Manganese Activation of Superoxide Dismutase 2 in the Mitochondria of Saccharomyces cerevisiae

Manganese-dependent superoxide dismutase 2 (SOD2) in the mitochondria plays a key role in protection against oxidative stress. Here we probed the pathway by which SOD2 acquires its manganese catalytic cofactor. We found that a mitochondrial localization is essential. A cytosolic version of Saccharomyces cerevisiae Sod2p is largely apo for manganese and is only efficiently activated when cells accumulate toxic levels of manganese. Furthermore, Candida albicans naturally produces a cytosolic manganese SOD (Ca SOD3), yet when expressed in the cytosol of S. cerevisiae, a large fraction of Ca SOD3 also remained manganese-deficient. The cytosol of S. cerevisae cannot readily support activation of Mn-SOD molecules. By monitoring the kinetics for metalation of S. cerevisiae Sod2p in vivo, we found that prefolded Sod2p in the mitochondria cannot be activated by manganese. Manganese insertion is only possible with a newly synthesized polypeptide. Furthermore, Sod2p synthesis appears closely coupled to Sod2p import. By reversibly blocking mitochondrial import in vivo, we noted that newly synthesized Sod2p can enter mitochondria but not a Sod2p polypeptide that was allowed to accumulate in the cytosol. We propose a model in which the insertion of manganese into eukaryotic SOD2 molecules is driven by the protein unfolding process associated with mitochondrial import. Manganese-dependent superoxide dismutase 2 (SOD2) in the mitochondria plays a key role in protection against oxidative stress. Here we probed the pathway by which SOD2 acquires its manganese catalytic cofactor. We found that a mitochondrial localization is essential. A cytosolic version of Saccharomyces cerevisiae Sod2p is largely apo for manganese and is only efficiently activated when cells accumulate toxic levels of manganese. Furthermore, Candida albicans naturally produces a cytosolic manganese SOD (Ca SOD3), yet when expressed in the cytosol of S. cerevisiae, a large fraction of Ca SOD3 also remained manganese-deficient. The cytosol of S. cerevisae cannot readily support activation of Mn-SOD molecules. By monitoring the kinetics for metalation of S. cerevisiae Sod2p in vivo, we found that prefolded Sod2p in the mitochondria cannot be activated by manganese. Manganese insertion is only possible with a newly synthesized polypeptide. Furthermore, Sod2p synthesis appears closely coupled to Sod2p import. By reversibly blocking mitochondrial import in vivo, we noted that newly synthesized Sod2p can enter mitochondria but not a Sod2p polypeptide that was allowed to accumulate in the cytosol. We propose a model in which the insertion of manganese into eukaryotic SOD2 molecules is driven by the protein unfolding process associated with mitochondrial import. Superoxide dismutase (SOD) 1The abbreviations used are: SOD, superoxide dismutase; YPR, raffinose based medium; CCCP, carbonyl cyanide m-chlorophenylhydrazone; β-ME, β-mercaptoethanol. enzymes represent a family of metalloproteins that have evolved to catalytically remove toxic superoxide anions. Most eukaryotes express two distinct forms, a copper- and zinc-containing enzyme (SOD1) that largely resides in the cytosol (1McCord J.M. Fridovich I. J. Biol. Chem. 1969; 244: 6049-6055Abstract Full Text PDF PubMed Google Scholar) but is also found in the intermembrane space of mitochondria (2Okado-Matsumoto A. Fridovich I. J. Biol. Chem. 2001; 276: 38388-38393Abstract Full Text Full Text PDF PubMed Scopus (807) Google Scholar, 3Weisiger R.A. Fridovich I. J. Biol. Chem. 1973; 248: 4793-4796Abstract Full Text PDF PubMed Google Scholar, 4Sturtz L.A. Diekert K. Jensen L.T. Lill R. Culotta V.C. J. Biol. Chem. 2001; 276: 38084-38089Abstract Full Text Full Text PDF PubMed Google Scholar) and a second SOD that contains manganese (SOD2) and is typically localized in the mitochondrial matrix (3Weisiger R.A. Fridovich I. J. Biol. Chem. 1973; 248: 4793-4796Abstract Full Text PDF PubMed Google Scholar, 5Ravindranath S.D. Fridovich I. J. Biol. Chem. 1975; 250: 6107-6112Abstract Full Text PDF PubMed Google Scholar). In both cases, enzymatic activity is reliant on the redox cycling of the bound copper or manganese ion cofactor. Hence, the post-translational insertion of the metal represents a key step in controlling enzymatic activity in vivo. Much is known about the mechanism by which SOD1 acquires copper in vivo. Copper is transported and trafficked to the site of SOD1 by the concerted action of cell surface and intracellular copper transporters and a copper chaperone known as CCS (6Culotta V.C. Klomp L. Strain J. Casareno R. Krems B. Gitlin J.D. J. Biol. 