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PKR1 Encodes an Assembly Factor for the Yeast V-Type ATPase

2006; Elsevier BV; Volume: 281; Issue: 42 Linguagem: Inglês

10.1016/s0021-9258(19)84116-9

1083-351X

Sandra R. Davis-Kaplan, Mark A. Compton, Andrew R. Flannery, Diane M. Ward, Jerry Kaplan, Tom H. Stevens, Laurie A. Graham,

RNA and protein synthesis mechanisms

Deletion of the yeast gene PKR1 (YMR123W) results in an inability to grow on iron-limited medium. Pkr1p is localized to the membrane of the endoplasmic reticulum. Cells lacking Pkr1p show reduced levels of the V-ATPase subunit Vph1p due to increased turnover of the protein in mutant cells. Reduced levels of the V-ATPase lead to defective copper loading of Fet3p, a component of the high affinity iron transport system. Levels of Vph1p in cells lacking Pkr1p are similar to cells unable to assemble a functional V-ATPase due to lack of a V0 subunit or an endoplasmic reticulum (ER) assembly factor. However, unlike yeast mutants lacking a V0 subunit or a V-ATPase assembly factor, low levels of Vph1p present in cells lacking Pkr1p are assembled into a V-ATPase complex, which exits the ER and is present on the vacuolar membrane. The V-ATPase assembled in the absence of Pkr1p is fully functional because the mutant cells are able to weakly acidify their vacuoles. Finally, overexpression of the V-ATPase assembly factor Vma21p suppresses the growth and acidification defects of pkr1Δ cells. Our data indicate that Pkr1p functions together with the other V-ATPase assembly factors in the ER to efficiently assemble the V-ATPase membrane sector. Deletion of the yeast gene PKR1 (YMR123W) results in an inability to grow on iron-limited medium. Pkr1p is localized to the membrane of the endoplasmic reticulum. Cells lacking Pkr1p show reduced levels of the V-ATPase subunit Vph1p due to increased turnover of the protein in mutant cells. Reduced levels of the V-ATPase lead to defective copper loading of Fet3p, a component of the high affinity iron transport system. Levels of Vph1p in cells lacking Pkr1p are similar to cells unable to assemble a functional V-ATPase due to lack of a V0 subunit or an endoplasmic reticulum (ER) assembly factor. However, unlike yeast mutants lacking a V0 subunit or a V-ATPase assembly factor, low levels of Vph1p present in cells lacking Pkr1p are assembled into a V-ATPase complex, which exits the ER and is present on the vacuolar membrane. The V-ATPase assembled in the absence of Pkr1p is fully functional because the mutant cells are able to weakly acidify their vacuoles. Finally, overexpression of the V-ATPase assembly factor Vma21p suppresses the growth and acidification defects of pkr1Δ cells. Our data indicate that Pkr1p functions together with the other V-ATPase assembly factors in the ER to efficiently assemble the V-ATPase membrane sector. The V-type ATPase (V-ATPase) 4The abbreviations used are: V-ATPase, V-type ATPase; BPS, bathophenanthroline disulfonate; ER, endoplasmic reticulum; YEPD, yeast extract-peptone-dextrose; Pkr, Pichia farnosia killer toxin resistance; HA, hemagglutinin; TRITC, tetramethylrhodamine isothiocyanate. 4The abbreviations used are: V-ATPase, V-type ATPase; BPS, bathophenanthroline disulfonate; ER, endoplasmic reticulum; YEPD, yeast extract-peptone-dextrose; Pkr, Pichia farnosia killer toxin resistance; HA, hemagglutinin; TRITC, tetramethylrhodamine isothiocyanate. is a ubiquitous multisubunit complex essential for cell viability in all eukaryotes except yeast (1Graham L.A. Flannery A.R. Stevens T.H. J. Bioenerg. Biomembr. 