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• W1992222348 abstract "J-proteins are molecular chaperones with a characteristic domain predicted to mediate interaction with Hsp70 proteins. We have previously isolated yeast mutants of the mitochondrial Hsp70, Ssq1p, in a genetic screen for mutants with altered iron homeostasis. Here we describe the isolation of mutants of the J-domain protein, Jac1p, using the same screen. Mutantjac1 alleles predicted to encode severely truncated proteins (lacking 70 or 152 amino acids) were associated with phenotypes strikingly similar to the phenotypes of ssq1mutants. These phenotypes include activation of the high affinity cellular iron uptake system and iron accumulation in mitochondria. In contrast to iron accumulation, Fe-S proteins of mitochondria were specifically deficient. In jac1 mutants, like inssq1 mutants, processing of the Yfh1p precursor protein from intermediate to mature forms was delayed. In the genetic backgrounds used in this study, jac1 null mutants were found to be viable, permitting analysis of genetic interactions. TheΔjac1 Δssq1 double mutant was more severely compromised for growth than either single mutant, suggesting a synthetic or additive effect of these mutations. Overexpression of Jac1p partially suppressed ssq1 slow growth and vice versa. Similar mitochondrial localization and similar mutant phenotypes suggest that Ssq1p and Jac1p are functional partners in iron homeostasis. J-proteins are molecular chaperones with a characteristic domain predicted to mediate interaction with Hsp70 proteins. We have previously isolated yeast mutants of the mitochondrial Hsp70, Ssq1p, in a genetic screen for mutants with altered iron homeostasis. Here we describe the isolation of mutants of the J-domain protein, Jac1p, using the same screen. Mutantjac1 alleles predicted to encode severely truncated proteins (lacking 70 or 152 amino acids) were associated with phenotypes strikingly similar to the phenotypes of ssq1mutants. These phenotypes include activation of the high affinity cellular iron uptake system and iron accumulation in mitochondria. In contrast to iron accumulation, Fe-S proteins of mitochondria were specifically deficient. In jac1 mutants, like inssq1 mutants, processing of the Yfh1p precursor protein from intermediate to mature forms was delayed. In the genetic backgrounds used in this study, jac1 null mutants were found to be viable, permitting analysis of genetic interactions. TheΔjac1 Δssq1 double mutant was more severely compromised for growth than either single mutant, suggesting a synthetic or additive effect of these mutations. Overexpression of Jac1p partially suppressed ssq1 slow growth and vice versa. Similar mitochondrial localization and similar mutant phenotypes suggest that Ssq1p and Jac1p are functional partners in iron homeostasis. open reading frame polymerase chain reaction succinate dehydrogenase matrix processing peptidase base pair(s) kilobase pair(s) p-iodonitrotetrazolium violet Iron is required as a cofactor for critical proteins involved in diverse biological processes including oxidation-reduction, cellular respiration, oxygen interaction, and metabolic conversions (1Brittenham G. Hoffman R. Benz E. Shattil S. Furie B. Cohen H. Silberstein L. McGlave P. Hematology: Basic Principles and Practice.in: Churchill Livingstone, Philadelphia2000: 397-428Google Scholar). However, iron can also be extremely toxic. The toxicity of iron results from interaction with reactive oxygen intermediates, generating highly toxic hydroxyl radicals that damage nearby macromolecules, including lipids, DNA, and proteins (2Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine. Clarendon, Oxford, United Kingdom1991Google Scholar). Cells cope with this dual nature of iron in part by homeostatic regulation of cellular iron uptake. InSaccharomyces cerevisiae and other organisms, iron uptake is induced by iron deprivation and repressed by iron sufficiency (3Askwith C.C. de Silva D. Kaplan J. Mol. Microbiol. 