Mitochondrial protein import: precursor oxidation in a ternary complex with disulfide carrier and sulfhydryl oxidase
Identifieur interne : 001121 ( Pmc/Corpus ); précédent : 001120; suivant : 001122Mitochondrial protein import: precursor oxidation in a ternary complex with disulfide carrier and sulfhydryl oxidase
Auteurs : Diana Stojanovski ; Dusanka Milenkovic ; Judith M. Müller ; Kipros Gabriel ; Agnes Schulze-Specking ; Michael J. Baker ; Michael T. Ryan ; Bernard Guiard ; Nikolaus Pfanner ; Agnieszka ChacinskaSource :
- The Journal of Cell Biology [ 0021-9525 ] ; 2008.
Abstract
The biogenesis of mitochondrial intermembrane space proteins depends on specific machinery that transfers disulfide bonds to precursor proteins. The machinery shares features with protein relays for disulfide bond formation in the bacterial periplasm and endoplasmic reticulum. A disulfide-generating enzyme/sulfhydryl oxidase oxidizes a disulfide carrier protein, which in turn transfers a disulfide to the substrate protein. Current views suggest that the disulfide carrier alternates between binding to the oxidase and the substrate. We have analyzed the cooperation of the disulfide relay components during import of precursors into mitochondria and identified a ternary complex of all three components. The ternary complex represents a transient and intermediate step in the oxidation of intermembrane space precursors, where the oxidase Erv1 promotes disulfide transfer to the precursor while both oxidase and precursor are associated with the disulfide carrier Mia40.
Url:
DOI: 10.1083/jcb.200804095
PubMed: 18852299
PubMed Central: 2568017
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PMC:2568017Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Mitochondrial protein import: precursor oxidation in a ternary complex with disulfide carrier and sulfhydryl oxidase</title><author><name sortKey="Stojanovski, Diana" sort="Stojanovski, Diana" uniqKey="Stojanovski D" first="Diana" last="Stojanovski">Diana Stojanovski</name><affiliation><nlm:aff wicri:cut=", and" id="N0x3a3fdd0N0x400fa70">Institut für Biochemie und Molekularbiologie, Zentrum für Biochemie und Molekulare Zellforschung</nlm:aff></affiliation></author><author><name sortKey="Milenkovic, Dusanka" sort="Milenkovic, Dusanka" uniqKey="Milenkovic D" first="Dusanka" last="Milenkovic">Dusanka Milenkovic</name><affiliation><nlm:aff wicri:cut=", and" id="N0x3a3fdd0N0x400fa70">Institut für Biochemie und Molekularbiologie, Zentrum für Biochemie und Molekulare Zellforschung</nlm:aff></affiliation></author><author><name sortKey="Muller, Judith M" sort="Muller, Judith M" uniqKey="Muller J" first="Judith M." last="Müller">Judith M. Müller</name><affiliation><nlm:aff wicri:cut=", and" id="N0x3a3fdd0N0x400fa70">Institut für Biochemie und Molekularbiologie, Zentrum für Biochemie und Molekulare Zellforschung</nlm:aff></affiliation></author><author><name sortKey="Gabriel, Kipros" sort="Gabriel, Kipros" uniqKey="Gabriel K" first="Kipros" last="Gabriel">Kipros Gabriel</name><affiliation><nlm:aff wicri:cut=", and" id="N0x3a3fdd0N0x400fa70">Institut für Biochemie und Molekularbiologie, Zentrum für Biochemie und Molekulare Zellforschung</nlm:aff></affiliation></author><author><name sortKey="Schulze Specking, Agnes" sort="Schulze Specking, Agnes" uniqKey="Schulze Specking A" first="Agnes" last="Schulze-Specking">Agnes Schulze-Specking</name><affiliation><nlm:aff wicri:cut=", and" id="N0x3a3fdd0N0x400fa70">Institut für Biochemie und Molekularbiologie, Zentrum für Biochemie und Molekulare Zellforschung</nlm:aff></affiliation></author><author><name sortKey="Baker, Michael J" sort="Baker, Michael J" uniqKey="Baker M" first="Michael J." last="Baker">Michael J. Baker</name><affiliation><nlm:aff id="aff3">Department of Biochemistry, La Trobe University, Melbourne 3086, Australia</nlm:aff></affiliation></author><author><name sortKey="Ryan, Michael T" sort="Ryan, Michael T" uniqKey="Ryan M" first="Michael T." last="Ryan">Michael