2003; 35: 301-312Crossref PubMed Scopus (84) Google Scholar). In the yeast Saccharomyces cerevisiae, the V-ATPase is localized to the membranes of the Golgi, endosomes, and vacuole where it plays a key role in cellular ion homeostasis (2Ramsay L.M. Gadd G.M. FEMS Microbiol. Lett. 1997; 152: 293-298Crossref PubMed Google Scholar). The acidification of organelles by the V-ATPase, due to proton translocation driven by ATP hydrolysis, is required for intracellular transporters to maintain cytosolic ion balance. The V-ATPase can be divided into two functionally distinct domains, the V0 proton translocating domain and the V1 ATP-hydrolytic domain. The yeast V-ATPase complex is composed of 14 subunits and requires several additional gene products to assemble a fully functional complex (1Graham L.A. Flannery A.R. Stevens T.H. J. Bioenerg. Biomembr. 2003; 35: 301-312Crossref PubMed Scopus (84) Google Scholar, 3Sambade M. Kane P.M. J. Biol. Chem. 2004; 279: 17361-17365Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 4Oluwatosin Y.E. Kane P.M. J. Biol. Chem. 1997; 272: 28149-28157Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 5Oluwatosin Y.E. Kane P.M. Mol. Cell. Biol. 1998; 18: 1534-1543Crossref PubMed Scopus (32) Google Scholar). One of the subunits is present as two isoforms and the isoform assembled into the complex dictates the localization of the V-ATPase in the cell (6Manolson M.F. Wu B. Proteau D. Taillon B.E. Roberts B.T. Hoyt M.A. Jones E.W. J. Biol. Chem. 1994; 269: 14064-14074Abstract Full Text PDF PubMed Google Scholar, 7Kawasaki-Nishi S. Bowers K. Nishi T. Forgac M. Stevens T.H. J. Biol. Chem. 2001; 276: 47411-47420Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). V-ATPase complexes assembled with the Vph1p isoform are localized to the vacuole and the Stv1p isoform-containing complex is found on the membranes of the Golgi and endosome. Yeast cells lacking the Stv1p Golgi/endosome-localized isoform complex display no obvious growth phenotype suggesting that the Stv1p containing V-ATPase complexes are the minor form present in the cells. Yeast cells lacking the vacuolar-localized V-ATPase isoform display an intermediate growth phenotype due to the decrease of the majority of the V-ATPase complexes present in the cell; they are able to grow in the presence of elevated calcium (100 mm) but are unable to grow in the presence of elevated zinc (4 mm). In addition to the genes encoding subunits of the V-ATPase complex, there are five previously characterized gene products that are not part of the complex but are required for its function. Three proteins (Vma12p, Vma21p, and Vma22p) are localized to the membrane of the endoplasmic reticulum (8Malkus P. Graham L.A. Stevens T.H. Schekman R. Mol. Biol. Cell. 2004; 15: 5075-5091Crossref PubMed Scopus (68) Google Scholar, 9Jackson D.D. Stevens T.H. J. Biol. Chem. 1997; 272: 25928-25934Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 10Hill K.J. Stevens T.H. Mol. Biol. Cell. 1994; 5: 1039-1050Crossref PubMed Scopus (95) Google Scholar, 11Hill K.J. Stevens T.H. J. Biol. Chem. 1995; 270: 22329-22336Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 12Hirata R. Umemoto N. Ho M.N. Ohya Y. Stevens T.H. Anraku Y. J. Biol. Chem. 1993; 268: 961-967Abstract Full Text PDF PubMed Google Scholar). These proteins play a role very early in the assembly of the V0 subcomplex of the V-ATPase following protein synthesis and insertion of the subunit proteins into the membrane of the ER. Vma21p may also function to escort the V-ATPase complex out of the ER (8Malkus P. Graham L.A. Stevens T.H. Schekman R. Mol. Biol. Cell. 2004; 15: 5075-5091Crossref PubMed Scopus (68) Google Scholar). Two additional non-subunit proteins, localized to the Golgi (Vma45p also known as Kex2p) and to the cytosol (Vma41p also called Cys4p), presumably function post-assembly (4Oluwatosin Y.E. Kane P.M. J. Biol. Chem. 