1996; 20: 27-34Crossref PubMed Scopus (103) Google Scholar). The components of the iron uptake system of yeast include a surface reductase encoded by FRE1 that is regulated by iron levels through changes in its transcription (4Dancis A. Roman D.G. Anderson G.J. Hinnebusch A.G. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3869-3873Crossref PubMed Scopus (282) Google Scholar). We used this property of theFRE1 gene to select mutants that failed to repressFRE1 expression in response to iron. The types of mutants selected included mutants with perturbed cellular iron uptake (5Dancis A. Yuan D.S. Haile D. Askwith C. Eide D. Moehle C. Kaplan J. Klausner R.D. Cell. 1994; 76: 393-402Abstract Full Text PDF PubMed Scopus (567) Google Scholar, 6Stearman R. Yuan D.S. Yamaguchi-Iwai Y. Klausner R.D. Dancis A. Science. 1996; 271: 1552-1557Crossref PubMed Scopus (582) Google Scholar), iron sensing (7Yamaguchi-Iwai Y. Dancis A. Klausner R.D. EMBO J. 1995; 14: 1231-1239Crossref PubMed Scopus (314) Google Scholar), or intracellular iron distribution (8Knight S.A. Sepuri N.B. Pain D. Dancis A. J. Biol. Chem. 1998; 273: 18389-18393Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 9Li J. Kogan M. Knight S. Pain D. Dancis A. J. Biol. Chem. 1999; 274: 33025-33034Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). This last category of mutants, which included ssq1 mutants, exhibited misregulated activation of high affinity cellular iron uptake and accumulation of iron within mitochondria (8Knight S.A. Sepuri N.B. Pain D. Dancis A. J. Biol. Chem. 1998; 273: 18389-18393Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). The SSQ1 gene encodes a low abundance Hsp70 chaperone of the mitochondrial matrix (10Schilke B. Forster J. Davis J. James P. Walter W. Laloraya S. Johnson J. Miao B. Craig E. J. Cell Biol. 1996; 134: 603-613Crossref PubMed Scopus (72) Google Scholar). We now describe the isolation of jac1 mutants with similar phenotypes using the same selection scheme. Jac1p contains a J-domain with a signature HPD (histidine, proline, aspartate) tripeptide motif (11Strain J. Lorenz C.R. Bode J. Garland S. Smolen G.A. Ta D.T. Vickery L.E. Culotta V.C. J. Biol. Chem. 1998; 273: 31138-31144Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar) predicted to mediate interaction with Hsp70s (12Johnson J.L. Craig E.A. Cell. 1997; 90: 201-204Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Thus, Jac1p may be the functional partner of Ssq1p with activity in controlling iron trafficking into the cell and mitochondria. Culotta and co-workers (11Strain J. Lorenz C.R. Bode J. Garland S. Smolen G.A. Ta D.T. Vickery L.E. Culotta V.C. J. Biol. Chem. 1998; 273: 31138-31144Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar) have identified jac1and ssq1 mutants by using a different genetic screen that selects for improved growth of a yeast strain deficient in cytosolic superoxide dismutase (Sod1p). Mutations of jac1 orssq1 were associated with deficient activities of Fe-S proteins (11Strain J. Lorenz C.R. Bode J. Garland S. Smolen G.A. Ta D.T. Vickery L.E. Culotta V.C. J. Biol. Chem. 1998; 273: 31138-31144Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Fe-S clusters are modular units in which iron and sulfur are coordinated in various combinations and linked to the peptide backbone of proteins via cysteine sulfurs. Because of their ability to donate or accept electrons with tremendously varied range of potentials, these clusters are involved