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• W2087946910 abstract "Saccharomyces cerevisiae must import copper into the mitochondrial matrix for eventual assembly of cytochrome c oxidase. This copper is bound to an anionic fluorescent molecule known as the copper ligand (CuL). Here, we identify for the first time a mitochondrial carrier family protein capable of importing copper into the matrix. In vitro transport of the CuL into the mitochondrial matrix was saturable and temperature-dependent. Strains with a deletion of PIC2 grew poorly on copper-deficient non-fermentable medium supplemented with silver and under respiratory conditions when challenged with a matrix-targeted copper competitor. Mitochondria from pic2Δ cells had lower total mitochondrial copper and exhibited a decreased capacity for copper uptake. Heterologous expression of Pic2 in Lactococcus lactis significantly enhanced CuL transport into these cells. Therefore, we propose a novel role for Pic2 in copper import into mitochondria.Background: Copper must enter the mitochondrial matrix prior to assembly into cytochrome c oxidase.Results: Pic2 transports mitochondrial copper in vivo and in vitro.Conclusion: Pic2 mediates copper import into the mitochondrial matrix.Significance: We have identified the first mitochondrial copper importer. Saccharomyces cerevisiae must import copper into the mitochondrial matrix for eventual assembly of cytochrome c oxidase. This copper is bound to an anionic fluorescent molecule known as the copper ligand (CuL). Here, we identify for the first time a mitochondrial carrier family protein capable of importing copper into the matrix. In vitro transport of the CuL into the mitochondrial matrix was saturable and temperature-dependent. Strains with a deletion of PIC2 grew poorly on copper-deficient non-fermentable medium supplemented with silver and under respiratory conditions when challenged with a matrix-targeted copper competitor. Mitochondria from pic2Δ cells had lower total mitochondrial copper and exhibited a decreased capacity for copper uptake. Heterologous expression of Pic2 in Lactococcus lactis significantly enhanced CuL transport into these cells. Therefore, we propose a novel role for Pic2 in copper import into mitochondria. Background: Copper must enter the mitochondrial matrix prior to assembly into cytochrome c oxidase. Results: Pic2 transports mitochondrial copper in vivo and in vitro. Conclusion: Pic2 mediates copper import into the mitochondrial matrix. Significance: We have identified the first mitochondrial copper importer. Metals are essential nutrients that pose a management quandary for cells. They must be directed to the correct proteins and organelles through a maze of cellular components and opportunistic metal-binding sites (1.Cobine P.A. Pierrel F. Winge D.R. Copper trafficking to the mitochondrion and assembly of copper metalloenzymes.Biochim. Biophys. Acta. 2006; 1763: 759-772Crossref PubMed Scopus (233) Google Scholar). Failure to control their delivery results in cellular stress, presumably due to inappropriate interactions and oxidative damage. Cells have adopted a protein-mediated delivery mechanism for copper within the cytosol. In the budding yeast Saccharomyces cerevisiae, copper enters the cell via specific (Ctr1) and nonspecific (e.g. Fet4) transporters and is then trafficked to points of utilization by copper chaperone proteins (2.Huffman D.L. O'Halloran T.V. Function, structure, and mechanism of intracellular copper trafficking proteins.Annu. Rev. Biochem. 2001; 70: 677-701Crossref PubMed Scopus (420) Google Scholar). Copper is used as a cofactor in three major enzymes: the multicopper oxidase Fet3 required for high affinity iron uptake (3.Dancis A. Yuan D.S. Haile D. Askwith C. Eide D. Moehle C. Kaplan J. Klausner R.D. Molecular characterization of a copper transport protein in S. cerevisiae: an unexpected role for copper in iron transport.Cell. 1994; 76: 393-402Abstract Full Text PDF PubMed Scopus (567) Google Scholar); Sod1, a Cu,Zn-superoxide dismutase required for protection against oxidative stress and regulation of glucose signaling in yeast (4.Reddi A.R. Culotta V.C. SOD1 integrates signals from oxygen and glucose to repress respiration.Cell. 2013; 152: 224-235Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar); and cytochrome c oxidase (CcO), 2The abbreviations used are: CcO, cytochrome c oxidase; IMS, intermembrane space; IM, inner membrane; CuL, copper ligand; MCF, mitochondrial carrier family; BCS, bathocuproinedisulfonic acid; AgL, silver ligand; ICP-OES, inductively coupled plasma optical emission spectroscopy; hSOD1, human SOD1. the terminal enzyme complex of the electron transport chain (5.Tsukihara T. Aoyama H. Yamashita E. Tomizaki T. Yamaguchi H. Shinzawa-Itoh K. Nakashima R. Yaono R. Yoshikawa S. The whole structure of the 13-subunit oxidized cytochrome c oxidase at 2.8 Å.Science. 