T. Ryan</name><affiliation><nlm:aff id="aff3">Department of Biochemistry, La Trobe University, Melbourne 3086, Australia</nlm:aff></affiliation></author><author><name sortKey="Guiard, Bernard" sort="Guiard, Bernard" uniqKey="Guiard B" first="Bernard" last="Guiard">Bernard Guiard</name><affiliation><nlm:aff id="aff4">Centre de Génétique Moléculaire, Centre National de la Recherche Scientifique, 91190 Gif-sur-Yvette, France</nlm:aff></affiliation></author><author><name sortKey="Pfanner, Nikolaus" sort="Pfanner, Nikolaus" uniqKey="Pfanner N" first="Nikolaus" last="Pfanner">Nikolaus Pfanner</name><affiliation><nlm:aff wicri:cut=", and" id="N0x3a3fdd0N0x400fa70">Institut für Biochemie und Molekularbiologie, Zentrum für Biochemie und Molekulare Zellforschung</nlm:aff></affiliation><affiliation><nlm:aff id="N0x3a3fdd0N0x400fa70">Centre for Biological Signalling Studies (<italic>bioss</italic>), Universität Freiburg, 79104 Freiburg, Germany</nlm:aff></affiliation></author><author><name sortKey="Chacinska, Agnieszka" sort="Chacinska, Agnieszka" uniqKey="Chacinska A" first="Agnieszka" last="Chacinska">Agnieszka Chacinska</name><affiliation><nlm:aff wicri:cut=", and" id="N0x3a3fdd0N0x400fa70">Institut für Biochemie und Molekularbiologie, Zentrum für Biochemie und Molekulare Zellforschung</nlm:aff></affiliation><affiliation><nlm:aff id="N0x3a3fdd0N0x400fa70">Centre for Biological Signalling Studies (<italic>bioss</italic>), Universität Freiburg, 79104 Freiburg, Germany</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">18852299</idno><idno type="pmc">2568017</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2568017</idno><idno type="RBID">PMC:2568017</idno><idno type="doi">10.1083/jcb.200804095</idno><date when="2008">2008</date><idno type="wicri:Area/Pmc/Corpus">001121</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">001121</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Mitochondrial protein import: precursor oxidation in a ternary complex with disulfide carrier and sulfhydryl oxidase</title><author><name sortKey="Stojanovski, Diana" sort="Stojanovski, Diana" uniqKey="Stojanovski D" first="Diana" last="Stojanovski">Diana Stojanovski</name><affiliation><nlm:aff wicri:cut=", and" id="N0x3a3fdd0N0x400fa70">Institut für Biochemie und Molekularbiologie, Zentrum für Biochemie und Molekulare Zellforschung</nlm:aff></affiliation></author><author><name sortKey="Milenkovic, Dusanka" sort="Milenkovic, Dusanka" uniqKey="Milenkovic D" first="Dusanka" last="Milenkovic">Dusanka Milenkovic</name><affiliation><nlm:aff wicri:cut=", and" id="N0x3a3fdd0N0x400fa70">Institut für Biochemie und Molekularbiologie, Zentrum für Biochemie und Molekulare Zellforschung</nlm:aff></affiliation></author><author><name sortKey="Muller, Judith M" sort="Muller, Judith M" uniqKey="Muller J" first="Judith M." last="Müller">Judith M. Müller</name><affiliation><nlm:aff wicri:cut=", and" id="N0x3a3fdd0N0x400fa70">Institut für Biochemie und Molekularbiologie, Zentrum für Biochemie und Molekulare Zellforschung</nlm:aff></affiliation></author><author><name sortKey="Gabriel, Kipros" sort="Gabriel, Kipros" uniqKey="Gabriel K" first="Kipros" last="Gabriel">Kipros Gabriel</name><affiliation><nlm:aff wicri:cut=", and" id="N0x3a3fdd0N0x400fa70">Institut für Biochemie und Molekularbiologie, Zentrum für Biochemie und Molekulare Zellforschung</nlm:aff></affiliation></author><author><name sortKey="Schulze Specking, Agnes" sort="Schulze Specking, Agnes" uniqKey="Schulze Specking A" first="Agnes" last="Schulze-Specking">Agnes Schulze-Specking</name><affiliation><nlm:aff wicri:cut=", and" id="N0x3a3fdd0N0x400fa70">Institut für Biochemie und Molekularbiologie, Zentrum für Biochemie und Molekulare Zellforschung</nlm:aff></affiliation></author><author><name sortKey="Baker, Michael J" sort="Baker, Michael J" uniqKey="Baker M" first="Michael J." last="Baker">Michael J. Baker</name><affiliation><nlm:aff id="aff3">Department of Biochemistry, La Trobe