in many fundamental biological processes, ranging from cellular respiration to metabolic conversions (13Beinert H. J. Biol. Inorg. Chem. 2000; 5: 2-15Crossref PubMed Scopus (535) Google Scholar). Although the clusters can be synthesized non-enzymaticallyin vitro (14Holm R.H. Adv. Inorg. Chem. 1992; 38: 1-71Crossref Scopus (306) Google Scholar), recent genetic and biochemical evidence suggests that their in vivo formation is catalyzed by specific enzymes (15Zheng L. White R. Cash V. Jack R. Dean D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2754-2758Crossref PubMed Scopus (501) Google Scholar, 16Zheng L. Cash V. Flint D. Dean D. J. Biol. Chem. 1998; 273: 13264-13272Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar, 17Schwartz C.J. Djaman O. Imlay J.A. Kiley P.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9009-9014Crossref PubMed Scopus (257) Google Scholar, 18Skovran E. Downs D.M. J. Bacteriol. 2000; 182: 3896-3903Crossref PubMed Scopus (67) Google Scholar). In bacteria, several of these proteins are associated together on the isc operon (16Zheng L. Cash V. Flint D. Dean D. J. Biol. Chem. 1998; 273: 13264-13272Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar), and their precise biological roles in Fe-S cluster synthesis are the subject of ongoing work (19Zheng L. Dean D. J. Biol. Chem. 1994; 269: 18723-18726Abstract Full Text PDF PubMed Google Scholar, 20Yuvaniyama P. Agar J. Cash V. Johnson M. Dean D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 599-604Crossref PubMed Scopus (275) Google Scholar, 21Hoff K.G. Silberg J.J. Vickery L.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7790-7795Crossref PubMed Scopus (200) Google Scholar, 22Kambampati R. Lauhon C.T. J. Biol. Chem. 2000; 275: 10727-10730Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Homologs of Ssq1p (called HscA or Hsc66) and Jac1p (called HscB or Hsc20) have been found on the iscoperon, suggesting a role in Fe-S cluster synthesis or maintenance (16Zheng L. Cash V. Flint D. Dean D. J. Biol. Chem. 1998; 273: 13264-13272Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar,23Vickery L.E. Silberg J.J. Ta D.T. Protein Sci. 1997; 6: 1047-1056Crossref PubMed Scopus (88) Google Scholar). Overexpression of these proteins is required for overexpression of Fe-S proteins in bacteria (24Nakamura M. Saeki K. Takahashi Y. J. Biochem. ( Tokyo ). 1999; 126: 10-18Crossref PubMed Scopus (168) Google Scholar). In a recent study, Vickery and co-workers (21Hoff K.G. Silberg J.J. Vickery L.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7790-7795Crossref PubMed Scopus (200) Google Scholar) demonstrated a three-way interaction of the Hsp70 (Hsc66), a J-protein co-chaperone (Hsc20), and another iscoperon-encoded protein, IscU. IscU may provide a scaffold for assembling intermediates of the Fe-S cluster assembly process (20Yuvaniyama P. Agar J. Cash V. Johnson M. Dean D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 599-604Crossref PubMed Scopus (275) Google Scholar). Here we characterize the phenotypes of jac1 mutants with altered iron homeostasis and their similarity to phenotypes ofssq1 mutants. We also demonstrate genetic interactions between JAC1 and SSQ1. Rich medium consisted of 1% yeast extract, 2% peptone, 100 μg/ml adenine, and various carbon sources. In some experiments 2% raffinose was used (YPAR), and in other experiments 2% glucose was used (YPAD). To induce the GAL10 promoter, the carbon source used was 2% raffinose and 0.5% galactose. Expression from theGAL10 promoter was turned off by growth in identical medium without galactose. For experiments with different concentrations of iron, standard defined medium with 2% raffinose was modified by omission of iron. Medium was autoclaved and iron