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CCS can insert copper into a pre-existing apopool of SOD1 with no need for new protein synthesis (13Schmidt P. Kunst C. Culotta V.C. J. Biol. Chem. 2000; 275: 33771-33776Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 14Bartnikas T.B. Gitlin J.D. J. Biol. Chem. 2003; 278: 33602-33608Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The copper chaperone can also act on newly synthesized molecules of SOD1 (14Bartnikas T.B. Gitlin J.D. J. Biol. Chem. 2003; 278: 33602-33608Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). In either case, oxygen is required for CCS activity, providing a means for regulating SOD1 activity in response to oxygen status (15Furukawa Y. Torres A.S. O'Halloran T.V. EMBO J. 2004; 23: 2872-2881Crossref PubMed Scopus (293) Google Scholar). The delivery of manganese to SOD2 should also involve a carefully controlled trafficking system. Using yeast genetics, we have identified two membrane proteins that help deliver manganese to the enzyme. One is the divalent metal transporter Smf2p that localizes in intracellular vesicles (16Cohen A. Nelson H. Nelson N. J. Biol. Chem. 2000; 275: 33388-33394Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 17Portnoy M.E. Liu X.F. Culotta V.C. Mol. Cell Biol. 2000; 20: 7893-7902Crossref PubMed Scopus (180) Google Scholar). Saccharomyces cerevisiae cells lacking Smf2p accumulate very low levels of manganese and show defects in manganese requiring enzymes of the Golgi and in Sod2p of mitochondria (18Luk E. Culotta V.C. J. Biol. Chem. 2001; 276: 47556-47562Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). A second transporter that affects yeast Sod2p is Mtm1p, a member of the mitochondrial carrier family of proteins (19Luk E. Carroll M. Baker M. Culotta V.C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10353-10357Crossref PubMed Scopus (115) Google Scholar). Although the precise substrate for transport by Mtm1p is not known, Mtm1p is needed for proper insertion of manganese into mitochondrial Sod2p (19Luk E. Carroll M. Baker M. Culotta V.C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10353-10357Crossref PubMed Scopus (115) Google Scholar). Despite the identification of these components, the mechanistic details of the post-translational events associated with activation of eukaryotic SOD2 are still poorly understood. The protein is encoded in the nucleus, transported across two mitochondrial membranes and once inside the mitochondrial matrix, and the polypeptide folds into a tetrameric enzyme. The stage at which manganese is introduced is not known. For example, can manganese be inserted into a pre-existing pool of apoSOD2, as is the case with copper containing SOD1? In addition, it is not clear whether SOD2 requires a mitochondrial location to acquire its metal cofactor. In certain organisms, Mn-SODs can be activated outside the mitochondria. The pathogenic fungi Candida albicans (20Lamarre C. LeMay J.D. Deslauriers N. Bourbonnais Y. J. Biol. Chem. 2001; 276: 43784-43791Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) and the blue crab Callinectes sapidus (21Brouwer M. Hoexum Brouwer T. Grater W. Brown-Peterson N. Biochem. J. 2003; 374: 219-228Crossref PubMed Scopus (97) Google Scholar) both express Mn-SODs in the cytosol. When the Mn-SODs from either C. albicans or from the bacterium Bacillus stearothermophilus were targeted to the cytosol of S. cerevisiae, they exhibited certain activity (20Lamarre C. LeMay J.D. Deslauriers N. Bourbonnais Y. J. Biol. Chem. 2001; 276: 43784-43791Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 22Wei J.P. Srinivasan C. Han H. Valentine J.S. Gralla E.B. J. Biol. Chem. 2001; 276: 44798-44803Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 23Bowler C. Van Kaer L. Van Camp W. Van Montagu M. Inze D. Dhaese P. J. Bacteriol. 