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A Fraction of Yeast Cu,Zn-Superoxide Dismutase and Its Metallochaperone, CCS, Localize to the Intermembrane Space of Mitochondria

A PHYSIOLOGICAL ROLE FOR SOD1 IN GUARDING AGAINST MITOCHONDRIAL OXIDATIVE DAMAGE*
  • Lori A. Sturtz
    Footnotes
    Affiliations
    From the Department of Environmental Health Sciences, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland 21205
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  • Kerstin Diekert
    Affiliations
    Institut fur Zytobiologie der Philipps-Universitat Marburg, Robert-Koch-Str. 5, 35033 Marburg, Germany
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  • Laran T. Jensen
    Affiliations
    From the Department of Environmental Health Sciences, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland 21205
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  • Roland Lill
    Affiliations
    Institut fur Zytobiologie der Philipps-Universitat Marburg, Robert-Koch-Str. 5, 35033 Marburg, Germany
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  • Valeria Cizewski Culotta
    Correspondence
    To whom correspondence should be addressed: Johns Hopkins University, 615 N. Wolfe Street, Room 7032, Baltimore, MD 21205. Tel.: 410-955-3029; Fax: 410-955-0116;
    Affiliations
    From the Department of Environmental Health Sciences, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland 21205
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  • Author Footnotes
    * This work was supported in part by the Johns Hopkins University NIEHS center, by National Institutes of Health Grant GM50016 (to V. C.), and by funding from Sonderforschungsbereich 286 of the Deutsche Forschungsgemeinschaft, the Volkswagen-Stiftung, and Chemischen Industrie (to R. L.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    § Supported by National Institutes of Health Training Grant ES 07141.
      Cu,Zn-superoxide dismutase (SOD1) is an abundant, largely cytosolic enzyme that scavenges superoxide anions. The biological role of SOD1 is somewhat controversial because superoxide is thought to arise largely from the mitochondria where a second SOD (manganese SOD) already resides. Using bakers' yeast as a model, we demonstrate that Cu,Zn-SOD1 helps protect mitochondria from oxidative damage, as sod1Δ mutants show elevated protein carbonyls in this organelle. In accordance with this connection to mitochondria, a fraction of active SOD1 localizes within the intermembrane space (IMS) of mitochondria together with its copper chaperone, CCS. Neither CCS nor SOD1 contains typical N-terminal presequences for mitochondrial uptake; however, the mitochondrial accumulation of SOD1 is strongly influenced by CCS. When CCS synthesis is repressed, mitochondrial SOD1 is of low abundance, and conversely IMS SOD1 is very high when CCS is largely mitochondrial. The mitochondrial form of SOD1 is indeed protective against oxidative damage because yeast cells enriched for IMS SOD1 exhibit prolonged survival in the stationary phase, an established marker of mitochondrial oxidative stress. Cu,Zn-SOD1 in the mitochondria appears important for reactive oxygen physiology and may have critical implications for SOD1 mutations linked to the fatal neurodegenerative disorder, amyotrophic lateral sclerosis.
      ROS
      reactive oxygen species
      SOD
      superoxide dismutase
      IMS
      intermembrane space
      PMS
      postmitochondrial supernatant
      DNP
      dinitrophenol
      DNPH
      dinitrophenylhydrazine
      CCHL
      cytochrome c heme lyase
      ER
      endoplasmic reticulum
      Oxygen is required for the sustenance of aerobic life. However, through sequential one-electron reduction of oxygen, its metabolism may lead to the production of reactive oxygen species (ROS)1 such as superoxide radical, hydrogen peroxide, and hydroxyl radical. When left unchecked, these can cause damage to DNA, lipids, and protein (
      • Fridovich I.
      ,
      • Fridovich I.
      ,
      • Halliwell B.
      ,
      • Berlett B.S.
      • Stadtman E.R.
      ,
      • Stadtman E.R.
      • Levine R.L.
      ). One primary source of superoxide is the electron transport chain located in the inner membrane of mitochondria; ∼2% of the oxygen consumed during respiration is incompletely reduced to ROS such as superoxide (
      • Boveris A.
      • Oshino N.
      • Chance B.