1997; 272: 28149-28157Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 5Oluwatosin Y.E. Kane P.M. Mol. Cell. Biol. 1998; 18: 1534-1543Crossref PubMed Scopus (32) Google Scholar). Cells lacking a functional V-ATPase display a distinct set of growth phenotypes related to their inability to maintain cellular homeostasis including the inability to grow at neutral pH, or in the presence of elevated concentrations of calcium or zinc (6Manolson M.F. Wu B. Proteau D. Taillon B.E. Roberts B.T. Hoyt M.A. Jones E.W. J. Biol. Chem. 1994; 269: 14064-14074Abstract Full Text PDF PubMed Google Scholar) or to grow on low iron medium (13Davis-Kaplan S.R. Ward D.M. Shiflett S.L. Kaplan J. J. Biol. Chem. 2004; 279: 4322-4329Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The ability of yeast to grow on low iron media requires the activity of the high affinity iron transport system. This system is comprised of two cell surface proteins, a multicopper oxidase Fet3p and a transmembrane permease Ftr1p (14Van Ho A. Ward D.M. Kaplan J. Annu. Rev. Microbiol. 2002; 56: 237-261Crossref PubMed Scopus (178) Google Scholar). The multicopper oxidase Fet3p is a Type I membrane protein that obtains its copper in a post-Golgi compartment. Copper loading of apoFet3p requires an acidic environment (15Davis-Kaplan S.R. Askwith C.C. Bengtzen A.C. Radisky D. Kaplan J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13641-13645Crossref PubMed Scopus (112) Google Scholar). The low pH of the vesicular apparatus is maintained by the V-ATPase and a voltage-regulated chloride channel (Gef1p). In the absence of these elements, apoFet3p fails to be copper loaded, and is localized to the cell surface in an inactive form. Using a genome-wide screen to identify genes required for growth on low iron we identified CWH36/VMA9 as a subunit of the V-ATPase (13Davis-Kaplan S.R. Ward D.M. Shiflett S.L. Kaplan J. J. Biol. Chem. 2004; 279: 4322-4329Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). In the absence of Vma9p the V-ATPase is not assembled, resulting in defective copper loading of apoFet3p. That screen also identified PKR1, a gene encoding a protein that when overexpressed confers Pichia farnosia killer toxin resistance (Saccharomyces Genome Database annotation), as being required for growth on low iron medium. In this work we show that Pkr1p is an integral membrane protein localized to the ER, similar to the V-ATPase assembly factors Vma12p, Vma21p, and Vma22p. Yeast cells lacking Pkr1p have greatly reduced levels of Vph1p, a subunit of the V-ATPase and component of the V0 subcomplex. The poor growth of pkr1Δ cells on low iron results from inefficient copper loading of apoFet3p. Unlike the other V-ATPase assembly factor mutants, cells lacking Pkr1p assemble some functional V-ATPase complex, consistent with the fact that pkr1Δ cells are able to grow on media containing 100 mm CaCl2 and these cells have weakly acidified vacuoles. Genetic interactions between PKR1 and VMA21 lead us to conclude that Pkr1p is required for efficient V0 assembly in the ER, but that a low level of V-ATPase assembly occurs in the absence of Pkr1p. Strains, Plasmids, and Culture Conditions—Strains and plasmids used in the study are listed in Tables 1 and 2. Yeast were cultured in YEPD either unbuffered or buffered to pH 5.0 using 50 mm succinate/phosphate, or yeast nitrogen base synthetic complete minimal medium with supplements as needed using standard techniques. Low iron growth medium was made by adding 40-90 μm bathophenanthroline disulfonate (BPS), an iron chelator, to complete minimal medium or YEPD and then adding back varying amounts of FeSO4. Media and procedures for growing and analyzing the phenotypes of strains on low iron media have been described (13Davis-Kaplan S.R. Ward D.M. Shiflett S.L. Kaplan J. J. Biol. Chem. 2004; 279: 4322-4329Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Exponentially growing cell cultures were diluted to 1.0 A600 and then serially diluted 10-fold to test the growth phenotype of various yeast strains. 