1996; 272: 1136-1144Crossref PubMed Scopus (1921) Google Scholar). Atx1 is the copper chaperone responsible for delivering copper to the trans-Golgi vesicles via Ccc2, a P-type ATPase (6.Pufahl R.A. Singer C.P. Peariso K.L. Lin S.J. Schmidt P.J. Fahrni C.J. Culotta V.C. Penner-Hahn J.E. O'Halloran T.V. Metal ion chaperone function of the soluble Cu(I) receptor Atx1.Science. 1997; 278: 853-856Crossref PubMed Scopus (590) Google Scholar), whereas Ccs1 serves as the copper donor for Sod1 and acts as a post-transfer modifying enzyme by facilitating the formation of an essential disulfide bond within the enzyme itself (7.Furukawa Y. Torres A.S. O'Halloran T.V. Oxygen-induced maturation of SOD1: a key role for disulfide formation by the copper chaperone CCS.EMBO J. 2004; 23: 2872-2881Crossref PubMed Scopus (291) Google Scholar). Although cytosolic copper trafficking has been well characterized, the pathway that delivers copper to mitochondria in yeast and in other eukaryotes is completely unknown. CcO is a multimeric protein complex that contains two copper centers, a binuclear CuA site and a heme a3-CuB site. A number of assembly factors act in concert to build both of these sites. The soluble intermembrane space (IMS) protein Cox17 delivers copper to both Sco1 and Cox11, which are integral inner membrane (IM) proteins that donate copper to the assembling holoenzyme (8.Horng Y.C. Cobine P.A. Maxfield A.B. Carr H.S. Winge D.R. Specific copper transfer from the Cox17 metallochaperone to both Sco1 and Cox11 in the assembly of yeast cytochrome c oxidase.J. Biol. Chem. 2004; 279: 35334-35340Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 9.Banci L. Bertini I. Ciofi-Baffoni S. Hadjiloi T. Martinelli M. Palumaa P. Mitochondrial copper(I) transfer from Cox17 to Sco1 is coupled to electron transfer.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 6803-6808Crossref PubMed Scopus (137) Google Scholar). Additionally, the IMS protein Cmc1 has been implicated in the control of copper flow within the IMS, potentially by directing copper to the Cox17-mediated CcO assembly pathway (10.Horn D. Al-Ali H. Barrientos A. Cmc1p is a conserved mitochondrial twin CX9C protein involved in cytochrome c oxidase biogenesis.Mol. Cell. Biol. 2008; 28: 4354-4364Crossref PubMed Scopus (45) Google Scholar). Organellar fractionation experiments showed that >70% of mitochondrial copper is present as a soluble anionic complex contained within a matrix-localized bioavailable pool (11.Cobine P.A. Ojeda L.D. Rigby K.M. Winge D.R. Yeast contain a non-proteinaceous pool of copper in the mitochondrial matrix.J. Biol. Chem. 2004; 279: 14447-14455Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). This complex has been defined as the copper ligand (CuL), and its existence and localization have since been confirmed by x-ray fluorescence imaging and copper chelation studies (12.Yang L. McRae R. Henary M.M. Patel R. Lai B. Vogt S. Fahrni C.J. Imaging of the intracellular topography of copper with a fluorescent sensor and by synchrotron x-ray fluorescence microscopy.Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 11179-11184Crossref PubMed Scopus (319) Google Scholar, 13.Dodani S.C. Leary S.C. Cobine P.A. Winge D.R. Chang C.J. A targetable fluorescent sensor reveals that copper-deficient SCO1 and SCO2 patient cells prioritize mitochondrial copper homeostasis.J. Am. Chem. Soc. 2011; 133: 8606-8616Crossref PubMed Scopus (235) Google Scholar). Copper-dependent human SOD1 localized to this mitochondrial compartment is able to rescue a range of phenotypic defects associated with SOD2 deletion, demonstrating the accessibility of this pool (11.Cobine P.A. Ojeda L.D. Rigby K.M. Winge D.R. Yeast contain a non-proteinaceous pool of copper in the mitochondrial matrix.J. Biol. Chem. 2004; 279: 14447-14455Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Expression of matrix-targeted human SOD1 or yeast Crs5, a copper-binding metallothionein, results in a specific loss of CcO activity that can be rescued by addition of copper or decreased expression of the competing cuproprotein (14.Cobine P.A. Pierrel F. Bestwick M.L. Winge D.R. Mitochondrial matrix copper complex used in metallation of cytochrome oxidase and superoxide dismutase.J. Biol. Chem. 2006; 281: 36552-36559Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). These observations led us to propose that the matrix copper pool is redistributed to the IMS, where it is made available to CcO. Although the exact structural identity of the CuL is unknown, we have characterized many aspects of its in vivo function. We propose that the ligand exists in a metal-free form in the cytosol, where it binds copper and delivers it to mitochondria, providing a non-proteinaceous trafficking system for copper delivery to the organelle. The IM is impermeable to most ions and molecules, so transporters must exist that facilitate matrix import of the CuL complex and its subsequent redistribution to the IMS. However, transporters required for the movement of copper across the IM have yet to be identified and remain a fundamental gap in our understanding of the mechanisms that provide for the assembly of CcO. Data from our previous studies suggest that the CuL is the molecule that is transported into the matrix and that this complex may resemble a metabolite or nucleotide. Therefore, copper transport across the IM may proceed through one or more of the mitochondrial carrier family (MCF) proteins. These proteins transport diverse metabolic substrates, such as oxaloacetate, citrate, GTP, and ATP, into and out of the matrix (15.Kunji E.R. Robinson A.J. The conserved substrate binding site of mitochondrial carriers.Biochim. Biophys. Acta. 2006; 1757: 1237-1248Crossref PubMed Scopus (105) Google Scholar). MCF proteins have been previously implicated in metal ion homeostasis (16.Froschauer E.M. Schweyen R.J. Wiesenberger G. The yeast mitochondrial carrier proteins Mrs3p/Mrs4p mediate iron transport across the inner mitochondrial membrane.Biochim. Biophys. Acta. 2009; 1788: 1044-1050Crossref PubMed Scopus (83) Google Scholar). High affinity iron uptake into mitochondria of S. cerevisiae is disrupted by simultaneous deletion of MRS3 and MRS4 (17.Mühlenhoff U. Stadler J.A. Richhardt N. Seubert A. Eickhorst T. Schweyen R.J. Lill R. Wiesenberger G. A specific role of the yeast mitochondrial carriers MRS3/4p in mitochondrial iron acquisition under iron-limiting conditions.J. Biol. Chem. 2003; 278: 40612-40620Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Studies of the Mrs3/4 homologs in vertebrate systems have demonstrated the conserved function of these proteins (18.Shaw G.C. Cope J.J. Li L. Corson K. Hersey C. Ackermann G.E. Gwynn B. Lambert A.J. Wingert R.A. Traver D. Trede N.S. Barut B.A. Zhou Y. Minet E. Donovan A. Brownlie A. Balzan R. Weiss M.J. Peters L.L. Kaplan J. Zon L.I. Paw B.H. Mitoferrin is essential for erythroid iron assimilation.Nature. 2006; 440: 96-100Crossref PubMed Scopus (438) Google Scholar, 19.Wang Y. Langer N.B. Shaw G.C. Yang G. Li L. Kaplan J. Paw B.H. Bloomer J.R. Abnormal mitoferrin-1 expression in patients with erythropoietic protoporphyria.Exp. Hematol. 2011; 39: 784-794Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 20.Chen W. Paradkar P.N. Li L. Pierce E.L. Langer N.B. Takahashi-Makise N. Hyde B.B. Shirihai O.S. Ward D.M. Kaplan J. Paw B.H. Abcb10 physically interacts with mitoferrin-1 (Slc25a37) to enhance its stability and function in the erythroid mitochondria.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 16263-16268Crossref PubMed Scopus (165) Google Scholar, 21.Paradkar P.N. Zumbrennen K.B. Paw B.H. Ward D.M. Kaplan J. Regulation of mitochondrial iron import through differential turnover of mitoferrin 1 and mitoferrin 2.Mol. Cell. Biol. 2009; 29: 1007-1016Crossref PubMed Scopus (224) Google Scholar, 22.Nilsson R. Schultz I.J. Pierce E.L. Soltis K.A. Naranuntarat A. Ward D.M. Baughman J.M. Paradkar P.N. Kingsley P.D. Culotta V.C. Kaplan J. Palis J. Paw B.H. Mootha V.K. Discovery of genes essential for heme biosynthesis through large-scale gene expression analysis.Cell Metab. 