      ). Two sites of the respiratory chain have been implicated, including the flavin mononucleotide contained within the NADH dehydrogenase and the ubisemiquinone anion of the cytochrome bc1complex (
      • Turrens J.F.
      • Boveris A.
      ,
      • Turrens J.F.
      • Alexandre A.
      • Lehninger A.L.
      ,
      • Zhang L.
      • Yu L.
      • Yu C.-A.
      ).
      A first line of defense against ROS include the superoxide dismutase (SOD) enzymes that catalyze the disproportionation of superoxide to hydrogen peroxide and water (
      • Fridovich I.
      ,
      • McCord J.M.
      • Fridovich I.
      ). Eukaryotes possess two intracellular SODs. A copper- and zinc-containing enzyme represents 90% of the total SOD activity and is located primarily in the cytosol (
      • Crapo J.D.
      • Oury T.
      • Rabouille C.
      • Slot J.W.
      • Chang L.Y.
      ). The other SOD (SOD2) is a manganese-containing enzyme located in the mitochondrial matrix. SOD2 is believed to represent the major means of protection against mitochondrial superoxide. However, the sources of ROS relevant to SOD1 are less clear because of the abundant location of this enzyme within the cytosol.
      In the case of SOD1, insertion of the copper cofactor in vivo requires an accessory protein, the so-called copper chaperone for superoxide dismutase or CCS (
      • Lyons T.J.
      • Nerissian A.
      • Goto J.J.
      • Zhu H.
      • Gralla E.B.
      • Valentine J.S.
      ,
      • Culotta V.C.
      • Klomp L.
      • Strain J.
      • Casareno R.
      • Krems B.
      • Gitlin J.D.
      ,
      • Wong P.C.
      • Waggoner D.
      • Subramaniam J.R.
      • Tessarollo L.
      • Bartnikas T.B.
      • Culotta V.C.
      • Price D.L.
      • Rothstein J.
      • Gitlin J.D.
      ). This protein was first discovered in the yeast Saccharomyces cerevisiae as the product of the LYS7 gene (
      • Culotta V.C.
      • Klomp L.
      • Strain J.
      • Casareno R.
      • Krems B.
      • Gitlin J.D.
      ) and has since been identified in a wide array of eukaryotes reviewed in Ref.
      • O'Halloran T.V.
      • Culotta V.C.
      . CCS interacts with SOD1 and directly inserts copper into the active site of the enzyme (
      • Casareno R.L.
      • Waggoner D.
      • Gitlin J.D.
      ,
      • Rae T.D.
      • Schmidt P.J.
      • Pufhal R.A.
      • Culotta V.C.
      • O'Halloran T.V.
      ,
      • Schmidt P.
      • Rae T.D.
      • Pufahl R.A.
      • Hamma T.
      • Strain J.
      • O'Halloran T.V.
      • Culotta V.C.
      ,
      • Schmidt P.
      • Kunst C.
      • Culotta V.C.
      ,
      • Rae T.D.
      • Torres A.S.
      • Pufahl R.A.
      • O'Halloran T.V.
      ,
      • Lamb A.L.
      • Torres A.S.
      • O'Halloran T.V.
      • Rosenzweig A.C.
      ). As expected, CCS co-localizes with SOD1 largely within the cytosol (
      • Culotta V.C.
      • Klomp L.
      • Strain J.
      • Casareno R.
      • Krems B.
      • Gitlin J.D.
      ,
      • Rothstein J.D.
      • Dykes-Hoberg M.
      • Corson L.B.
      • Becker M.
      • Cleveland D.W.
      • Price D.
      • Culotta V.C.
      • Wong P.C.
      ).
      Although considered primarily a “cytosolic” enzyme, SOD1 has also been identified in peroxisomes, lysosomes, and the nucleus (
      • Chang L.
      • Slot J.W.
      • Geuza H.J.
      • Crapo J.D.
      ,
      • Keller G.A.
      • Warner T.G.
      • Steimer K.S.
      • Hallewell R.A.
      ,
      • Geller B.L.
      • Winge D.R.
      ). Moreover, Fridovich and colleagues reported that a fraction of Cu,Zn-SOD localizes within mitochondria (
      • Weisiger R.A.
      • Fridovich I.
      ). Although an intriguing notion, a mitochondrial form of Cu,Zn-SOD was subjected to debate because of the possible contamination of mitochondrial preparations with other organelles (
      • Geller B.L.
      • Winge D.R.
      ).
      In the present study, we have revisited the possible mitochondrial localization of Cu,Zn-SOD using yeast as a model system. We demonstrate here that a fraction of both Cu,Zn-SOD and its metallochaperone, CCS, localize to the intermembrane space (IMS) of mitochondria. Interestingly, the accumulation of yeast Cu,Zn-SOD within mitochondria is greatly affected by the mitochondrial form of CCS. Evidence is provided herein for a physiological role of Cu,Zn-SOD in protecting against mitochondrial oxidative damage.