University, Melbourne 3086, Australia</nlm:aff></affiliation></author><author><name sortKey="Ryan, Michael T" sort="Ryan, Michael T" uniqKey="Ryan M" first="Michael T." last="Ryan">Michael T. Ryan</name><affiliation><nlm:aff id="aff3">Department of Biochemistry, La Trobe University, Melbourne 3086, Australia</nlm:aff></affiliation></author><author><name sortKey="Guiard, Bernard" sort="Guiard, Bernard" uniqKey="Guiard B" first="Bernard" last="Guiard">Bernard Guiard</name><affiliation><nlm:aff id="aff4">Centre de Génétique Moléculaire, Centre National de la Recherche Scientifique, 91190 Gif-sur-Yvette, France</nlm:aff></affiliation></author><author><name sortKey="Pfanner, Nikolaus" sort="Pfanner, Nikolaus" uniqKey="Pfanner N" first="Nikolaus" last="Pfanner">Nikolaus Pfanner</name><affiliation><nlm:aff wicri:cut=", and" id="N0x3a3fdd0N0x400fa70">Institut für Biochemie und Molekularbiologie, Zentrum für Biochemie und Molekulare Zellforschung</nlm:aff></affiliation><affiliation><nlm:aff id="N0x3a3fdd0N0x400fa70">Centre for Biological Signalling Studies (<italic>bioss</italic>), Universität Freiburg, 79104 Freiburg, Germany</nlm:aff></affiliation></author><author><name sortKey="Chacinska, Agnieszka" sort="Chacinska, Agnieszka" uniqKey="Chacinska A" first="Agnieszka" last="Chacinska">Agnieszka Chacinska</name><affiliation><nlm:aff wicri:cut=", and" id="N0x3a3fdd0N0x400fa70">Institut für Biochemie und Molekularbiologie, Zentrum für Biochemie und Molekulare Zellforschung</nlm:aff></affiliation><affiliation><nlm:aff id="N0x3a3fdd0N0x400fa70">Centre for Biological Signalling Studies (<italic>bioss</italic>), Universität Freiburg, 79104 Freiburg, Germany</nlm:aff></affiliation></author></analytic><series><title level="j">The Journal of Cell Biology</title><idno type="ISSN">0021-9525</idno><idno type="eISSN">1540-8140</idno><imprint><date when="2008">2008</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>The biogenesis of mitochondrial intermembrane space proteins depends on specific machinery that transfers disulfide bonds to precursor proteins. The machinery shares features with protein relays for disulfide bond formation in the bacterial periplasm and endoplasmic reticulum. A disulfide-generating enzyme/sulfhydryl oxidase oxidizes a disulfide carrier protein, which in turn transfers a disulfide to the substrate protein. Current views suggest that the disulfide carrier alternates between binding to the oxidase and the substrate. We have analyzed the cooperation of the disulfide relay components during import of precursors into mitochondria and identified a ternary complex of all three components. The ternary complex represents a transient and intermediate step in the oxidation of intermembrane space precursors, where the oxidase Erv1 promotes disulfide transfer to the precursor while both oxidase and precursor are associated with the disulfide carrier Mia40.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">J Cell Biol</journal-id><journal-id journal-id-type="publisher-id">jcb</journal-id><journal-title>The Journal of Cell Biology</journal-title><issn pub-type="ppub">0021-9525</issn><issn pub-type="epub">1540-8140</issn><publisher><publisher-name>The Rockefeller University Press</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">18852299</article-id><article-id pub-id-type="pmc">2568017</article-id><article-id pub-id-type="publisher-id">200804095</article-id><article-id pub-id-type="doi">10.1083/jcb.200804095</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Articles</subject><subj-group><subject>Report</subject></subj-group></subj-group></article-categories><title-group><article-title>Mitochondrial protein import: precursor oxidation in a ternary complex with disulfide carrier and sulfhydryl oxidase</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Stojanovski</surname><given-names>Diana</given-names></name><xref ref-type="aff" rid="N0x3a3fdd0N0x400fa70">1</xref></contrib><contrib contrib-type="author"><name><surname>Milenkovic</surname><given-names>Dusanka</given-names></name><xref ref-type="aff" rid="N0x3a3fdd0N0x400fa70">1</xref></contrib><contrib contrib-type="author"><name><surname>Müller</surname><given-names>Judith M.