2009; 10: 119-130Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Other members of this family have also been associated with iron transport with varying levels of specificity (23.Yang M. Cobine P.A. Molik S. Naranuntarat A. Lill R. Winge D.R. Culotta V.C. The effects of mitochondrial iron homeostasis on cofactor specificity of superoxide dismutase 2.EMBO J. 2006; 25: 1775-1783Crossref PubMed Scopus (122) Google Scholar, 24.Gordon D.M. Lyver E.R. Lesuisse E. Dancis A. Pain D. GTP in the mitochondrial matrix plays a crucial role in organellar iron homoeostasis.Biochem. J. 2006; 400: 163-168Crossref PubMed Scopus (34) Google Scholar, 25.Yoon H. Zhang Y. Pain J. Lyver E.R. Lesuisse E. Pain D. Dancis A. Rim2, pyrimidine nucleotide exchanger, is needed for iron utilization in mitochondria.Biochem. J. 2011; 440: 137-146Crossref PubMed Scopus (38) Google Scholar, 26.Lin H. Li L. Jia X. Ward D.M. Kaplan J. Genetic and biochemical analysis of high iron toxicity in yeast. Iron toxicity is due to the accumulation of cytosolic iron and occurs under both aerobic and anaerobic conditions.J. Biol. Chem. 2011; 286: 3851-3862Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Therefore, a precedent exists for the involvement of multiple MCF proteins in modulating mitochondrial metal ion homeostasis. Herein, we present evidence that the MCF protein Pic2 transports copper across the mitochondrial IM, allowing for its accumulation within the matrix. The yeast strains used in this study were BY4741 (MATa leu2Δ met15Δ ura3Δ his3Δ) and the isogenic kanMX4-containing mutant from Invitrogen. ccs1Δ::IMhSOD1 was created in the Y7092 background (MATα can1Δ::STE2pr-Sp_his5 lyp1Δ his3Δ leu2Δ ura3Δ met15Δ) (27.Tong A.H. Boone C. Synthetic genetic array analysis in Saccharomyces cerevisiae.Methods Mol. Biol. 2006; 313: 171-192PubMed Google Scholar). All cultures were grown in YP medium (1% yeast extract and 2% peptone) or synthetic defined media (with selective amino acids excluded) with the appropriate filter-sterilized carbon source added. Metal concentrations were varied using BIO 101 yeast nitrogen base (Sunrise Science Products) plus added 0.1 mm ferrous chloride to give copper-deficient conditions. If required, further copper chelation was achieved by adding bathocuproinedisulfonic acid (BCS). Exogenous copper was provided by adding CuSO4. All of the growth tests were performed at 30 °C with 1:10 serial dilutions of pre-cultures grown under permissive conditions. Matrix-targeted Crs5 was described previously (14.Cobine P.A. Pierrel F. Bestwick M.L. Winge D.R. Mitochondrial matrix copper complex used in metallation of cytochrome oxidase and superoxide dismutase.J. Biol. Chem. 2006; 281: 36552-36559Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). The PIC2/YER053C ORF plus 300 bp upstream (to include the endogenous promoter) was cloned into pRS415. The PIC2 ORF was also cloned into pNZ8148 (MoBiTec) under the control of the nisin-inducible promoter. The fidelity of each construct was verified by dideoxynucleotide sequencing prior to use. Intact mitochondria were prepared, and the resultant soluble contents were fractionated as described previously (11.Cobine P.A. Ojeda L.D. Rigby K.M. Winge D.R. Yeast contain a non-proteinaceous pool of copper in the mitochondrial matrix.J. Biol. Chem. 2004; 279: 14447-14455Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Anionic fractions for reverse phase chromatography were prepared by adding DEAE resin (Whatman) in batch. The resin was washed with 25 bed volumes of 20 mm ammonium acetate (pH 8.0) and eluted with 5 volumes of 1 m ammonium acetate (pH 8.0). The samples were loaded directly onto a Phenomenex C18 column. Unbound fractions were removed with 50 mm ammonium acetate (pH 5.0) or with 0.1% trifluoroacetic acid to isolate the apo-ligand. A 60-min gradient to 100% acetonitrile was used, and 1-ml fractions were collected. The final fractions were analyzed for copper by inductively coupled plasma optical emission spectroscopy (ICP-OES; PerkinElmer Life Sciences 9300-DV system) and for fluorescence (PerkinElmer Life Sciences LS55 fluorometer). Excitation and emission scans of