      MATERIALS AND METHODS

       Yeast Strains and Culture Conditions

      Most of the yeast strains were derived from EG103 (MATα leu2–3, 112 his3Δ1 trp1–289 ura3–52) (
      • Liu X.F.
      • Elashvili I.
      • Gralla E.B.
      • Valentine J.S.
      • Lapinskas P.
      • Culotta V.C.
      ) and include KS107 (EG103sod1Δ::TRP1) (
      • Slekar K.H.
      • Kosman D.
      • Culotta V.C.
      ), LS101 (EG103sod1Δ::TRP1 lys7Δ::URA3) and AS001 (EG103sod2Δ::URA3). LS101 was derived from the sod1Δ strain KS107 by disrupting LYS7(encoding CCS) with plasmid pPS003 (
      • Schmidt P.J.
      • Ramos-Gomez M.
      • Culotta V.C.
      ). SOD2 was disrupted in strain AS001 using plasmid pGSOD2 (
      • Culotta V.C.
      • Klomp L.
      • Strain J.
      • Casareno R.
      • Krems B.
      • Gitlin J.D.
      ). The lys7Δstrain SY2950 was previously described (MATα leu2–3, 112 ura-523 ade1 his3 lys7Δ::LEU2) (
      • Horecka J.
      • Kinsey P.T.
      • Sprague G.F.
      ), as was W303 (MATA ura3–1 ade2–1 trp1–1 his3–11, 15 leu2–3, 112 CANR) (
      • Diekert K.
      • de Kroon A.I.P.M.
      • Kispal G.
      • Lill R.
      ). Cells were propagated at 30 °C in enriched yeast extract, peptone-based medium with 2% glucose (YPD) or 2% raffinose (YPR), or in minimal SD (synthetic dextrose) medium (
      • Sherman F.
      • Fink G.R.
      • Lawrence C.W.
      ).
      To monitor cell viability in the stationary phase, strains were inoculated at an A600 = 0.1 in 1.5 ml of liquid SD medium and allowed to reach stationary phase by aerobic growth at 30 °C for 24 h. Incubation was continued aerobically for up to 5 days at 30 °C. At designated times the viability of serially diluted cells was examined by anaerobic growth on YPD plates.
      The methionine repression experiments utilized the SY2950 background because our studies indicated that this strain provided the strongest repression of the MET25 promoter. For methionine repression experiments, strain SY2950 (
      • Horecka J.
      • Kinsey P.T.
      • Sprague G.F.
      ) was transformed with the URA3 CEN plasmid pPS029-encoding sequences for yeast CCS (theLYS7 gene) under the control of the methionine-repressibleMET25 promoter (
      • Mumberg D.
      • Muller R.
      • Funk M.
      ). Gene repression was achieved by growth in SD medium containing 4.0 mm methionine.

       Plasmids

      To create vectors for expressing wild type yeast CCS and a cytochrome b2-CCS fusion, nucleotides −453 to +1011 of the yeast LYS7 gene were removed from vector pSL2502 (
      • Horecka J.
      • Kinsey P.T.
      • Sprague G.F.
      ) by XbaI/HindIII digestion and were ligated into pBluescript (Stratagene), generating vector pLS100. By site-directed mutagenesis (Quikchange kit, Stratagene),BglII and NdeI sites were introduced at the translational start site for yeast CCS. The mitochondrial targeting sequence of S. cerevisiae cytochromeb2 (codons for amino acids 1 to 88) was amplified by polymerase chain reaction and inserted in-frame at the N terminus of yeast CCS at the BglII and NdeI sites, creating plasmid pLS116. Plasmids pLS100 and pLS116 were digested with SalI and NotI to mobilize the respective yeast CCS-containing inserts, which were then ligated into pRS313 (HIS3 CEN) (
      • Sikorski R.S.
      • Hieter P.
      ) generating vectors pLS113 and pLS117, respectively. The sequence integrity of the plasmids was verified by double-stranded DNA sequencing (Core facility, Johns Hopkins University).
      A SOD1 expression plasmid (pLS101) was obtained by liberating SOD1 gene sequences −674 to +584 as aBamHI/SalI fragment from vector pPS008 (
      • Schmidt P.
      • Kunst C.
      • Culotta V.C.
      ) and ligating this into pRS315 (LEU2 CEN) (
      • Sikorski R.S.
      • Hieter P.
      ).