1990; 172: 1539-1546Crossref PubMed Google Scholar). These results alone would suggest that a mitochondrial location may not be essential for manganese activation of SOD2. In this study, we explored the pathway for inserting manganese into mitochondrial SOD2 using S. cerevisiae Sod2p as a model. We found that efficient metalation of Sod2p requires a mitochondrial localization of the protein; a cytosolic version of Sod2p is poorly activated with the metal. Furthermore, only newly synthesized molecules of Sod2p that are freshly imported into mitochondria can acquire the metal in vivo. Manganese cannot be readily inserted into a pool of Sod2p that is apo for manganese. We provide a model in which manganese insertion into Sod2p is driven by the protein unfolding process associated with mitochondrial import. Yeast Strains and Growth Conditions—The yeast strains used in this study, including the wild-type BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) and the isogenic smf2Δ::kanMX4 (1878) and sod2Δ::kanMX4 (6605) mutant variants, were purchased from Research Genetics (Huntsville, AL). Yeast cultures were maintained in enriched yeast-peptone-based medium supplemented with 2% glucose (YPD) or 2% raffinose (YPR) or in minimal synthetic medium supplemented with 2% glucose (24Sherman F. Fink G.R. Lawrence C.W. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York1978Google Scholar). Plasmids—The pEL111 was by the of (18Luk E. Culotta V.C. J. Biol. Chem. 2001; 276: 47556-47562Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) containing to of the S. cerevisiae SOD2 into the R.S. M. P. PubMed Scopus Google Scholar) with the the a a site was introduced in the (18Luk E. Culotta V.C. J. Biol. Chem. 2001; 276: 47556-47562Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) the of the SOD2 by was with and and the containing the SOD2 and its was of a in the using the was by The for cytosolic Sod2p to the was by the of (18Luk E. Culotta V.C. J. Biol. Chem. 2001; 276: 47556-47562Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) into R.S. M. P. PubMed Scopus Google Scholar) at the enzyme The for cytosolic Sod2p contains the SOD2 to and to but the mitochondrial The C. albicans SOD3 was (20Lamarre C. LeMay J.D. Deslauriers N. Bourbonnais Y. J. Biol. Chem. 2001; 276: 43784-43791Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). of cell S. cerevisiae strains were in of at a of and allowed to at for In cell were by as (18Luk E. Culotta V.C. J. Biol. Chem. 2001; 276: 47556-47562Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). mitochondrial was yeast cells were to with a and into mitochondrial and cell by as L. Culotta V.C. Mol. Cell Biol. 2000; 20: PubMed Scopus Google Scholar). cell or were for SOD activity by and with L. F. L. Methods in in New Scholar). S. cerevisiae Sod2p and C. albicans SOD3 polypeptide levels were by cell or cell to and with against S. cerevisiae Sod2p (18Luk E. Culotta V.C. J. Biol. Chem. 2001; 276: 47556-47562Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) that with C. albicans against from and cytosolic were used as (18Luk E. Culotta V.C. J. Biol. Chem. 2001; 276: 47556-47562Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). S. cerevisiae Sod2p the mitochondrial and C. albicans SOD3 were as proteins as (19Luk E. Carroll M. Baker M. Culotta V.C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10353-10357Crossref PubMed Scopus (115) Google Scholar, C. LeMay J.D. Deslauriers N. Bourbonnais Y. J. Biol. Chem. 2001; 276: 43784-43791Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, M.E. 2000; PubMed Scopus Google Scholar). of these SOD molecules were by A the of manganese activation of in manganese were in medium for of was and of were for of cell by and for monitoring Sod2p activity and polypeptide levels as was to cultures to manganese In a of