copper-containing fractions were performed at an excitation maximum of 220 nm and an emission maximum of 360 nm using 5-nm slit widths. L. lactis cells transformed with vector (pNZ8148) alone or carrying the PIC2 gene were grown overnight at 30 °C in M17 medium with 0.5% glucose and 10 μg/ml chloramphenicol. Cells were diluted into fresh medium at an A600 of 0.1, grown to an A600 of 0.4, and induced using 1 ng/ml nisin for 5 h. Protein expression was confirmed using SDS-PAGE, followed by SYPRO staining or immunoblotting for Pic2. Isolated mitochondria suspended in 0.6 m sorbitol were incubated with the CuL for 30-s intervals and removed from the solution by centrifugation. Uptake was measured by ICP-OES as an increase in copper over time. Copper uptake was assayed in L. lactis using a modified method in which whole cells were resuspended in soluble matrix copper, purified ligand, or copper salts in either water or potassium phosphate buffer (pH 7.5). Cells were incubated for different time points at room temperature, removed by centrifugation, and washed with water, and total metals were measured by ICP-OES. Uptake is reported as the increase in copper over time. CcO and malate dehydrogenase activities were measured as described previously (11.Cobine P.A. Ojeda L.D. Rigby K.M. Winge D.R. Yeast contain a non-proteinaceous pool of copper in the mitochondrial matrix.J. Biol. Chem. 2004; 279: 14447-14455Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar) using a Shimadzu UV-2450 system. Superoxide dismutase (SOD1) activity was measured using a xanthine oxidase-linked assay kit (Sigma). The anti-human SOD1 monoclonal antibody was purchased from Santa Cruz Biotechnology. Antisera for Cox2 (cytochrome c oxidase subunit 2) and porin were purchased from Invitrogen. Antiserum for yeast Pic2 was raised against a synthetic peptide consisting of the 20 N-terminal residues (GenScript). We assume that the transported form of copper into mitochondria is the CuL complex. To assess transport characteristics into yeast mitochondria, we isolated the soluble matrix copper contents using anion exchange resin and incubated purified intact mitochondria with variable concentrations of the stable CuL complex. Mitochondria were removed by centrifugation and then assayed for their total copper content by ICP-OES. The CuL was imported into mitochondria in a time-dependent manner (Fig. 1A). The observed increase in copper content was not due to membrane association, as lysis of mitochondria via sonication before assay prevented accumulation (Fig. 1A), and lysis after uptake released the copper into the soluble fraction (80 ± 5% soluble). Increasing concentrations of the CuL complex saturated the initial rate of copper uptake (Fig. 1B). Half-maximal transport was observed at ∼15 μm CuL complex. Uptake was temperature-dependent, as incubation of mitochondria at 4 °C prevented CuL accumulation, and identical initial rates were obtained in mitoplasts lacking the outer membrane (Fig. 1C), suggesting that the transport occurred at the IM. Addition of the uncoupling ionophore carbonyl cyanide m-chlorophenylhydrazone did not affect the initial rates of uptake (Fig. 1C). Therefore, we conclude that the mitochondrial IM has a saturable temperature-dependent copper transport system. External chelators are often used to deplete the medium of copper. However, we sought to find a competitor that could more directly affect mitochondrial copper. Silver shares similar electronic properties with copper, and it is often used as a toxic mimetic of copper in biological systems (28.Jin Y.H. Dunlap P.E. McBride S.J. Al-Refai H. Bushel P.R. Freedman J.H. Global transcriptome and deletome profiles of yeast exposed to transition metals.PLoS Genet. 2008; 4: e1000053Crossref PubMed Scopus (118) Google Scholar, 29.Zatulovskiy E.A. Skvortsov A.N. Rusconi P. Ilyechova E.Y. Babich P.S. Tsymbalenko N.V. Broggini M. Puchkova L.V. Serum depletion of holo-ceruloplasmin induced by silver ions in vivo reduces uptake of cisplatin.J. Inorg. Biochem. 