5 μl of the starting culture and each dilution were spotted onto a YEPD, pH 5.0, agar, YEPD + 100 mm CaCl2 agar, or YEPD + 90 μm BPS + FeSO4 and incubated 48 h at 30 °C.TABLE 1S. cerevisiae strains and plasmids used in this studyStrainGenotypeSourceSF838-1DαMATα, ade6, leu2–3,112, ura3–52, pep4–3, his4–519, gal 2Ref. 33Rothman J.H. Stevens T.H. Cell. 1986; 47: 1041-1051Abstract Full Text PDF PubMed Scopus (298) Google ScholarKEBY4MATα, ade6, leu2–3,112, ura3–52, pep4–3, his4–519, gal 2, stv1Δ::KanrRef. 7Kawasaki-Nishi S. Bowers K. Nishi T. Forgac M. Stevens T.H. J. Biol. Chem. 2001; 276: 47411-47420Abstract Full Text Full Text PDF PubMed Scopus (158) Google ScholarKHY31MATα, ade6, leu2–3,112, ura3–52, pep4–3, his4–519, gal 2, vph1Δ::LEU2Ref. 29Graham L.A. Hill K.J. Stevens T.H. J. Cell Biol. 1998; 142: 39-49Crossref PubMed Scopus (81) Google ScholarLGY113MATα, ade6, leu2–3,112, ura3–52, pep4–3, his4–519, gal 2, vma3Δ::KanrRef. 17Compton M.A. Graham L.A. Stevens T.H. J. Biol. Chem. 2006; 281: 15312-15319Abstract Full Text Full Text PDF PubMed Scopus (24) Google ScholarLGY116MATα, ade6, leu2–3,112, ura3–52, pep4–3, his4–519, gal 2, pkr1Δ::KanrThis studyLGY117MATα, ade6, leu2–3,112, ura3–52, pep4–3, his4–519, gal 2, pkr1Δ::NatrThis studyLGY118MATα, ade6, leu2–3,112, ura3–52, pep4–3, his4–519, gal 2, pkr1Δ::Natr stv1Δ::KanrThis studyLGY119MATα, ade6, leu2–3,112, ura3–52, pep4–3, his4–519, gal 2, pkr1Δ::Natr vph1Δ::LEU2This studyLGY120MATα, ade6, leu2–3,112, ura3–52, pep4–3, his4–519, gal 2,vph1Δ::KanrThis studyLGY136MATα, ade6, leu2–3,112, ura3–52, pep4–3, his4–519, gal 2,vph1Δ::Natr stv1Δ::KanrThis studyLGY146MATα, ade6, leu2–3,112, ura3–52, pep4–3, his4–519, gal 2, vma1Δ::KanrThis studyLGY147MATα, ade6, leu2–3,112, ura3–52, pep4–3, his4–519, gal 2, vma1Δ::Kanr pkr1Δ::NatrThis studyTASY006MATα, ade6, leu2–3,112, ura3–52, pep4–3, his4–519, gal 2, vma21Δ::KanrRef. 17Compton M.A. Graham L.A. Stevens T.H. J. Biol. Chem. 2006; 281: 15312-15319Abstract Full Text Full Text PDF PubMed Scopus (24) Google ScholarBY4743MATa/MATα his3Δ1/his3Δ1, ura3Δ0/ura3Δ0, leu2Δ0/leu2Δ0, lys2Δ0/+, met15Δ/+Research Geneticspkr1ΔMATa/MATα his3Δ1/his3Δ1, ura3Δ0/ura3Δ0, leu2Δ0/leu2Δ0, lys2Δ0/+, met15Δ/+, pkr1Δ::Kanr/pkr1Δ::KanrResearch Geneticsvma2ΔMATa/MATα his3Δ1/his3Δ1, ura3Δ0/ura3Δ0, leu2Δ0/leu2Δ0, lys2Δ0/+, met15Δ/+, vma2Δ::Kanr/vma2Δ::KanrResearch Geneticsvma21ΔMATa/MATα his3Δ1/his3Δ1, ura3Δ0/ura3Δ0, leu2Δ0/leu2Δ0, lys2Δ0/+, met15Δ/+, vma21Δ::Kanr/vma21Δ::KanrResearch GeneticsDY1640MATa/MATα, his3–11/his3–11, leu2–3,112/leu2–3,112, trp1–1/trp1–1, ura3–52/ura3–52, ade2–1/ade2-, can1–100(oc)/can1–100(oc)Ref. 13Davis-Kaplan S.R. Ward D.M. Shiflett S.L. Kaplan J. J. Biol. Chem. 2004; 279: 4322-4329Abstract Full Text Full Text PDF PubMed Scopus (54) Google ScholarSDK13-5Chis3–11 leu2–3,112, trp1–1, ura3–52, ade2–1, can1–100(oc)This studySDK13-5Dhis3–11 leu2–3,112, trp1–1, ura3–52, ade2–1, can1–100(oc), pkr1Δ::KanrThis studySDK13-6Ahis3–11 leu2–3,112, trp1–1, ura3–52, ade2–1, can1–100(oc)This studySDK13-6Dhis3–11 leu2–3,112, trp1–1, ura3–52, ade2–1, can1–100(oc), pkr1Δ::KanrThis studyfet3ΔMATa/MATα his3Δ1/his3Δ1, ura3Δ0/ura3Δ0, leu2Δ0/leu2Δ0, lys2Δ0/+, met15Δ/+, fet3Δ::Kanr/fet3Δ::KanrResearch Genetics Open table in a new tab TABLE 2Plasmids used in this studyPlasmidsDescriptionSourcepAG25pAF6 natMX4Ref. 34Goldstein A.L. McCusker J.H. Yeast. 