were from and mitochondria were L. Culotta V.C. Mol. Cell Biol. 2000; 20: PubMed Scopus Google Scholar). The mitochondria were for manganese as in E. Carroll M. Baker M. Culotta V.C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10353-10357Crossref PubMed Scopus (115) Google by using a in mitochondrial import of yeast mutant cells were with the and for in medium to of 2% was to SOD2 of the carbonyl cyanide was to mitochondrial import J. Biol. Chem. Full Text PDF PubMed Google Scholar). of to as J. Biol. Chem. Full Text PDF PubMed Google Scholar). Cell were by and mitochondrial and were as of Sod2p whether a mitochondrial localization of Sod2p was needed for manganese insertion into the enzyme. A cytosolic version of S. cerevisiae Sod2p was by the mitochondrial C. P. L. E. J. Biochem. PubMed Scopus Google Scholar). The Sod2p was expressed in a mutant of S. lacking the mitochondrial in with the cytosolic and is largely from the mitochondria by the mitochondrial matrix protein By of Sod2p the in a mitochondrial localization of the enzyme as for enzymatic activity, from cells or were to a and for SOD activity by in the cytosolic Sod2p was largely with the mitochondrial enzyme and The activity of was by manganese in vivo, that the of activity results from a manganese in the enzyme. is that the of manganese required to is is a that is toxic to the as by E. Carroll M. Baker M. Culotta V.C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10353-10357Crossref PubMed Scopus (115) Google Scholar) and The cytosolic of Sod2p is only when cells manganese. Sod2p to be inside the mitochondria to be efficiently SOD3 from C. albicans When in S. pathogenic fungi C. albicans a SOD in the cytosol (Ca that is to be when expressed in the cytosol of S. cerevisiae (20Lamarre C. LeMay J.D. Deslauriers N. Bourbonnais Y. J. Biol. Chem. 2001; 276: 43784-43791Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). We whether Ca SOD3 a to acquire manganese in the cytosol. with (19Luk E. Carroll M. Baker M. Culotta V.C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10353-10357Crossref PubMed Scopus (115) Google Scholar, C. LeMay J.D. Deslauriers N. Bourbonnais Y. J. Biol. Chem. 2001; 276: 43784-43791Abstract Full Text Full Text PDF PubMed Scopus (100) Google Ca SOD3 expressed in S. cerevisiae activity 2 and was in both a and a the mitochondrial Sod2p of S. cerevisiae of Ca SOD3 expressed in S. cerevisiae is by manganese intracellular manganese a of the Smf2p manganese transporter (18Luk E. Culotta V.C. J. Biol. Chem. 2001; 276: 47556-47562Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) Ca SOD3 activity and activity was by cells in the of manganese In manganese also a on Ca SOD3 activity in wild-type cells and Hence, appears to be a large pool of Ca SOD3 that is and can be activated at intracellular of the with S. cerevisiae The of Ca SOD3 in S. cerevisiae is driven by a and the (20Lamarre C. LeMay J.D. Deslauriers N. Bourbonnais Y. J. Biol. Chem. 2001; 276: 43784-43791Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Ca SOD3 is to Sod2p and Ca SOD3 proteins of known were used as in a against from cells Sod2p and Ca in Ca SOD3 is expressed in S. cerevisiae on a at levels that are the of Ca SOD3 protein may activity can be in S. cerevisiae, the that a large fraction of the protein manganese. the with Ca SOD3 and with cytosolic Sod2p in S. cerevisiae, efficient activation of Sod2p requires a mitochondrial is a from the cytosol that is required for efficient activation of of Manganese into Sod2p New is mitochondrial Sod2p activated with We in the case of a pre-existing apopool of the enzyme is activated with copper in the of new protein synthesis (13Schmidt P. Kunst C. Culotta V.C. J. Biol. Chem. 2000; 275: 33771-33776Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 14Bartnikas T.B. Gitlin J.D. J. Biol. Chem. 2003; 278: 33602-33608Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). We whether the was for Sod2p of the mitochondria. activation of a pool of Sod2p that is largely apo for we the In these the Sod2p polypeptide still in the but is largely of low mitochondrial manganese (18Luk E. Culotta V.C. J. Biol. Chem. 2001; 276: 47556-47562Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Sod2p activity is in this mutant by cells in the of manganese A and We the required to Sod2p the of manganese to the in A and Sod2p was activated very by manganese and required at of with the metal. By activation of cytosolic by copper in S. cerevisiae cells is in (13Schmidt P. Kunst C. Culotta V.C. J. Biol. Chem. 2000; 275: 33771-33776Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). The activation of mitochondrial Sod2p is not a of trafficking of the metal to the as mitochondrial manganese was to wild-type levels of with manganese a in metalation of the enzyme that new protein synthesis may be the for protein the for Sod2p activation was in which in protein was by that and the in Sod2p activity with manganese of manganese into the was by as by manganese insertion requires new protein The metal is not readily inserted into a pre-existing pool of and only a freshly synthesized Sod2p appears for and of Sod2p of the Sod2p polypeptide outside the metalation of Sod2p mitochondria. on these distinct would polypeptide synthesis be required for manganese a the synthesis of Sod2p may be closely coupled with mitochondrial and it is the import process that manganese In it that certain mitochondrial proteins are imported K. Biochem. Sci. Full Text PDF PubMed Scopus Google Scholar, M. K. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, A. L. H. Biochem. J. 2004; PubMed Scopus Google of the polypeptide in the cytosol would mitochondrial We whether this was the case for was in which Sod2p synthesis was controlled the S. cerevisiae A was with Sod2p and of with newly synthesized Sod2p of the newly synthesized Sod2p as a the mitochondrial Sod2p is in the that the mitochondrial import of Sod2p or Sod2p this we mitochondrial import and protein was of the CCCP, which import by the mitochondrial membrane J. Biol. Chem. Full Text PDF PubMed Google Scholar). that import of Sod2p into the mitochondria. a the Sod2p in the cytosolic fraction The of can be by J. Biol. Chem. Full Text PDF PubMed Google and when is CCCP, is no of and newly synthesized Sod2p was into mitochondria and Using this we whether pre-existing cytosolic Sod2p can be into mitochondria. In the of Sod2p synthesis was for in the of to of cytosolic Sod2p was for the of this the of Sod2p mitochondrial Sod2p the Sod2p that to was when mitochondrial import was with in and Furthermore, the of mitochondrial Sod2p required new protein as of Sod2p in cells these are with the that mitochondrial import requires freshly the mitochondrial and manganese insertion for Sod2p are closely in The mitochondrial SOD2 enzyme is known for its role in and A. A. M. A. Genetics. 2003; PubMed Google Scholar, K. J. J.P. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, Y. S. C. Genet. PubMed Scopus Google Scholar, Y. T.D. L. D. PubMed Scopus Google Scholar, W. S. L. 2004; PubMed Scopus Google Scholar, H. Y. M. M. N. Nelson J. R. A. 2003; PubMed Scopus Google Scholar, Full Text PDF PubMed Scopus Google Scholar, A. J. PubMed Google Scholar). this is known about the of the SOD2 polypeptide in vivo. is the protein encoded by the into enzyme in the mitochondrial We have that activation of S. cerevisiae Sod2p insertion of the manganese the mitochondria. When expressed in the cytosol of S. cerevisiae, Mn-SOD molecules are poorly manganese activation also requires new protein synthesis and mitochondrial import. are with a model in which the mitochondrial and manganese activation of Sod2p are closely coupled in Although Sod2p is largely when expressed in the cytosol of S. activity be by cells to toxic of manganese. the of manganese in the appears low to newly synthesized may be a eukaryotes not express a cytosolic are as in the case of the cytosolic Mn-SOD of C. albicans (20Lamarre C. LeMay J.D. Deslauriers N. Bourbonnais Y. J. Biol. Chem. 2001; 276: 43784-43791Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) and of (21Brouwer M. Hoexum Brouwer T. Grater W. Brown-Peterson N. Biochem. 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