC2HA (10.Ellis C.D. Wang F. MacDiarmid C.W. Clark S. Lyons T. Eide D.J. J. Cell Biol. 2004; 166: 325-335Crossref PubMed Scopus (152) Google Scholar). Briefly, PCR products containing the ZRG17 promoter and open reading frame (and terminator in the case of pZRG17) were put into pFL38 or YCpZRC1-HA by homologous recombination to generate pZRG17 and pZRG17HA, respectively (27.Ma H. Kunes S. Schatz P.J. Botstein D. Gene (Amst.). 1987; 58: 201-216Crossref PubMed Scopus (425) Google Scholar). To generate the pGAL-ZRG17HA construct, the ZRG17 open reading frame starting at the fourth in-frame ATG plus 3× hemagglutinin antigen (HA) tags and termination sequence from pZRG17HA was amplified by PCR. This fragment was then inserted into the SacI site of the vector pRS316GAL1LEU2 by homologous recombination. We previously determined that the annotation in the Saccharomyces Genome Data base is likely to be incorrect regarding the ATG translation start site of the ZRG17 gene (17.Lyons T.J. Gasch A.P. Gaither L.A. Botstein D. Brown P.O. Eide D.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7957-7962Crossref PubMed Scopus (250) Google Scholar). All of the above plasmids encoding ZRG17 constructs were confirmed to be functional by complementation of the zrg17 mutant growth phenotype on YPGE plates at 37 °C. pGEV-TRP1 was co-transformed with pRK315, pZnT5L, or pZnT6 so that expression levels of Pma1p-HA, ZnT5, and ZnT6 could be controlled by the addition of β-estradiol (Sigma) (10-7 to 10-6 m) (28.Gao C.Y. Pinkham J.L. BioTechniques. 2000; 29: 1226-1231Crossref PubMed Scopus (42) Google Scholar).Table IPlasmids used in this studyPlasmidRelevant genotypeReference/SourcepTF63High copy vectorRef. 46YEpMSC2-myc (pMsc2ox)MSC2-myc in pTF63Ref. 21pRS316GAL1Low copy expression vectorRef. 47pRS316GAL1LEU2Low copy expression vector (LEU2-marked)This workpGAL-ZRG17HA (pZRG17ox)ZRG17-3×HA driven by GAL1 promoterThis workYEp24High copy vectorRef. 48pHSP150HSP150 in YEp24This workpMCZ-Y (UPRE-lacZ)UPRE-lacZ reporterRef. 49pDg2L (ZRE-lacZ)ZRE-lacZ reporterRef. 14pRK315PMA1-HA driven by GAL1 promoterRef. 50pGEV-TRP1GAL4/estrogen recepor/VP16 hybrid activatorRef. 28pZRG17ZRG17 in low copy vectorThis workpZRG17HAZRG17-3×HA in low copy vectorThis workpMSC2MSC2 in low copy vectorRef. 10pMSC2HAMSC2-3×HA in low copy vectorRef. 10pYES2High copy expression vectorInvitrogenpYES2LHigh copy expression vector (LEU2-marked)This workpYES2-ZnT5 (pZnT5)ZnT5 driven by GAL1 promoterRef. 24pZnT5LZnT5 driven by GAL1 promoter (LEU2-marked)This workpYES2-ZnT6 (pZnT6)ZnT6 driven by GAL1 promoterRef. 25 Open table in a new tab Isolation of pHSP150—In a screen to identify suppressors of the msc2 37 °C growth defect, the msc2 mutant was transformed with a yeast genomic library in the high copy vector YEp24. After initial selection for the plasmids, transformed cells were harvested and subsequently plated onto YPGE plates at a concentration of ∼10,000 cells/plate. YPGE plates were incubated at 37 °C for 3 or 4 days and screened for colonies. Plasmids were isolated from these colonies and sequenced. One of the plasmids isolated was pHSP150, whose insert consists of a genomic fragment from yeast chromosome X and contains the HSP150 gene. Growth Assays—The desired strains were grown overnight in YPD, SD galactose, or SD glucose with the appropriate auxotrophies and β-estradiol where indicated. These cultures were subsequently diluted into the same medium, and 5 μl of diluted culture, yielding 104 or 103 cells, were spotted onto YPGE or YPGEgal plates. Where indicated, different concentrations of ZnCl2 and/or β-estradiol were added to the plates. The plates were incubated 3-4 days and photographed. β-Galactosidase Assays and Subcellular Fractionation—β-Galactosidase assays, with specific activity normalized to protein content, and subcellular fractionation, where protein extracts were fractionated on sucrose gradients with or without