       Biochemical Procedures

      Yeast cells grown to mid-late log phase in either YPD or YPR were converted to spheroplasts, and mitochondria and postmitochondrial supernatant (PMS) were isolated as described (
      • Daum G.
      • Bohni P.C.
      • Schatz G.
      ). The PMS fraction represents the supernatant from 12,000 × g centrifugation and is largely cytosolic. As needed, mitochondria were further purified by Nycodenz density gradient centrifugation (
      • Diekert K.
      • de Kroon A.I.P.M.
      • Kispal G.
      • Lill R.
      ,
      • Lewin A.S.
      • Hines V.
      • Small G.M.
      ). Subfractionation of mitochondria by hypotonic treatment (
      • Glick B.S.
      • Pon L.A.
      ) and salt extraction of intact mitochondria and mitochondria with ruptured outer membranes (
      • Diekert K.
      • de Kroon A.I.P.M.
      • Kispal G.
      • Lill R.
      ) followed published protocols. Outer membranes were selectively ruptured by swelling of the mitochondrial matrix during incubation in a hypotonic solution.
      Immunoblot analysis of yeast SOD1 and CCS proteins was conducted as described (
      • Schmidt P.
      • Rae T.D.
      • Pufahl R.A.
      • Hamma T.
      • Strain J.
      • O'Halloran T.V.
      • Culotta V.C.
      ). The integrity of mitochondrial and PMS fractions was monitored by antibodies directed against cytochromeb2 (IMS), Mas2p and Mge1p (mitochondrial matrix), cytochrome c1 heme lyase (IMS), Kar2p, Sec61p, and Sbh1p (endoplasmic reticulum), and Pgk1p (cytosol), as described (
      • Kispal G.
      • Csere P.
      • Prohl C.
      • Lill R.
      ). Detection required use of the ECL kit (Amersham Pharmacia Biotech). In the various experiments, either equal amounts of PMS and mitochondrial protein were analyzed (e.g. Figs. 1and 2) or the PMS and mitochondria were analyzed according to cell equivalents where 25- or 100-fold more cell equivalents of mitochondria were analyzed compared with the PMS for immunodetection of CCS or SOD1 (e.g. Figs. Figure 3, Figure 4, Figure 5). With either approach, roughly equivalent signals were obtained for PMS and mitochondrial CCS and SOD1, yielding an estimate of 1–5% of the total SOD1 and CCS that was mitochondrial.
      Figure thumbnail gr1
      Figure 1Yeast lacking Cu,Zn-SOD exhibit increased carbonylation of both cytosolic and mitochondrial proteins. Yeast cell lysates were prepared from EG103 (WT) and the isogenicsod1Δ strain KS107 grown aerobically and fractionated by differential centrifugation into PMS and mitochondria (Mito). 20 μg of protein from each were reacted with DNPH and DNPH-reactive carbonyl groups were detected by immunoblotting with an anti-DNP antibody. Arrows indicate protein bands enhanced in the sod1Δ strain.
      Figure thumbnail gr2
      Figure 2Yeast Cu,Zn-SOD and its metallochaperone, CCS, localize to the mitochondrial intermembrane space. A, yeast cell lysates were prepared from strain W303 and were fractionated as in Fig. . 50 μg of protein of each fraction were analyzed by immunostaining for yeast proteins SOD1, CCS, CC1HL, and cytosolic phosphoglycerol kinase (Pgk1p). B, mitochondria isolated in A(DGP−) were further purified by density gradient centrifugation (DGP+) and analyzed by immunostaining. Sec61p and Sbh1p are ER proteins both involved in ER protein translocation; Mge1p (molecular chaperone involved in mitochondrial import) is in the mitochondrial matrix.C, intact mitochondria (left) or mitochondria with ruptured outer membranes (right) were incubated in buffer containing 100 mm NaCl and then subjected to centrifugation to isolate the mitochondria or mitoplasts as indicated. The resultant pellet (P) and supernatant (S) fractions were analyzed by immunostaining. Cytochromeb2 and Mge1p are soluble mitochondrial IMS and matrix proteins, respectively. Kar2p, a molecular chaperone in the ER lumen, is not solubilized, indicating that microsomes remain intact.
      Figure thumbnail gr3
      Figure 3The fraction of mitochondrial Cu,Zn-SOD is active. Mitochondria and PMS fractions were prepared from wild type (WT) (EG103) and isogenic sod2Δ (AS001) strains. A, SOD activity was assayed by native gel electrophoresis and nitro blue tetrazolium staining; positions of SOD1 and SOD2 are indicated. B, fractions were analyzed for SOD1 protein levels by immunostaining.
      Figure thumbnail gr4
      Figure 4CCS influences the partitioning of Cu,Zn-SOD between the cytosol and mitochondria. A, thelys7Δ strain SY2950 was transformed with plasmid pPS029 expressing yeast CCS under the control of the methionine-repressibleMET25 promoter. Cells were grown in the absence or presence of 4.0 mm methionine, and mitochondrial and PMS levels of SOD1 and CCS were analyzed by immunostaining. B andC, strain LS101 was transformed with the yeast SOD1 expression plasmid pLS101 and with either pLS113 expressing wild type yeast CCS or with pLS117 expressing the cytochromeb2-CCS fusion. Cells were grown in YPR, and either whole cell lysates (C) or PMS, mitochondrial, IMS, and matrix fractions (B) were analyzed by immunoblotting as in A. B, top, shows that in cells expressing the cytochrome b2-CCS fusion, the cytochrome b2 portion of the fusion was effectively processed. Cyt b2 = immunoblot analysis of cytochrome b2, an IMS marker. For analysis of SOD1 and CCS, PMS was diluted 1:100 and 1:25, respectively. The apparent increase in molecular weight of mitochondrial CCS (as inA) is not seen in all experiments and may represent proteolytic cleavage of the cytosolic protein.
      Figure thumbnail gr5
      Figure 5Mitochondrial Cu,Zn-SOD protects against cell death in stationary phase. A and B, mitochondrial and PMS fractions were obtained from the cells described in Fig. , B and C, but grown in YPD medium.A, fractions were analyzed for SOD activity as in Fig.B. B, mitochondria were further fractionated into IMS and matrix, and fractions were subjected to immunoblot analysis. Cyt b2, IMS marker; Mas2p, mitochondrial peptidase in the matrix. native SOD1, cells expressing wild type yeast CCS and exhibiting normal distribution of SOD1 between the mitochondria and PMS; IMS-enriched SOD1, cells expressing the cytochrome b2-CCS fusion and exhibiting high levels of mitochondrial SOD1. C, the KS107 (sod1Δ) strain transformed where indicated with the SOD1 expression plasmid (WT) was grown aerobically for 2 days in stationary phase and viability-tested by spotting 1 × 105, 1 × 104, 1 × 103, and 1 × 102 cells onto YPD plates. D, stationary phase survival at 2 or 5 days was measured as inC using the cells described in A.
      SOD enzymatic activity in mitochondrial and PMS fractions was monitored by non-denaturing gel electrophoresis and nitro blue tetrazolium staining as described (
      • Schmidt P.
      • Rae T.D.
      • Pufahl R.A.
      • Hamma T.
      • Strain J.
      • O'Halloran T.V.
      • Culotta V.C.
      ,
      • Flohe L.
      • Otting F.
      ). Samples contained 0.5% Tween 20 and were filtered through a 0.22-μm membrane prior to electrophoresis.
      Carbonylated proteins were detected using the OxyBlot kit (Intergen). Briefly, lysates were prepared from cells grown by shaking in YPD medium to log phase and were separated into PMS and mitochondrial fractions. 20 μg of protein from each were reacted with 2,4-dinitrophenylhydrazine (DNPH) for 15 min at 25 °C. Samples were resolved on 12% denaturing polyacrylamide gels, and DNP-derivatized proteins were identified by immunoblot using an anti-DNP antibody.