1999; 15: 1541-1553Crossref PubMed Scopus (1376) Google ScholarpLG174pCR4Blunt containing PKR1This studypLG221pRS416 PKR1::3xHAThis studypSMY92pUC19 containing 123-bp 3XHA BglII fragmentRef 6Manolson M.F. Wu B. Proteau D. Taillon B.E. Roberts B.T. Hoyt M.A. Jones E.W. J. Biol. Chem. 1994; 269: 14064-14074Abstract Full Text PDF PubMed Google ScholarpRS315PKR1-FLAGpRS315 PKR1::FLAGThis studypKH28pRS316 VMA21::HARef. 10Hill K.J. Stevens T.H. Mol. Biol. Cell. 1994; 5: 1039-1050Crossref PubMed Scopus (95) Google Scholar Open table in a new tab Cloning and Construction of Disruption Strains—A pkr1Δ::Kanr fragment was amplified by PCR from genomic DNA of the pkr1Δ yeast genomic deletion strain (Open Biosystems), including ∼300 base pairs of the 5′ and 3′ flanking sequence in addition to the drug resistance cassette. The PCR fragment was transformed into either SF838-1Dα or DY1640 using a standard technique (16Ito H. Fukuda Y. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar), replacing the genomic copy of PKR1 and generating LGY116 (pkr1Δ::Kanr) and SDK13-6D or SDK13-5D, haploids obtained following sporulation of drug-resistant diploid transformant. SDK13-5C and SDK13-6A were wild-type haploid strains obtained following transformation and sporulation. A PCR fragment containing the Natr sequence using pAG25 as the template also possessing sequence homologous to 40 bp 5′ and 3′ of the PKR1 open reading frame was generated and used to replace the genomic deletion of pkr1Δ::Kanr generating the pkr1Δ::Natr deletion strain (LGY117). A pkr1Δ::Natr fragment was generated using genomic DNA prepared from LGY117 by PCR that included ∼300 base pairs of 5′ and 3′ flanking sequence in addition to the open reading frame. The PCR fragment was transformed into yeast strains (KEBY4, KHY31, and LGY146) replacing the genomic copy of PKR1 generating LGY118, LGY119, and LGY147. A similar strategy was used to generate LGY120 and LGY146 using PCR fragments with an ∼300-bp flanking sequence amplified from genomic DNA prepared from the appropriate genomic deletion collection strain. A PCR fragment was generated containing the Natr sequence using pAG25 as a template along with primers that also possessed sequences homologous to the 40-bp 5′ and 3′ of the VPH1 open reading frame. The vph1Δ::Natr PCR fragment was transformed into KEBY4 to generate LGY136. A PCR fragment containing the PKR1 open reading frame and 300-bp flanking DNA was generated from genomic DNA prepared from wild-type yeast strain SF838-1Dα. The fragment was subcloned into pCR4Blunt vector (Invitrogen) generating pLG174, which was then digested with EcoRI to generate a PKR1 fragment that was subcloned into pRS416 also digested with EcoRI. A BglII site was introduced in the PKR1 open reading frame immediately prior to the stop codon by PCR. A BglII fragment from pSMY92 containing a triple HA epitope tag was subcloned into PKR1 to generate pLG221. The construct PKR1::FLAG was generated by PCR amplification from genomic DNA using primers that added a diglycine hinge and a single FLAG epitope at the C terminus prior to the stop codon. The purified PCR product was digested with BamHI and SacI and subcloned into pRS315 generating pRS315PKR1-FLAG. Immunoblot Analyses of Whole Cell Extracts and Purified Vacuoles—Whole cell protein extracts from various yeast strains were prepared as previously described (17Compton M.A. Graham L.A. Stevens T.H. J. Biol. Chem. 2006; 281: 15312-15319Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Briefly, yeast cells were resuspended in Thorner buffer minus β-mercaptoethanol (8 m urea + 10% SDS + 40 mm Tris, pH 6.8) and solubilized by mixing aggressively in the presence of glass beads (0.5 mm diameter). Cell debris and glass beads were collected by centrifugation at 16,000 × g for 10 min and the supernatant was transferred to a fresh tube. Protein concentrations were determined using the method of Markwell (18Markwell M.A. Haas S.M. Bieber L.L. Tolbert N.E. Anal. Biochem. 