</given-names></name><xref ref-type="aff" rid="N0x3a3fdd0N0x400fa70">1</xref></contrib><contrib contrib-type="author"><name><surname>Gabriel</surname><given-names>Kipros</given-names></name><xref ref-type="aff" rid="N0x3a3fdd0N0x400fa70">1</xref></contrib><contrib contrib-type="author"><name><surname>Schulze-Specking</surname><given-names>Agnes</given-names></name><xref ref-type="aff" rid="N0x3a3fdd0N0x400fa70">1</xref></contrib><contrib contrib-type="author"><name><surname>Baker</surname><given-names>Michael J.</given-names></name><xref ref-type="aff" rid="aff3">3</xref></contrib><contrib contrib-type="author"><name><surname>Ryan</surname><given-names>Michael T.</given-names></name><xref ref-type="aff" rid="aff3">3</xref></contrib><contrib contrib-type="author"><name><surname>Guiard</surname><given-names>Bernard</given-names></name><xref ref-type="aff" rid="aff4">4</xref></contrib><contrib contrib-type="author"><name><surname>Pfanner</surname><given-names>Nikolaus</given-names></name><xref ref-type="aff" rid="N0x3a3fdd0N0x400fa70">1</xref><xref ref-type="aff" rid="N0x3a3fdd0N0x400fa70">2</xref></contrib><contrib contrib-type="author"><name><surname>Chacinska</surname><given-names>Agnieszka</given-names></name><xref ref-type="aff" rid="N0x3a3fdd0N0x400fa70">1</xref><xref ref-type="aff" rid="N0x3a3fdd0N0x400fa70">2</xref></contrib></contrib-group><aff id="N0x3a3fdd0N0x400fa70"><label>1</label>Institut für Biochemie und Molekularbiologie, Zentrum für Biochemie und Molekulare Zellforschung, and<label>2</label>Centre for Biological Signalling Studies (<italic>bioss</italic>), Universität Freiburg, 79104 Freiburg, Germany</aff><aff id="aff3"><label>3</label>Department of Biochemistry, La Trobe University, Melbourne 3086, Australia</aff><aff id="aff4"><label>4</label>Centre de Génétique Moléculaire, Centre National de la Recherche Scientifique, 91190 Gif-sur-Yvette, France</aff><author-notes><fn><p>Correspondence to Nikolaus Pfanner: <email>nikolaus.pfanner@biochemie.uni-freiburg.de</email>; or Agnieszka Chacinska: <email>agnieszka.chacinska@biochemie.uni-freiburg.de</email></p></fn></author-notes><pub-date pub-type="ppub"><day>20</day><month>10</month><year>2008</year></pub-date><volume>183</volume><issue>2</issue><fpage>195</fpage><lpage>202</lpage><history><date date-type="received"><day>17</day><month>4</month><year>2008</year></date><date date-type="accepted"><day>15</day><month>9</month><year>2008</year></date></history><permissions><copyright-statement>© 2008 Stojanovski et al.</copyright-statement><license><p>This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see <ext-link ext-link-type="uri" xlink:href="http://www.jcb.org/misc/terms.shtml">http://www.jcb.org/misc/terms.shtml</ext-link>). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by-nc-sa/3.0/">http://creativecommons.org/licenses/by-nc-sa/3.0/</ext-link>).</p></license></permissions><self-uri xlink:title="pdf" xlink:href="jcb1830195.pdf"></self-uri><abstract><p>The biogenesis of mitochondrial intermembrane space proteins depends on specific machinery that transfers disulfide bonds to precursor proteins. The machinery shares features with protein relays for disulfide bond formation in the bacterial periplasm and endoplasmic reticulum. A disulfide-generating enzyme/sulfhydryl oxidase oxidizes a disulfide carrier protein, which in turn transfers a disulfide to the substrate protein. Current views suggest that the disulfide carrier alternates between binding to the oxidase and the substrate. We have analyzed the cooperation of the disulfide relay components during import of precursors into mitochondria and identified a ternary complex of all three components. The ternary complex represents a transient and intermediate step in the oxidation of intermembrane space precursors, where the oxidase Erv1 promotes disulfide transfer to the precursor while both oxidase and precursor are associated with the disulfide carrier Mia40.