      RESULTS

       Yeast Cu,Zn-SOD1 Guards Mitochondrial Proteins Against Oxidative Damage

      During oxidative stress, proteins are susceptible to metal-catalyzed oxidation, resulting in formation of protein carbonyl groups (
      • Stadtman E.R.
      ). To address whether Cu,Zn-SOD1 can protect against this type of oxidative damage, we monitored protein carbonylation in aerobically grown wild type and sod1Δ mutant yeast. In Fig. 1, cell lysates were resolved into mitochondrial and PMS (largely cytosolic) fractions; protein carbonyls were then derivatized to dinitrophenol (DNP) hydrazone and detected by immunoblotting with an anti-DNP antibody. Consistent with previous studies (
      • Cabiscol E.
      • Piulats E.
      • Echave P.
      • Herrero E.
      • Ros J.
      ), wild type cells exhibited limited protein oxidation (Fig.1). In the sod1Δ strain, a number of protein species in the PMS showed evidence for increased oxidative damage (Fig. 1,arrows), as expected for strains lacking cytosolic SOD. Surprisingly, however, oxidation of mitochondrial proteins was also elevated in the sod1Δ strain (Fig. 1). As one possibility, a fraction of SOD1 may actually reside within the mitochondria, as originally proposed by Weisiger and Fridovich (
      • Weisiger R.A.
      • Fridovich I.
      ).

       Yeast SOD1 and Its Copper Chaperone Are in the Mitochondrial IMS

      To address the localization of yeast Cu,Zn-SOD, cell lysates were fractionated into mitochondrial and PMS fractions, and equal protein amounts from each were analyzed by immunoblotting. As expected, both yeast Cu,Zn-SOD and the CCS copper chaperone were observed in the PMS (Fig. 2A), consistent with the predominant cytosolic localization of these proteins (
      • Crapo J.D.
      • Oury T.
      • Rabouille C.
      • Slot J.W.
      • Chang L.Y.
      ,
      • Culotta V.C.
      • Klomp L.
      • Strain J.
      • Casareno R.
      • Krems B.
      • Gitlin J.D.
      ). However, both polypeptides were also detected in mitochondria (Fig.2A). To verify the mitochondrial localization of SOD1 and CCS, crude mitochondria were purified using Nycodenz density gradient centrifugation (
      • Glick B.S.
      • Pon L.A.
      ). As shown in Fig. 2B, yeast SOD1 and CCS co-purified with mitochondrial markers (Mge1p and CC1HL) following gradient purification, whereas the endoplasmic reticulum markers Sec61p and Sbh1p were largely removed. Additionally, the vacuolar marker carboxypeptidase Y was selectively depleted by this treatment (not shown). To exclude the possibility that yeast SOD1 and CCS adhere nonspecifically to mitochondria, a salt extraction was performed. Treatment of intact mitochondria with 100 mmNaCl failed to release yeast SOD1 and CCS from this organelle (Fig.2C). However, when the outer membrane of the mitochondria was ruptured prior to salt treatment, SOD1, CCS, and the IMS protein cytochrome b2 were all released into the supernatant. Therefore, yeast Cu,Zn-SOD and its copper chaperone CCS are located inside the mitochondria, specifically in the IMS fraction, as they are only solubilized upon breakage of the outer membrane. The localization of SOD1 and CCS to mitochondria is highly reproducible and has been observed with three different strain backgrounds (used in Figs. 2, 3, and 4) and under a variety of growth conditions including both fermentable and non-fermentable carbon sources.
      We estimate that ≈1–5% of total yeast SOD1 and its copper chaperone are located within the mitochondria (see “Materials and Methods”). At first glance, this may seem to be a low amount; however, when considering the very small volume of the mitochondrial IMS,
      The volume of the IMS is difficult to measure. However, considering that mitochondria represent approximately 10% of the cell volume and that the IMS portion can be no more than 5% of this, the IMS is likely to represent ≤0.5% of the total cell volume.
      SOD1 and CCS actually appear to be quite concentrated in the mitochondrial compartment.
      To test whether mitochondrial SOD1 is active, PMS and mitochondrial fractions were analyzed for superoxide scavenging activity by native gel electrophoresis and nitro blue tetrazolium) staining (
      • Flohe L.
      • Otting F.
      ). An activity band corresponding to SOD1 was observed in both the PMS and mitochondrial fractions (Fig.3A). The activity in mitochondria is not due to manganese SOD2, as the activity band corresponding to SOD1 was not affected by a sod2Δmutation.
      How does copper reach CCS and SOD1 in the mitochondria? Copper activation of mitochondrial cytochrome oxidase relies on Cox17p, a protein that appears to deliver copper from the cytosol to the mitochondrial IMS (
      • Beers J.
      • Glerum D.M.
      • Tzagoloff A.
      ,
      • Glerum D.M.
      • Shtanko A.
      • Tzagoloff A.
      ,
      • Heaton D.
      • Nittis T.
      • Srinivasan C.
      • Winge D.R.
      ,
      • Srinivasan C.
      • Posewitz M.C.
      • George G.N.
      • Winge D.R.
      ). However, we noted that in acox17Δ mutant, mitochondrial Cu,Zn-SOD still retained activity (data not shown). Therefore copper activation of mitochondrial SOD1 and cytochrome oxidase appear to occur through separate pathways.