Mg2+, were performed as described previously (10.Ellis C.D. Wang F. MacDiarmid C.W. Clark S. Lyons T. Eide D.J. J. Cell Biol. 2004; 166: 325-335Crossref PubMed Scopus (152) Google Scholar). Protein Lysates, Co-immunoprecipitation, and Immunoblotting—Protein lysates for co-immunoprecipitation were obtained as follows. Yeast cultures were grown to an A600 = ∼ 1.0 in LZM medium containing 1 or 1000 μm zinc. The cells were harvested, washed once with water, and resuspended in 50 mm NaCl. Protein lysates were obtained using glass beads in the presence of protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 50 μg/ml leupeptin, 10 μg/ml pepstatin A, 1 mm EDTA (all from Sigma), mini-EDTA-free protease inhibitor mixture pellets (Roche Applied Science)) and vortexing 10 × 30 s, with 30 s on ice between pulses. The cell debris was pelleted by centrifuging for 2 min at 500 × g at 4 °C. The resulting supernatant was the total protein lysate. The lysates were then solubilized with 3% LM (n-dodecyl-β-d-maltoside) (MP Biomedicals) on ice for 2 h. The insoluble proteins were pelleted by centrifuging for 15 min at 15,000 × g at 4 °C. The detergent-soluble protein lysates were diluted 1:3 into IPP150 buffer (10 mm Tris-Cl, pH 8.0, 150 mm NaCl), and then 50 μl of IgG-Sepharose beads (Amersham Biosciences) were added to each lysate. The lysates plus beads were rotated at 4 °C for 30 min, after which the beads were washed four times with IPP150 buffer plus 0.1% LM. The beads were resuspended in elution buffer (50 mm Tris-Cl, pH 8.0, 10 mm EDTA, 1% SDS) and incubated at 65 °C for 30 min to elute the proteins off of the beads. Equal volumes of eluted protein lysates were loaded onto SDS-PAGE for immunoblotting. When denatured by boiling, epitope-tagged Zrg17p exhibited high molecular weight aggregates on immunoblots (data not shown). Therefore, lysates containing epitope-tagged Zrg17p were always denatured for 30 min at either 37 °C or 65°C prior to SDS-PAGE. Immunoblots were done by standard techniques (29.Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, NY1988: 471-510Google Scholar). Blots were visualized with POD (Roche Applied Science). Band quantitation to determine -fold difference in protein levels was performed using NIH Image 1.61. Antibodies used were rabbit anti-HA (Sigma), mouse anti-Dpm1p (Molecular Probes, Inc., Eugene, OR), rabbit anti-Kex2p (gift of Steven Nothwehr, University of Missouri-Columbia), goat anti-mouse horseradish peroxidase-conjugated secondary (Pierce), and goat anti-rabbit horseradish peroxidase-conjugated secondary (Pierce). Zrg17p Is a Distant Member of the CDF Family of Metal Ion Transporters—The Zrg17p protein is 504 amino acids in length. While originally estimated to have seven transmembrane domains (22.Yuan D.S. Genetics. 2000; 156: 45-58Crossref PubMed Google Scholar), the consensus of several algorithms (e.g. TOPPRED2, SOSUI, DAS, HMMTOP, and TMPRED) predicted only six such domains in the Zrg17p protein (Fig. 1A). Membrane topology predictions of Zrg17p consistently placed both the amino- and carboxyl-terminal ends on the cytosolic surface of cellular membranes. This topology is common among many members of the CDF family of metal ion transporters (18.Gaither L.A. Eide D.J. Biometals. 2001; 14: 251-270Crossref PubMed Scopus (420) Google Scholar). Sequence data base comparisons indicated that Zrg17p is closely related to gene products found in a number of other fungal species, including Aspergillus nidulans, Gibberella zeae, Neurospora crassa, Schizosaccharomyces pombe and Ashbya gossypii. When the amino acid sequences of these orthologs were used to identify other related proteins in the sequence data bases using PSI-BLAST, several members of the CDF family were identified in the first iteration. For example, the human ZnT3 and ZnT6 CDF proteins were detected with E-values of 1 × 10-6 and 8 × 10-6, respectively. These results suggested that Zrg17p