was added back from a filter sterilized stock of 100 mm ferric ammonium sulfate. To achieve severe iron deprivation, defined medium without added iron was supplemented with 10 μm chelator bathophenanthrolene disulfonate (Fluka). Methods for growing yeast, crossing strains, sporulation, tetrad dissection, and yeast transformation have been described (25Sherman F. Fink G.R. Hicks J.B. Laboratory Course Manual for Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1986Google Scholar). Haploid strain 61q (MATα, trp1–63,leu2–3,112, gcn4–101, his3–609,ura3–52, FRE1-HIS3::URA3,SSQ1::LEU2) was derived from strain 61 by insertion of an additional copy of SSQ1 carried on plasmid pRS405 and integrated at the BstEII site of theleu2–3,112 locus. 61q was subjected to mutagenesis by UV irradiation to produce a mortality of 40–70%. The mutagenized population was diluted and allowed to form colonies from single cells on YPAD agar plates. These colonies were replicated to agar plates containing defined medium supplemented with 10 μm copper sulfate and 20 μm ferric ammonium sulfate. After 4 days, some of the colonies developed papillae, which were streaked for single clones on YPAD plates and evaluated further. The mutants derived from 61q by this procedure included UV6.3 (jac1–3,rho°) and UV6.30 (jac1–30, rho°). The related parental strains CM3260 (MATα,trp1–63, leu2–3,112, gcn4–101,his3–609, ura3–52) and CM3262 (MATa , ino1–13,leu2–3,112, gcn4–101, his3–609,ura3–52) were used in crosses (5Dancis A. Yuan D.S. Haile D. Askwith C. Eide D. Moehle C. Kaplan J. Klausner R.D. Cell. 1994; 76: 393-402Abstract Full Text PDF PubMed Scopus (567) Google Scholar). UV6.3 and UV6.30 were backcrossed with CM3262, and spore clones were evaluated. Backcross of UV6.3 yielded spore clones 634–3C (FRE1-HIS3::URA3, jac1–3,rho+) and 8A (ura3–52, jac1–3,rho+). Backcross of UV6.30 yielded spore clones 725–3D (FRE1-HIS3::URA3, jac1–30,rho+) and 2C (ura3–52, jac1–30,rho+). In prior work, mutant selection using strain 61 yielded the mutant 191–33C (FRE1-HIS3::URA3, ssq1–4). This strain carries a mutant allele of ssq1, which is truncated at amino acid 220 of the coding region and was previously called ssc2–1 (8Knight S.A. Sepuri N.B. Pain D. Dancis A. J. Biol. Chem. 1998; 273: 18389-18393Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). In strains CM3260, 8A, and 2C, theAFT1 open reading frame (ORF)1 was interrupted by insertion of a URA3 marker gene at a uniqueHindIII site in the genome (7Yamaguchi-Iwai Y. Dancis A. Klausner R.D. EMBO J. 1995; 14: 1231-1239Crossref PubMed Scopus (314) Google Scholar), creating strains CM3260Δaft1, 8AΔaft1, and 2CΔaft1. Plasmid pRS406-JAC1 was inserted at theMscI site of the JAC1 locus in CM3262, creating strain JM1 (URA3::JAC1). JM1 was crossed with 8A (jac1–3, ura3–52) and sporulated, and the tetrads were scored for non-repressed ferric reductase (mutant iron regulatory phenotype) and Ura+growth. All Ura+ tetrads lacked the mutant iron regulatory phenotype. A shuffle strain for JAC1 was constructed as follows. A diploid resistant to cycloheximide, YPH501 (cyh2/cyh2), was transformed with the construct jac1KOhis. This heterozygous knockout (JAC1/Δjac1::HIS3,cyh2/cyh2) was transformed with plasmid pRS318-JAC1, carrying the JAC1 ORF, 500 bp of 5′- and 3′-flanking region on a centromere-based plasmid with LEU2 and CYH2genes. A tetrad clone with Leu+ and His+growth, 729–4B, was identified and designated as a shuffle strain (MATa , ura3–52,lys2–801(amber), ade2–101(ochre),trp1-Δ63, his3-Δ200,leu2-Δ1,Δjac1::HIS3(pRS318-JAC1)). A shuffle strain for SSQ1 was constructed in analogous fashion. The SSQ1 gene was interrupted withHIS3 in the YPH501 (cyh2/cyh2) diploid. This diploid was transformed with plasmid pRS318-SSQ1 sporulated, and a tetrad clone with Leu+ and His+ growth, MK4–6, was identified and used as a shuffle strain (MATα ,ura3–52, lys2–801(amber),ade2–101(ochre), trp1-Δ63,his3-Δ200, leu2-Δ1,Δjac1::HIS3(pRS318-SSQ1)). For counterselection against the presence of the plasmid pRS318, cells were exposed to 10 μg/ml cycloheximide added to agar plates containing growth media (26Sikorski R.S. Boeke J.D. Methods Enzymol. 