       The Role of the Copper Chaperone in Mitochondrial Accumulation of SOD1

      In a lys7Δ yeast strain that lacks CCS, the mitochondrial form of Cu,Zn-SOD was difficult to detect (not shown), suggesting that CCS may control mitochondrial accumulation of SOD1. To more rigorously address this, we manipulated yeast CCS levels by placing the corresponding gene under the control of the methionine-repressible MET25 promoter (
      • Mumberg D.
      • Muller R.
      • Funk M.
      ). As seen in Fig.4A (top), growth in the presence of methionine resulted in greater than 90% depletion of CCS in cells expressing the MET25-CCS fusion. Mitochondrial levels of CCS seemed particularly affected (Fig. 4A,top). Notably, this lowering of CCS levels resulted in a dramatic loss of mitochondrial SOD1, whereas cytosolic SOD1 was largely unaffected (Fig. 4A, bottom). To address further the impact of CCS on mitochondrial SOD1, we conducted the converse study and greatly increased mitochondrial CCS by introducing the targeting sequence of cytochrome b2 at its N terminus. As seen in Fig. 4B (top), this cytochrome b2-CCS fusion was properly targeted to the mitochondrial IMS. Notably, this increase in mitochondrial CCS resulted in a dramatic increase of SOD1 within the mitochondrial IMS (Fig. 4B, bottom). The expression of cytochromeb2-CCS did not alter total levels of SOD1 (Fig.4C), only its localization. This effect of targeting CCS to the IMS on mitochondrial SOD1 is highly reproducible and has been observed under numerous growth conditions. Together, these results demonstrate that the partitioning of Cu,Zn-SOD between the cytosol and mitochondria is greatly influenced by the cellular location of its copper chaperone, CCS.

       A Physiological Role for Mitochondrial SOD1 in Guarding Against Oxidative Damage

      We estimated that yeast cells expressing the cytochrome b2-CCS fusion (as in Fig. 4) have ∼20-fold higher levels of mitochondrial SOD1. This elevation in mitochondrial SOD1 protein and SOD1 activity was seen when cells were grown either under non-fermentable carbon sources (Fig. 4B) or when cells were grown in glucose (Fig.5, A and B). These cells provided us with a unique opportunity to investigate the role of mitochondrial Cu,Zn-SOD in guarding against oxidative stress.
      Yeast sod1Δ mutants exhibit a number of metabolic defects when grown aerobically in glucose-containing medium. For example,sod1Δ cells show oxygen-dependent blocks in methionine and lysine biosynthesis (
      • Chang E.
      • Kosman D.
      ,
      • Bilinski T.
      • Krawiec Z.
      • Liczmanski L.
      • Litwinska J.
      ), and we noted that both defects are complemented in cells expressing high levels of IMS SOD1 (not shown). Additionally, sod1Δ mutants exhibit rapid death in the stationary phase (
      • Longo V.D.
      • Gralla E.B.
      • Valentine J.S.
      ). As seen in Fig. 5C, the viability of our sod1Δ mutant after only 2 days in stationary phase is two to three orders of magnitude lower than that of a corresponding wild type cell. This poor survival is complemented by the IMS-enriched form of SOD1 (Fig. 5D, left). More importantly, cells expressing high levels of IMS SOD1 showed improved viability in stationary phase when compared with a wild type yeast cell. After 5 days in stationary phase, viability of the IMS-enriched SOD1 cells was 1–2 orders of magnitude greater than that of wild type cells expressing native SOD1 (Fig. 5D,right). The effect of increasing mitochondrial SOD1 on yeast stationary phase viability is very reproducible over numerous experimental trials. It is noteworthy that death in the stationary phase has been correlated with increased ROS production from the mitochondria (
      • Jakubowski W.
      • Bilinski T.
      • Bartosz G.
      ,
      • Longo V.D.
      • Liou L.
      • Valentine J.S.
      • Gralla E.B.
      ) and with the aging process in yeast (
      • Ashrafi K.
      • Sinclair D.
      • Gordon J.L.
      • Guarente L.
      ). As such, the observed increase in survival of cells expressing IMS-enriched SOD1 strongly indicates that mitochondrial SOD1 performs a physiological role in scavenging mitochondrial ROS. This may be particularly relevant for long term survival of the cell.