and its fungal orthologs are distant members of the CDF family of metal transporters. Zrg17p and its closely related proteins appear to form a distinct subfamily of CDF proteins, as indicated by a multiple sequence alignment/tree-building analysis depicted in Fig. 1B. Unlike many other CDF proteins, however, Zrg17p has a histidine-rich domain (... HDHDEINEQIPHSH...) located between predicted transmembrane domains III and IV (Fig. 1A). Similar domains are found between transmembrane domains IV and V in most other eukaryotic CDF proteins. Studies Implicating Zrg17p in ER Zinc Transport—Zrg17p was previously proposed to be involved in zinc uptake across the plasma membrane (22.Yuan D.S. Genetics. 2000; 156: 45-58Crossref PubMed Google Scholar). However, the apparent relationship of Zrg17p to members of the CDF family suggested that this protein was more likely to transport zinc either outside of the cell or into an intracellular compartment. Furthermore, the zinc-suppressible large cell phenotype previously observed for zrg17 mutants was similar to that seen with msc2 mutants. This suggested that Zrg17p may also be involved in transporting zinc into the ER lumen. If so, we predicted that zrg17 mutants would share other phenotypes with msc2 mutants. We generated an isogenic strain where the ZRG17 gene was deleted from the genome. We also generated an msc2 zrg17 double deletion strain. If Msc2p and Zrg17p functioned separately to supply zinc to the ER, we predicted that these phenotypes would be more severe in the double mutant than in either single mutant. However, if Msc2p and Zrg17p functioned in the same pathway, no additivity of their effects would be expected. Mutant msc2 cells grow well at 30 °C on rich YPGE medium plates containing the respired carbon sources glycerol and ethanol but grow poorly at 37 °C (10.Ellis C.D. Wang F. MacDiarmid C.W. Clark S. Lyons T. Eide D.J. J. Cell Biol. 2004; 166: 325-335Crossref PubMed Scopus (152) Google Scholar). This temperature-sensitive growth phenotype is suppressible by high zinc added to the medium (21.Li L. Kaplan J. J. Biol. Chem. 2000; 276: 5036-5043Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). As seen in Fig. 2A, the zrg17 mutant also exhibited a temperature-sensitive growth defect on YPGE, and it was also suppressible by added zinc. To determine whether the msc2 and zrg17 single mutations differed in their severity, we assessed suppression of the growth defect over a range of zinc concentrations added to these plates. The single mutants each showed partial suppression with the addition of 100-250 μm zinc and full suppression with 500 μm zinc added. Thus, msc2 and zrg17 mutants show very similar zinc-suppressible growth defects at 37 °C. As expected, the msc2 zrg17 double mutant also showed poor growth on YPGE at the higher temperature. Similar concentrations of added zinc suppressed the double mutant to the same degree as the single mutants, indicating that the two mutations are not additive for this phenotype. Another previously observed phenotype of the msc2 mutant is the up-regulation of the UPR in zinc-limiting conditions (10.Ellis C.D. Wang F. MacDiarmid C.W. Clark S. Lyons T. Eide D.J. J. Cell Biol. 2004; 166: 325-335Crossref PubMed Scopus (152) Google Scholar). The UPR is a response to misfolded proteins in the ER of cells whereby protein chaperones and degradation systems are up-regulated to refold or degrade the aberrant proteins. The increased level of UPR in msc2 mutants probably reflects, at least in part, the zinc requirement of luminal protein chaperones (i.e. Scj1p) (10.Ellis C.D. Wang F. MacDiarmid C.W. Clark S. Lyons T. Eide D.J. J. Cell Biol. 2004; 166: 325-335Crossref PubMed Scopus (152) Google Scholar). We introduced a UPR reporter construct (UPRE-lacZ) into the msc2, zrg17, and msc2 zrg17 mutants and determined the activity of this reporter over a range of zinc concentrations. As observed previously, wild