1991; 194: 302-318Crossref PubMed Scopus (495) Google Scholar). The JAC1 shuffle strain 729–4B and SSQ1 shuffle strain MK4–6 were mated, and a zygote was picked by micromanipulation. The diploid was streaked on cycloheximide plates to eject the pRS318 plasmids. The diploid, which became auxotrophic for leucine, was then sporulated. Tetrads were dissected and photographed after 5 days. The spore clones were transferred to YPAD plates, and genotypes were analyzed for presence ofSSQ1,Δssq1::HIS3,JAC1, andΔjac1::HIS3 alleles using genomic PCR. The primers for this analysis were as follows. ForJAC1, primer 011700B (5′-GACGCAAGAACAGACCTC-3′) and primer 041100A (5′-CGACCACAAGGAATTTGGAAC-3′) produced a product of 802 bp. ForΔjac1, primer 101499E (5′-CGCTCACCAAGCTCTTAAAACG-3′) and primer 041100A (see above) produced a 539-bp product. For SSQ1, primer 021198C (5′-CCTGAAACAAACTTCAG-3′) and 013098B (5′-CACCCGCTTGCCCATCAACA-3′) produced a 928-bp product. For Δssq1, primer 101499E (see above) and 013098B (see above) produced a 275-bp product. F1 was identified by screening a yeast genomic library (27Rose M.D. Novick P. Thomas J.H. Botstein D. Fink G.R. Gene ( Amst .). 1987; 60: 237-243Crossref PubMed Scopus (830) Google Scholar) for correction of the slow growth and non-repressed reductase of 8A (jac1–3). F1 contained a 5.3-kb insert in the vector YCp50. The insert consisted of bp 458483–463769 of chromosome VII and included JAC1. The JAC1 ORF with 500 bp of flanking DNA was amplified from genomic DNA isolated from YPH501 by PCR using Pfu polymerase and inserted into the vector pCR2.1-TOPO (Invitrogen). Similarly, the ORFs and flanking regions were amplified from the 8A (jac1–3) and 2C (jac1–30) strains and inserted into the same vector. Several clones of each were subjected to DNA sequencing. The wild-type and mutant JAC1genomic fragments with SstI and XhoI ends were subcloned into pRS406 for integration into the genome atStuI in the ura3–52 locus. The wild-typeJAC1 genomic fragment was also subcloned into the same sites in pRS318 for use in plasmid shuffling. For obtaining high levels of regulated expression, the JAC1 ORF and truncated variants (with amino acids 1–114 or amino acids 1–32) were amplified from the F1 plasmid with 5′ BamHI and 3′ XhoI sites and cloned into pEMBlyex4-i (9Li J. Kogan M. Knight S. Pain D. Dancis A. J. Biol. Chem. 1999; 274: 33025-33034Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). The plasmids, called GAL10-Jac1, GAL10-jac1(1–114), and GAL10-jac1(1–32) could be linearized with a unique StuI restriction site for integration at theura3–52 site. The SSQ1 ORF was inserted into theBamHI site of the same vector and could also be linearized with the unique StuI site. The JAC1 deletion construct, jac1KOhis, was made by inserting a genomic fragment containing JAC1 and flanking region between SalI and KpnI sites into plasmid YCplac22. BamHI sites were introduced at the start and stop of the open reading frame, allowing for replacement of the entire ORF with a cassette containing the HIS3 gene. The knockout construct could be released by digestion with NotI and SstI. To deleteJAC1, diploids CM3263, YPH501 (28Brachmann C.B. Davies A. Cost G.J. Caputo E. Li J. Hieter P. Boeke J.D. Yeast. 