1978; 87: 206-210Crossref PubMed Scopus (5326) Google Scholar). Vacuolar isolations were performed as previously described (19Li L. Chen O.S. McVey Ward D. Kaplan J. J. Biol. Chem. 2001; 276: 29515-29519Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). Briefly, cells were converted to spheroplasts, resuspended in 15% Ficoll + 0.2 m sorbitol, lysed by the addition of 100 μg/ml DEAE-dextran, transferred to a centrifuge tube, and overlaid with 8, 4, and 0% Ficoll in sorbitol, centrifuged for 90 min at 110,000 × g, and vacuoles were collected from the 0/4% interface. SDS-PAGE analysis was performed on the various protein preparations. Proteins were transferred to 0.2-μm nitrocellulose or polyvinylidene difluoride membrane and probed with antibodies recognizing Vph1p (Molecular Probes 10D7A), Vma1p (Molecular Probes 8B1), Vma2p (Molecular Probes 13D11), Dpm1p (Molecular Probes 5C5), carboxypeptidase Y (Molecular Probes 10A5), or Ccc1p. Bands were visualized by incubation with anti-mouse or anti-rabbit horseradish peroxidase (Jackson ImmunoResearch Laboratories, Inc.) conjugated secondary antibodies and chemiluminescence reagents. Pulse-Chase Immunoprecipitation of Vph1p—Denaturing immunoprecipitations were performed as previously described (10Hill K.J. Stevens T.H. Mol. Biol. Cell. 1994; 5: 1039-1050Crossref PubMed Scopus (95) Google Scholar). Prior to metabolic labeling with [35S]methionine, yeast cells were cultured in synthetic defined medium containing all amino acids except methionine. Briefly, cells (0.5 A600) for each time point were labeled for 10 min with 100 μCi of 35S-Express label (PerkinElmer Life Sciences), unlabeled cysteine/methionine mixture was added and samples were removed at the time points indicated. Cell pellets were denatured by SDS solubilization of spheroplasted cells, diluted in IP buffer (10 mm Tris, pH 8.0, 0.1% SDS, 0.1% Triton X-100 final concentration), precleared, and proteins immunoprecipited using rabbit anti-Vph1p serum (10Hill K.J. Stevens T.H. Mol. Biol. Cell. 1994; 5: 1039-1050Crossref PubMed Scopus (95) Google Scholar) and fixed Staphylococcus aureus cells (IgG Sorb, The Enzyme Center). Immunocomplexes were denatured and solubilized in SDS-PAGE-Thorner buffer (8 m urea + 10% SDS + 40 mm Tris, pH 6.8, + 5% β-mercapthoethanol + 5% glycerol + 0.001% bromphenol blue). Samples were separated by SDS-PAGE, gels were fixed and then dried onto chromatography paper, and exposed to a PhosphorScreen. Data were collected using a STORM PhosphorImager (GE Healthcare), and analyzed using Quality One software (Bio-Rad). Microscopy—The acidification of vacuoles in various yeast strains was visualized using the lysosomotropic fluorescent dye quinacrine or LysoSensor Green DND-189 (Molecular Probes). Quinacrine staining of live yeast cells was conducted as previously described (20Uchida E. Ohsumi Y. Anraku Y. J. Biol. Chem. 1985; 260: 1090-1095Abstract Full Text PDF PubMed Google Scholar, 21Flannery A.R. Graham L.A. Stevens T.H. J. Biol. Chem. 2004; 279: 39856-39862Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Quinacrine was used at a final concentration of 200 μm to stain acidified vacuoles, whereas concanavalin A TRITC (Molecular Probes) was added at a final concentration of 50 μg/ml to allow for fluorescent visualization of the cell surface. Samples for indirect immunofluorescence were prepared as previously described and probed with anti-HA antibody (Covance Research Products) (22Conibear E. Stevens T.H. Methods Enzymol. 