      DISCUSSION

      Eukaryotic cells express two distinct intracellular SOD enzymes: the mitochondrial Mn-SOD and the largely cytosolic Cu,Zn-SOD. Based on studies in yeast, Cu,Zn-SOD curiously seems to protect both cytosolic and mitochondrial components from oxidative damage. Yeast cells lacking Cu,Zn-SOD show defects in the cytosolic methionine biosynthetic pathway (
      • Slekar K.H.
      • Kosman D.
      • Culotta V.C.
      ,
      • Chang E.
      • Kosman D.
      ), yet also exhibitmitochondrial defects including poor growth on non-fermentable carbon sources (
      • Longo V.D.
      • Gralla E.B.
      • Valentine J.S.
      ), a deficiency in mitochondrial aconitase (
      • Strain J.
      • Lorenz C.R.
      • Bode J.
      • Smolen G.A.
      • Garland S.A.
      • Vickery L.E.
      • Culotta V.C.
      ), and rapid death in stationary phase (
      • Longo V.D.
      • Gralla E.B.
      • Valentine J.S.
      ). Herein, we provide direct biochemical evidence that Cu,Zn-SOD protects against mitochondrial oxidative damage. In particular, yeast cells lacking Cu,Zn-SOD show evidence of increased carbonylation damage to mitochondrial proteins when compared with their wild type counterparts. Although the identity of these carbonylated proteins is presently unknown, they may be analogous to those damaged in yeast mitochondria following exposure to menedione (
      • Cabiscol E.
      • Piulats E.
      • Echave P.
      • Herrero E.
      • Ros J.
      ). In any case, the evidence points to a striking connection between so-called cytosolic SOD1 and the mitochondria.
      We noted that a fraction of both yeast Cu,Zn-SOD and its metallochaperone, CCS, reside within the mitochondrial IMS. Similar findings on yeast SOD1 and CCS have recently been observed by Gralla and colleagues.
      E. Gralla, personal communication.
      The notion of a mitochondrial SOD1 is not unique to fungi. Mammalian SOD1 has recently been observed in the mitochondria of mammalian neuronal tissues,
      Z. Xu, manuscript in preparation.
      and the mitochondrial form of mammalian SOD1 is active.
      Okado-Matsumoto, A., and Fridovich, I. (2001)J. Biol. Chem. 276, in press.
      Furthermore, human Cu,Zn-SOD expressed in yeast partitions similarly between the cytosol and mitochondria.
      P. J. Schmidt and V. C. Culotta, unpublished observations.
      The accumulation of SOD1 within the mitochondrial IMS is strongly influenced by its copper chaperone, CCS. Cu,Zn-SOD accumulates poorly in the mitochondria of yeast depleted of CCS, and conversely SOD1 shows high mitochondrial accumulation when IMS CCS is abundant. This effect of CCS on mitochondrial SOD1 is remarkably similar to how cytochromec heme lyase (CCHL) affects the uptake of cytochromec into the mitochondrial IMS. CCHL catalyzes insertion of the heme cofactor into cytochrome c, and studies in the yeast Neurospora crassa have shown a direct correlation between the levels of mitochondrial CCHL and accumulation of cytochromec in the IMS (
      • Mayer A.
      • Neupert W.
      • Lill R.
      ,
      • Dumont M.E.
      • Ernst J.F.
      • Sherman F.
      ,
      • Dumont M.E.
      • Cardillo T.S.
      • Hayes M.K.
      • Sherman F.
      ,
      • Nargang F.E.
      • Drygas M.E.
      • Kwong P.L.
      • Nicholson D.W.
      • Neupert W.
      ). Like apocytochrome c (
      • Mayer A.
      • Neupert W.
      • Lill R.
      ), the apo form of SOD1 may reversibly pass through the mitochondrial outer membrane. Once in the IMS, SOD1 may become trapped through association with CCS, because the two proteins can interact (
      • Casareno R.L.
      • Waggoner D.
      • Gitlin J.D.
      ,
      • Schmidt P.
      • Kunst C.
      • Culotta V.C.
      ,
      • Lamb A.L.
      • Torres A.S.
      • O'Halloran T.V.
      • Rosenzweig A.C.
      ). CCS may therefore serve as a “trans side” receptor, as postulated for CCHL function during import of apocytochromec (
      • Mayer A.
      • Neupert W.
      • Lill R.
      ). Furthermore, metallation of SOD1 by mitochondrial CCS may shift the equilibrium of the import reaction further and prevent retrotranslocation. Conversely, the bulk metallation of SOD1 in the cytosol may preclude its entry into mitochondria, explaining the degree of partitioning of SOD1 between cytosolic and mitochondrial pools. The polypeptide sequences that mediate mitochondrial localization of SOD1 and CCS are not obvious, because neither contain typical N-terminal presequences. However, not all IMS proteins utilize such presequences, e.g. cytochrome c and CCHL (
      • Nye S.H.
      • Scarpulla R.C.
      ,
      • Wang W.
      • Dumont M.E.