1998; 14: 115-132Crossref PubMed Scopus (2618) Google Scholar), and W303 (29Meyers A.M. Pape L.K. Tzagoloff A. EMBO J. 1985; 4: 2087-2092Crossref PubMed Scopus (246) Google Scholar) were transformed with this fragment, and transformants were selected for growth in the absence of histidine. In each case, correct integration of the knockout was verified by PCR. For interruption/deletion ofSSQ1, a HIS3 cassette with BamHI ends was inserted into BglII sites within a 2.2-kbKpnI-EcoRI SSQ1 genomic fragment carried on the vector pCRscript. For disruption of SSQ1, digestion of this plasmid with KpnI and EcoRI was performed, releasing a 3.5-kb fragment prior to transformation. The pRS318-SSQ1 plasmid carried a 3461-bp EcoRI-SalI genomic fragment containing SSQ1. For expression in bacteria, the JAC1 ORF was amplified with 5′ NdeI and 3′ XhoI sites and cloned into the corresponding sites of pET21b (Novagen), forming an in-frame fusion with 6 histidines (His6). Expression in BL21(DE3) was induced by 1 mmisopropyl-β-d-1-thiogalactopyranoside for 3 h at 37 °C. The overexpressed Jac1p-His6 fusion protein was isolated from inclusion bodies, purified on a nickel-nitrilotriacetic acid column (Qiagen), and eluted with 50 mm Tris-HCl, pH 8.0, 8 m urea, 0.4 m imidazole, and 5 mm β-mercaptoethanol. The eluate was analyzed by SDS-polyacrylamide gel electrophoresis and found to contain a major band by staining with Coomassie Blue. The band was excised from the gel and used to inject rabbits for generation of polyclonal antibodies. The carboxyl-terminal 109 amino acids of the SSQ1 ORF was expressed in pET21b with His6 tag, purified from the soluble fraction of the bacterial lysate using the nickel-nitrilotriacetic acid column as above, and used to immunize rabbits. Antibodies directed against Aco1p (30Li J. Saxena S. Pain D. Dancis A. J. Biol. Chem. 2001; 276: 1503-1509Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), Mir1p, and Por1p (31Schulke N. Sepuri N.B. Gordon D.M. Saxena S. Dancis A. Pain D. J. Biol. Chem. 1999; 274: 22847-22854Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) have been described previously. Commercially available antibodies used in some experiments included mouse monoclonal antibodies directed against Cox3p or Pgk1p (3-phosphoglycerate kinase) (Molecular Probes). Mitochondria were isolated as described (32Murakami H. Pain D. Blobel G. J. Cell Biol. 1988; 107: 2051-2057Crossref PubMed Scopus (279) Google Scholar). The post-mitochondrial fraction was centrifuged at 386,000 ×g for 20 min, and the supernatant was used as a cytosolic fraction. Intact mitochondria were resuspended in 20 mmTris-HCl, pH 7.5, 0.6 m sorbitol. For determining the iron content of mitochondria, wild-type or mutant yeast cells were grown to steady state for 16 h in defined medium supplemented with varying concentrations of iron. A small amount (100 nm) of tracer55Fe (Amersham Pharmacia Biotech) was added during growth. Mitochondria were isolated in the usual fashion, and an amount equivalent to 250 μg of protein was exposed to 2% SDS in 50 μl for 10 min at 55 °C and suspended in 1 ml of scintillation mixture for counting in a Beckman scintillation counter. Radiolabeled precursors (Jac1p, Yfh1p, Put2p) were synthesized in a cell-free translation in the presence of a mixture of [35S]methionine and [35S]cysteine (31Schulke N. Sepuri N.B. Gordon D.M. Saxena S. Dancis A. Pain D. J. Biol. Chem. 1999; 274: 22847-22854Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Import reactions contained mitochondria (100 μg), radiolabeled preprotein, and 4 mm ATP and 1 mm GTP (31Schulke N. Sepuri N.B. Gordon D.M. Saxena S. Dancis A. Pain D. J. Biol. Chem. 1999; 274: 22847-22854Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Following import at 20 °C for 15 min, reaction mixtures were treated with trypsin (0.1 mg/ml) for 30 min at 0 °C. The protease was inactivated, and the samples were analyzed by SDS-polyacrylamide gel electrophoresis and exposed to film. Use of purified matrix processing protease (MPP) to test processing of mitochondrial precursor proteins was as described (33Gordon D.M. Qi S. Dancis A. Pain D. Hum. Mol. Genet. 