2002; 351: 408-432Crossref PubMed Scopus (45) Google Scholar). These images were acquired on a Zeiss Axioplan 2 fluorescence microscope using ×100 objective and manipulated using AxioVision software (Zeiss). Staining with LysoSensor Green DND-189 was performed as described (23Perzov N. Padler-Karavani V. Nelson H. Nelson N. J. Exp. Biol. 2002; 205: 1209-1219Crossref PubMed Google Scholar). Cells where incubated in YEPD + 100 mm HEPES, pH 7.6, + 4 μm LysoSensor Green for 5 min at 30 °C, washed once with buffer minus dye, resuspended in YEPD, and visualized. Immunofluorescence for Fet3p or colocalization of Pkr1p-FLAG and Dpm1p was performed as described previously (19Li L. Chen O.S. McVey Ward D. Kaplan J. J. Biol. Chem. 2001; 276: 29515-29519Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar) using either rabbit anti-Fet3p (24Felice M.R. De Domenico I. Li L. Ward D.M. Bartok B. Musci G. Kaplan J. J. Biol. Chem. 2005; 280: 22181-22190Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) with Alexa 488-conjugated goat anti-rabbit IgG for Fet3p immunofluorescence or rabbit anti-FLAG antibody (1:500) (Sigma) and mouse anti-Dpm1p (1:200) (Molecular Probes) followed by Alexa 594-conjugated goat anti-rabbit IgG and Alexa 488-conjugated goat anti-mouse IgG for colocalization immunofluorescence. Cells were imaged using an Olympus epifluorescence microscope with MagnaFire software. Localization of Proteins by Subcellular Fractionation—Yeast cells were spheroplasted with zymolyase and lysed in phosphate-buffered saline + 200 mm sorbitol as previously described (17Compton M.A. Graham L.A. Stevens T.H. J. Biol. Chem. 2006; 281: 15312-15319Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Unbroken cells were removed by brief centrifugation at 500 × g and supernatant (S5) was re-centrifuged for 15 min at 13,000 rpm (16,000 × g) generating a pellet (P13) and supernatant fraction (S13). Samples were solubilized in SDS-PAGE-Thorner buffer, separated by SDS-PAGE, transferred to nitrocellulose, and probed with antibodies recognizing Dpm1, HA, and 3-phosphoglycerate kinase (Molecular Probes 22C5). Samples were visualized as described above. Extraction of Peripheral Membrane Proteins with Carbonate—P13 membranes were prepared and treated with TE (10 mm Tris, pH 7.4, 1 mm EDTA), 100 mm sodium carbonate, or 1% Triton X-100 as previously described (9Jackson D.D. Stevens T.H. J. Biol. Chem. 1997; 272: 25928-25934Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Samples were analyzed using antibodies against HA, Vph1p, or Vma2p. Iron Transport Activity—Measurement of iron transport activity was performed as previously described (13Davis-Kaplan S.R. Ward D.M. Shiflett S.L. Kaplan J. J. Biol. Chem. 2004; 279: 4322-4329Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Briefly, cells where grown in YEPD + 90 μm BPS + 7.5 μm FeSO4, cells were collected by centrifugation, and incubated in assay buffer either with or without 2.5 μm CuSO4 + 1.0 mm ascorbate for 30 min on ice. Cells were collected, washed, resuspended to 4-6 × 107 cells, then incubated with 0.50 μm 59Fe for 10 min at 30 °C, placed at 0 °C and washed, onto filter paper, and iron uptake was measured by counting using a Packard 5000 series γ-counter. Deletion of PKR1 Leads to a Low Iron Growth Phenotype Due to Defective Copper Loading of apoFet3p—A homozygous diploid strain BY4743 (S288c derivative) with a deletion in PKR1 showed a growth defect on iron-limited medium (BPS + 2.5 μm Fe) that could be overcome with additional iron (BPS + 100 μm Fe; Fig. 1A). Transformation of pkr1Δ cells with either a low or high copy plasmid containing PKR1 under the control of its endogenous promoter complemented the low iron growth deficit (data not shown). Addition of a carboxyl-terminal