      • Sherman F.
      ,
      • Diekert K.
      • Kispal G.
      • Guiard B.
      • Lill R.
      ).
      What is the physiological role of Cu,Zn-SOD in the mitochondrial IMS? The respiratory chain NADH dehydrogenase and ubisemiquinone anion can both release superoxide to the mitochondrial matrix (
      • Turrens J.F.
      • Boveris A.
      ,
      • Turrens J.F.
      • Alexandre A.
      • Lehninger A.L.
      ,
      • Zhang L.
      • Yu L.
      • Yu C.-A.
      ). However, the ubisemiquinone anion can also effect release of superoxide in the IMS (
      • Zhang L.
      • Yu L.
      • Yu C.-A.
      ,
      • Trumpower B.L.
      ). Nevertheless, experimental evidence for the exit of superoxide from intact mitochondria has been difficult to obtain, and it has been proposed that this is because of the short half-life of the radical (
      • Zhang L.
      • Yu L.
      • Yu C.-A.
      ,
      • Loschen G.
      • Azzi A.
      • Richter C.
      • Flohe L.
      ). As an alternative explanation, Cu,Zn-SOD in the IMS may preclude exit of mitochondrial superoxide, and as such, protect extramitochondrial cell components from oxidative damage. The IMS form of Cu,Zn-SOD indeed protects against mitochondrially derived oxidants. Specifically, yeast cells expressing high levels of IMS SOD1 display prolonged survival during the stationary phase. In stationary phase, yeast mitochondria exhibit a burst of ROS production (
      • Jakubowski W.
      • Bilinski T.
      • Bartosz G.
      ,
      • Longo V.D.
      • Liou L.
      • Valentine J.S.
      • Gralla E.B.
      ), and yeast undergo rapid aging during this stage (
      • Ashrafi K.
      • Sinclair D.
      • Gordon J.L.
      • Guarente L.
      ). Survival during yeast stationary phase can be prolonged through expression of human Bcl-2, an anti-apoptosis protein (
      • Longo V.D.
      • Ellerby L.M.
      • Bredesen D.E.
      • Valentine J.S.
      • Gralla E.B.
      ). Our observed role of IMS SOD1 in protecting against stationary phase death strongly suggests that mitochondrial SOD1 helps promote long term survival of the aerobic cell.
      The presence of Cu,Zn-SOD in the mitochondria may be relevant to amyotrophic lateral sclerosis (ALS), a fatal, adult-onset neurodegenerative disease. A fraction of inherited ALS cases (familial ALS or FALS) are due to toxic gain of function mutations in human Cu,Zn-SOD (
      • Deng H.X.
      • Hentati A.
      • Tainer J.A.
      • Iqbal Z.
      • Cayabyabi A.
      • Hung W.Y.
      • Getzoff E.D.
      • Hu P.
      • Herzfeld B.
      • Roos R.P.
      • Warner C.
      • Deng G.
      • Soriano E.
      • Smyth C.
      • Parge H.
      • Ahmed A.
      • Roses A.D.
      • Hallewell R.A.
      • Pericak-Vance M.A.
      • Siddique T.
      ,
      • Gurney M.E.
      • Pu H.
      • Chiu A.U.
      • Canto M.C.D.
      • Polchow C.Y.
      • Alexander D.D.
      • Caliendo J.
      • Hentati A.
      • Kwon Y.
      • Deng H.S.
      • Ehen W.
      • Zhai P.
      • Sufit R.L.
      • Siddique T.
      ). The mechanism by which SOD1 mutants cause motor neuron death is still unclear (
      • Cleveland D.W.
      • Liu J.
      ); however, many studies have implicated mitochondria. Various mitochondrial pathologies have been associated with ALS including damage to mitochondrial DNA, defects in respiratory chain enzymes, and abnormal mitochondrial morphology (
      • Beal M.F.
      ,
      • Dhaliwal G.K.
      • Grewal R.P.
      ,
      • Borthwick G.M.
      • Johnson M.A.
      • Ince P.G.
      • Shaw P.J.
      • Turnbull D.M.
      ,
      • Wong P.C.
      • Pardo C.A.
      • Borchelt D.R.
      • Lee M.k.
      • Copeland N.G.
      • Jenkins N.J.
      • Sisodia S.S.
      • Cleveland D.W.
      • Price D.L.
      ,
      • Canto M.C.D.
      • Gurney M.E.
      ). It has been proposed that disease involves an interplay between oxidative damage and mitochondrial dysfunction (
      • Klivenyi P.
      • Ferrante R.J.
      • Matthews R.T.
      • Bogdanov M.B.
      • Klein A.M.
      • Andreassen O.A.
      • Mueller G.
      • Wermer M.
      • Kaddurah-Daouk R.
      • Beal M.F.
      ). As such, the fraction of mutant SOD1 molecules that reside within the mitochondria may be critical in the etiology of FALS.

      ACKNOWLEDGEMENTS

      We thank A. Sullivan for strain AS001, P. Schmidt for plasmid pPS029, and D. Winge for thecox17Δ strain. Antibodies were generous gifts of D. Kosman (yeast SOD1), T. O'Halloran (yeast CCS), and R. Jensen (cytochrome b2 and Mas2p). We also thank R. Jensen, R. Poyton, and D. Winge for thoughtful discussions.

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