type cells show a small induction in UPRE-lacZ expression under low zinc conditions, and the msc2 mutation exacerbates this effect (Fig. 2B). Like the msc2 mutant, the zrg17 mutant also exhibited elevated UPRE-lacZ activity in low zinc (LZM + 0.3-10 μm ZnCl2) compared with wild type cells. The double mutant showed an increase similar to that seen in both single mutants, again demonstrating that these mutations are not additive with respect to this phenotype. We have shown previously that activity of a HIS4-lacZ reporter, which is not responsive to either zinc or unfolded proteins, showed high expression in both wild type and mutant strains at all zinc concentrations (10.Ellis C.D. Wang F. MacDiarmid C.W. Clark S. Lyons T. Eide D.J. J. Cell Biol. 2004; 166: 325-335Crossref PubMed Scopus (152) Google Scholar). This indicates that the results presented here are specific to the UPRE-lacZ reporter and not a general effect on lacZ activity. Therefore, the UPRE-lacZ results suggest that Zrg17p, like Msc2p, is needed to maintain protein folding in the ER. Previous studies suggested that loss of Msc2p function results in an increased level of labile cytosolic/nuclear zinc, and this is consistent with the role of Msc2p in transporting cytosolic zinc into the ER lumen (10.Ellis C.D. Wang F. MacDiarmid C.W. Clark S. Lyons T. Eide D.J. J. Cell Biol. 2004; 166: 325-335Crossref PubMed Scopus (152) Google Scholar, 21.Li L. Kaplan J. J. Biol. Chem. 2000; 276: 5036-5043Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). This phenotype was observed using a Zap1p-regulated ZRE-lacZ reporter construct. Zap1p activity and ZRE-lacZ expression is high when labile zinc levels decrease and is reduced when labile zinc levels rise. If Msc2p and Zrg17p are involved in the same process, we predicted that the zrg17 mutant would also have altered Zap1p-mediated regulation. The ZRE-lacZ reporter was transformed into the msc2, zrg17, and msc2 zrg17 mutants to determine whether these strains have altered zinc homeostasis. In wild type cells, Zap1p is more active in low zinc, resulting in high expression of the ZRE-lacZ reporter (Fig. 2C). With increasing medium zinc concentrations, Zap1p becomes less active, resulting in decreased expression of the reporter. The reporter expression profile in the msc2, zrg17, and msc2 zrg17 mutants was quite different from the wild type profile. All three mutants had reporter activity in LZM plus 1-30 μm ZnCl2 lower than that seen in wild type cells. These results suggest that Zap1p is sensing higher amounts of zinc in the cytosol of these mutants and are consistent with Zrg17p, like Msc2p, being needed to transport zinc into an intracellular compartment. Once more, the phenotypes of the single mutants were not additive in the double mutant. Finally, we tested whether zrg17 mutants also show a genetic interaction that we discovered for msc2. We performed a genetic screen for genes that, when overexpressed from a high copy plasmid, could suppress the msc2 temperature-sensitive growth defect (see "Materials and Methods"). One gene identified in this screen was HSP150. Hsp150p is a cell wall protein whose exact function is unknown. However, overexpression of Hsp150p completely suppresses the temperature-sensitive msc2 growth defect. As seen in Fig. 3, HSP150 overexpressed from a high copy plasmid (pHSP150) also suppressed the growth defect of the zrg17 mutant as well as the msc2 zrg17 double mutant. Thus, the zrg17 and msc2 mutant defects are alleviated by the same overexpression suppressor. These results, combined with shared and nonadditive phenotypes of the zrg17 and msc2 mutants described in Fig. 2 argue that both proteins are involved in the same pathway supplying zinc to the endoplasmic reticulum of yeast. Zrg17p and Msc2p Co-localize to the ER—We next addressed the question of whether Msc2p and Zrg17p were fu
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