FLAG epitope to Pkr1p did not affect the ability of the plasmid to complement the phenotype of the deletion strain (Fig. 1A). The low iron growth defect of pkr1Δ was not specific to the S288c homozygous diploid strain. A haploid W303 derivative strain with a deletion in PKR1 (SDK13-6D) also showed a low iron growth defect (Fig. 1B). The low iron growth deficit could be complemented by transformation of SDK13-6D yeast cells with a low copy PKR1 containing plasmid. An inability to grow on low iron medium may reflect a defect in iron transport activity or an inability to utilize iron once transported. Growth of cells in low iron medium results in an induction of the high affinity iron transport system (25Askwith C. Eide D. Van Ho A. Bernard P.S. Li L. Davis-Kaplan S. Sipe D.M. Kaplan J. Cell. 1994; 76: 403-410Abstract Full Text PDF PubMed Scopus (587) Google Scholar). Wild-type yeast cells grown in iron-limited medium showed high levels of 59Fe transport activity (Fig. 2A, open bars). Measurement of 59Fe transport in pkr1Δ cells grown in iron-limited medium showed much lower rates of 59Fe transport than wild-type cells. Cells with deletions in genes that encode a structural subunit of the V-ATPase (vma2Δ) or a protein required for the assembly of the V-ATPase (vma21Δ) possess even lower iron transport activities compared with pkr1Δ cells. In the absence of copper loading, an apoFet3p can still localize to the cell surface (26Yuan D.S. Stearman R. Dancis A. Dunn T. Beeler T. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2632-2636Crossref PubMed Scopus (393) Google Scholar). The presence of apoFet3p at the cell surface can be determined by measuring iron transport activity in cells that had been incubated with reduced copper, low pH, and chloride at 0 °C (15Davis-Kaplan S.R. Askwith C.C. Bengtzen A.C. Radisky D. Kaplan J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13641-13645Crossref PubMed Scopus (112) Google Scholar). Wild-type or pkr1Δ cells were copper-loaded at 0 °C and then assayed for 59Fe transport activity. Wild-type cells treated this way did not show increased transport activity, showing that cell surface Fet3p is fully copper loaded (Fig. 2A, gray bars). In contrast, copper loading of pkr1Δ cells resulted in increased 59Fe transport activity. Copper loading of apoFet3p in vma2Δ and vma21Δ cells also increased the 59Fe transport activity of these mutant cells. The decrease in induction of the iron transport system is a reflection of the low levels of cell surface apoFet3 in these deletion strains relative to pkr1Δ cells (see below). To confirm that pkr1Δ cells express Fet3p on their cell surface, cells grown in iron-limited (BPS + 5 μm iron) and iron-replete media (YEPD) were fixed and stained with an antibody to Fet3p. Both wild-type and pkr1Δ cells grown in iron-limited medium showed high levels of Fet3p on the cell surface (Fig. 2B, top panels). In contrast, when grown in iron-replete medium only pkr1Δ cells showed cell surface Fet3p (Fig. 2B, middle panels). These results demonstrate that the defect in copper loading of Fet3p in pkr1Δ cells leads to the presence of enzymatically inactive apoFet3p on the cell surface. Cells Lacking Pkr1p Demonstrate Compromised Growth Suggesting Reduced V-ATPase Function—Yeast cells lacking a functional V-ATPase are unable to grow on media containing 100 mm CaCl2, yet are able to grow on media buffered to an acidic pH 5.0. The growth phenotype of cells lacking Pkr1p was compared with wild-type yeast cells and to cells lacking a required V-ATPase assembly factor Vma21p (8Malkus P. Graham L.A. Stevens T.H. Schekman R. Mol. Biol. Cell. 2004; 15: 5075-5091Crossref PubMed Scopus (68) Google Scholar, 10Hill K.J. Stevens T.H. Mol. Biol.