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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.
Open AccessPublished:October 12, 2001DOI:https://doi.org/10.1074/jbc.M105296200
      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.

      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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