1999; 8: 2255-2262Crossref PubMed Scopus (37) Google Scholar). High affinity cellular iron uptake was measured as described (5Dancis A. Yuan D.S. Haile D. Askwith C. Eide D. Moehle C. Kaplan J. Klausner R.D. Cell. 1994; 76: 393-402Abstract Full Text PDF PubMed Scopus (567) Google Scholar). Aconitase was assayed by measuring the formation ofcis-aconitate at 240 nm as described (34Kennedy M.C. Emptage M.H. Dreyer J.L. Beinert H. J. Biol. Chem. 1983; 258: 11098-11105Abstract Full Text PDF PubMed Google Scholar). Succinate dehydrogenase was assayed on mitochondria lysed in 0.5% Triton X-100 by following the reduction of p-iodonitrotetrazolium violet (INT) to INT-formazan as described (35Munujos P. Coll-Canti J. Gonzalez-Sastre F. Gella F.J. Anal. Biochem. 1993; 212: 506-509Crossref PubMed Scopus (102) Google Scholar). For detection of heme, mitochondrial proteins (100 μg) separated by polyacrylamide gel electrophoresis and transferred to nitrocellulose were incubated with peroxide developer and chemiluminescent substrates (SuperSignal ECL, Pierce) for 5 min. The covalently bound heme group of cytochromec (Cyc1p) and the heme group of thebc1 complex (Cyt1p) were visualized by exposing the blot to film (36Vargas C. McEwan A.G. Downie J.A. Anal. Biochem. 1993; 209: 323-326Crossref PubMed Scopus (106) Google Scholar). FRE1 encodes the major cell surface reductase and is required for iron acquisition from ferric iron chelates (4Dancis A. Roman D.G. Anderson G.J. Hinnebusch A.G. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3869-3873Crossref PubMed Scopus (282) Google Scholar). The FRE1 promoter, which is repressed in response to iron, was fused to the HIS3 coding region. Mutants that failed to respond to iron-mediated repression were selected for their ability to grow in the presence of iron and in the absence of histidine (5Dancis A. Yuan D.S. Haile D. Askwith C. Eide D. Moehle C. Kaplan J. Klausner R.D. Cell. 1994; 76: 393-402Abstract Full Text PDF PubMed Scopus (567) Google Scholar). The most commonly selected mutants were defective in the SSQ1 gene. Therefore, in order to avoid selecting more of these mutants, the screen was modified by insertion of an additional copy of SSQ1 in the haploid genome. Two new mutants, UV6.3 and UV6.30 (Fig.1 A), were selected and found to exhibit small colony size, non-repressing surface ferric reductase, and increased high affinity ferrous iron uptake. The mutant phenotypes were found to be recessive. The mutants were backcrossed with a wild-type parental strain, and the slow growth and iron related phenotypes segregated in a 2+:2− pattern in the tetrads, consistent with involvement of a single nuclear locus. Crosses revealed that the two independent isolates contained allelic mutations. The wild-type allele for the mutant gene in 8A was then cloned by complementation of the slow growth and reductase phenotypes. The complementing activity was further localized to a minimal construct containing only the JAC1 ORF and 500 bp of 5′- and 3′-flanking DNA. To rule out the possibility that complementation by the JAC1-containing genomic DNA fragment could be indirect, the JAC1 ORF with its native promoter and a URA3marker gene were inserted into the unique MscI site of the chromosomal JAC1 locus in a wild-type strain. This strain JM1 (URA3::JAC1) was crossed with 8A (jac1–3, ura3–52) and sporulated. All Ura− tetrad clones exhibited slow growth and non-repressing reductase (mutant phenotype), indicating absence of recombination between the marked wild-type JAC1 and the 8A mutant. Hence, meiotic mapping also supported that the mutation in 8A responsible for the mutant phenotypes was in JAC1 or closely linked to this locus. The mutant alleles were rescued from strains 8A (jac1–3) and 2C (jac1–30) by PCR of theJAC1 ORF and flanking regions. DNA sequence analysis revealed single nucleotide changes in each case. For thejac1–3 allele, nucleotide 344 was changed from T to G, altering codon 115 from TTA (leucine) to TGA (Stop). Thejac1–3 mutant allele codes for a truncated protein lacking its carboxyl one-third. For the jac1–30 allele, nucleotide 97 was changed from C to T, altering codon 33 from CAA (glutamine) to TAA (Stop), and is thereby predicted to code for a severely truncated protein (Fig. 1). The severity of the truncation of the predictedJAC1 ORF in allele jac1–30 suggested that it would be non-functional or null. The viability of this mutant was surprising in view of an earlier work indicating that JAC1was an essential gene required for vegetative growth (11Strain J. Lorenz C.R. Bode J. Garland S. Smolen G.A. Ta D.T. Vickery L.E. Culotta V.C. J. Biol. Chem. 1998; 273: 31138-31144Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). To directly address the question of essentiality of JAC1 in the CM3263 genetic background in which the mutants were selected, the entire ORF was deleted in this diploid, and the correctness of the deletion was verified by genomic PCR. The heterozygous knockout was sporulated and spore clones were dissected on rich (YPAD) medium and incubated at 30 °C. All four spores formed colonies, although the histidine prototrophs carrying the knockout constructs formed much smaller colonies than the wild-type spore clones (Fig.2 A). The tetrad clones containing the jac1 deletions could be maintained in culture, although they were often overgrown by more rapidly growing suppressor mutants. Similarly, in another yeast genetic background, YPH501, jac1 knockouts germinated and formed small colonies (Fig. 2 A). This slow growth phenotype of the jac1deletion was more severe when incubated at elevated (37 °C) or lower (23 °C) temperatures (data not shown). Thus, in both of these yeast strains, jac1 deletion was deleterious but still allowed germination and vegetative growth. We also examined the deletion phenotype in W303, another commonly used parental strain. In this background, the jac1 deletion underwent germination and formed tiny macroscopic colonies consisting of several thousand cells (Fig. 2 A, enlarged panel). However, these clones could not be propagated. Perhaps small amounts of Jac1p carried over from the diploid into the deletion spore clones supported a finite number of doublings prior to growth arrest. In summary,JAC1 is required for vegetative growth in some yeast genetic backgrounds and not in others. In genetic backgrounds in which the jac1 null strain was viable, it was extremely slow growing and accumulated suppressor mutations. To accurately assess the phenotype of mutants and to more easily work with strains carrying mutated forms of JAC1, we used a plasmid shuffle strategy as is used to study essential genes (26Sikorski R.S. Boeke J.D. Methods Enzymol. 1991; 194: 302-318Crossref PubMed Scopus (495) Google Scholar). The phenotype of the deletion (Fig. 2 B,line 1) uncovered aft" @default.
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• W1992222348 date "2001-05-01" @default.
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• W1992222348 title "J-domain Protein, Jac1p, of Yeast Mitochondria Required for Iron Homeostasis and Activity of Fe-S Cluster Proteins" @default.
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