2012; 116: 88-96Crossref PubMed Scopus (16) Google Scholar). We therefore isolated intact mitochondria from yeast grown in glucose-containing medium with or without 185 μm AgNO3. Mitochondria from cells grown in the presence of silver accumulated 17 ± 0.4 mmol of silver/mol of sulfur and contained 3.3 ± 0.2 mmol of copper/mol of sulfur, whereas mitochondria from untreated cells contained 5.4 ± 0.1 mmol of copper/mol of sulfur. Although the mitochondrial copper content was reduced in silver-treated cultures, the other mineral element concentrations were comparable to those in the untreated cultures (Fig. 2A). The decrease in copper was associated with a decrease in CcO activity and oxygen consumption (Fig. 2B). Separate cultures were grown in medium containing either 150 μm silver or 150 μm copper. Under these identical conditions, silver accumulated to ∼5-fold higher concentrations in mitochondria compared with copper (data not shown). Moreover, addition of 185 μm silver to rich medium containing a non-fermentable carbon source limited the growth of wild-type cells (Fig. 2C). Mitochondria from these silver-treated cells were fractionated into soluble and insoluble components, and the soluble contents were separated by anion exchange chromatography (Fig. 2D). The fractions containing the CuL also contained an anionic silver complex (AgL). Anionic AgL was used for in vitro uptake assays. Like the CuL, the AgL complex was imported into mitochondria in a time- and concentration-dependent manner (Fig. 3A). On the basis of the in vivo observation that silver affected copper accumulation in mitochondria, we attempted to mimic this in vitro. The AgL complex was added to mitochondria in 10-fold excess compared with the CuL, and the initial rate of uptake was monitored. The excess AgL acted as a competitor, greatly decreasing CuL uptake (Fig. 3B). Conversely, a 10-fold excess of the CuL slowed uptake of the AgL into mitochondria (Fig. 3C). Yeast lacking PIC2, which encodes a MCF protein, had a growth defect on non-fermentable medium in the presence of the cell-impermeable copper chelator BCS. This defect was exacerbated by addition of silver to the medium and reversed upon addition of copper (Fig. 4A). The decrease in growth was accompanied by a silver-dependent depletion of Cox2 protein in these mitochondria (Fig. 4B). The pic2Δ strain also showed a defect on copper-limited synthetic medium with a non-fermentable carbon source without addition of silver (Fig. 5A). In agreement with the growth phenotype, we observed a 50% reduction in CcO activity and oxygen consumption in the pic2Δ mutant (Fig. 5B). To exaggerate the growth defect in the pic2Δ strain, we transformed the cells with a matrix-targeted copper metallothionein (CRS5) that we have used previously to biochemically deplete the bioavailable matrix copper pool (14.Cobine P.A. Pierrel F. Bestwick M.L. Winge D.R. Mitochondrial matrix copper complex used in metallation of cytochrome oxidase and superoxide dismutase.J. Biol. Chem. 2006; 281: 36552-36559Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). The pic2Δ strain expressing matrix-targeted CRS5 exhibited a greater growth defect compared with the pic2Δ strain alone on copper-replete non-fermentable medium, a defect that became more severe upon depletion of available copper from the medium by addition of increasing BCS concentrations (Fig. 5C).FIGURE 5Growth phenotypes of pic2Δ yeast strains upon copper depletion. A, serial dilutions of BY4741 and pic2Δ strains grown in synthetic medium with a non-fermentable carbon source (glycerol; SC Gly) with or without 20 μm BCS. B, left panel, CcO activity in mitochondria isolated from parental and pic2Δ strains grown in synthetic medium containing galactose as a carbon source. MDH, malate dehydrogenase. Right panel, oxygen consumption read as percent air/A600/s of whole cells from each strain grown in galactose-containing rich medium (n = 3). C, serial dilutions of parental and pic2Δ strains transformed with either empty vector (vec) or matrix-targeted CRS5 (mCRS5) on rich medium with a fermentable carbon source (YPD medium), with a non-fermentable carbon source (YPLG medium), or YPLG medium with limited copper availability (+50 μm BCS and +100 μm BCS).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To monitor mitochondrial copper homeostasis without confounding factors related to translation or the activity of other chaperone proteins, we used the previously described copper/IMS biomarker strain (14.Cobine P.A. Pierrel F. Bestwick M.L. Winge D.R. Mitochondrial matrix copper complex used in metallation of cytochrome oxidase and superoxide dismutase.J. Biol. Chem. 2006; 281: 36552-36559Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). In this biomarker strain, the gene for the copper chaperone for Sod1, CCS1, has been deleted, rendering the yeast Sod1 inactive and causing a lysine auxotrophy. An IM-tethered human SOD1 (IM-hSOD1) is then stably expressed and rescues the Sod1 deficit of the ccs1Δ strain in a manner that is dependent on matrix copper being made available within the IMS. Although deletion of PIC2 did not change the steady-state levels of IM-hSOD1 (Fig. 6A), SOD1 activity in BCS-supplemented medium was decreased to 39% of the parental strain (Fig. 6B). Supplementation of the medium with silver progressively decreased the activity of IM-hSOD1 in ccs1Δ mitochondria, and deletion of PIC2 exacerbated this defect (Fig. 6C). These data strongly suggest that mitochondrial copper required for the metallation of IMS cuproenzymes is a limiting factor in pic2Δ cells. Mitochondria isolated from pic2Δ cells grown in synthetic medium were analyzed for total metals by ICP-OES and showed a mild decrease in copper to 0.7-fold of the parental strain (1.9 ± 0.2 mmol of copper/mol of sulfur in the parental strain versus 1.2 ± 0.2 mmol of copper/mol of sulfur in pic2Δ mitochondria). To exaggerate this phenotypic effect, pic2Δ cells were grown in synthetic medium with added 0.5 mm CuSO4, and the mineral profile of the mitochondria was compared with that of parental yeast cultured under the same conditions (Fig. 7A). Although both strains had increased mitochondrial copper under these conditions, the pic2Δ mitochondria accumulated less copper compared with the parental strain (∼0.4-fold). There was no observable change in the content of phosphorus, iron, manganese, calcium, and magnesium, but we observed an ∼1.5-fold increase in zinc and potassium. These data suggest a specific defect in copper uptake in pic2Δ mitochondria (Fig. 7A). Intact mitochondria from pic2Δ cells were incubated with purified CuL complex and assayed for uptake. Initial rates of CuL uptake were decreased in pic2Δ mitochondria relative to those in parental cells (Fig. 7B). Moreover, pic2Δ mitochondria showed lower maximal rates of uptake. This change in saturation suggests a decreased capacity for CuL uptake in the pic2Δ strain, consistent with the lower total copper accumulation. This uptake defect was evident across a range of CuL concentrations (Fig. 7B). Because Pic2 has been previously identified as a secondary phosphate carrier, mitochondria from pic2Δ cells were assayed for phosphate uptake using an established swelling assay (30.Hamel P. Saint-Georges Y. de Pinto B. Lachacinski N. Altamura N. Dujardin G. Redundancy in the function of mitochondrial phosphate transport in Saccharomyces cerevisiae and Arabidopsis thaliana.Mol. Microbiol. 2004; 51: 307-317Crossref PubMed Scopus (68) Google Scholar). Consistent with previous observations, no defect in phosphate uptake was detected in pic2Δ mitochondria (data not shown). To account for possible indirect effects of other proteins contributing to the observed defects in mitochondrial copper uptake, we cloned PIC2 into a nisin-inducible vector for expression in the bacterium L. lactis. MCF proteins expressed in L. lactis are folded correctly in the cytoplasmic membrane, and transport can be assayed directly in whole cells (31.Kunji E.R. Chan K.W. Slotboom D.J. Floyd S. O'Connor R. Monné M. Eukaryotic membrane protein overproduction in Lactococcus lactis.Curr. Opin. Biotechnol. 2005; 16: 546-551Crossref PubMed Scopus (56) Google Scholar). The presence of Pic2 in L. lactis induced with nisin was confirmed by Western blotting using a Pic2-specific anti" @default.
• W2087946910 created "2016-06-24" @default.
• W2087946910 creator A5026524021 @default.
• W2087946910 creator A5048383846 @default.
• W2087946910 creator A5052419395 @default.
• W2087946910 creator A5060468901 @default.
• W2087946910 date "2013-08-01" @default.
• W2087946910 modified "2023-10-17" @default.
• W2087946910 title "Copper Import into the Mitochondrial Matrix in Saccharomyces cerevisiae Is Mediated by Pic2, a Mitochondrial Carrier Family Protein" @default.
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