</p></abstract></article-meta><notes><fn-group><fn><p>D. Stojanovski and D. Milenkovic contributed equally to this paper.</p></fn><fn><p>K. Gabriel's present address is Department of Genetics, University of Melbourne, Parkville 3010, Australia</p></fn><fn><p>Abbreviations used in this paper: AMS, 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid; Dsb, disulfide bond formation protein; IMS, intermembrane space; MIA, mitochondrial IMS import and assembly; Tim, translocase of the inner mitochondrial membrane.</p></fn></fn-group></notes></front><body><sec><title>Introduction</title><p>Mitochondria use diverse mechanisms to import precursor protein (<xref ref-type="bibr" rid="bib16">Jensen and Johnson, 2001</xref>; <xref ref-type="bibr" rid="bib7">Endo et al., 2003</xref>; <xref ref-type="bibr" rid="bib19">Koehler, 2004</xref>; <xref ref-type="bibr" rid="bib27">Oka and Mihara, 2005</xref>; <xref ref-type="bibr" rid="bib6">Dolezal et al., 2006</xref>; <xref ref-type="bibr" rid="bib26">Neupert and Herrmann, 2007</xref>). The mitochondrial intermembrane space (IMS) contains an import and assembly (MIA) machinery, which transfers disulfide bonds to precursors and promotes their import and maturation (<xref ref-type="bibr" rid="bib2">Chacinska et al., 2004</xref>; <xref ref-type="bibr" rid="bib19">Koehler, 2004</xref>; <xref ref-type="bibr" rid="bib22">Mesecke et al., 2005</xref>; <xref ref-type="bibr" rid="bib36">Tokatlidis, 2005</xref>). The MIA pathway was reported to operate as a protein relay for disulfide bond formation like the disulfide relay systems of the bacterial periplasm (disulfide bond formation protein B [DsbB]–DsbA) and the ER (Ero1–protein disulfide isomerase [PDI]; <xref ref-type="bibr" rid="bib3">Collet and Bardwell, 2002</xref>; <xref ref-type="bibr" rid="bib18">Kadokura et al., 2004</xref>; <xref ref-type="bibr" rid="bib38">Tu and Weissman, 2004</xref>; <xref ref-type="bibr" rid="bib30">Sevier and Kaiser, 2006</xref>; <xref ref-type="bibr" rid="bib11">Herrmann and Köhl, 2007</xref>). In each case, a disulfide-generating enzyme/sulfhydryl oxidase (DsbB, Ero1, and Erv1) oxidizes a disulfide carrier protein (DsbA, PDI, and Mia40). The oxidized disulfide carrier subsequently transfers the disulfide to the substrate, typically a newly synthesized protein arriving in the periplasm, ER, or IMS.</p><p>Mixed disulfides are generated between oxidase and disulfide carriers, as well as between carrier protein and substrate as direct means for the transfer of disulfide bonds. The current models of disulfide relays suggest an alternating association of the carrier protein with the substrate (reduction of the carrier) and the sulfhydryl oxidase (reoxidation of the carrier; <xref ref-type="bibr" rid="bib3">Collet and Bardwell, 2002</xref>; <xref ref-type="bibr" rid="bib18">Kadokura et al., 2004</xref>; <xref ref-type="bibr" rid="bib38">Tu and Weissman, 2004</xref>; <xref ref-type="bibr" rid="bib22">Mesecke et al., 2005</xref>; <xref ref-type="bibr" rid="bib30">Sevier and Kaiser, 2006</xref>; <xref ref-type="bibr" rid="bib11">Herrmann and Köhl, 2007</xref>). This implies that substrates, which receive more than one disulfide bond, require multiple cycles of the disulfide relay. An alternative, efficient mechanism would be that oxidative protein folding involves a direct cooperation of all three partners in a ternary complex to promote the flow of electrons. However, such a ternary complex has not been shown experimentally.</p><p>We addressed the cooperation of sulfhydryl oxidase and disulfide carrier during oxidation of mitochondrial IMS precursors. Mia40 (Tim40), which contains three essential pairs of cysteines, forms a mixed disulfide with precursors in translocation across the outer membrane, drives their import into the IMS, and transfers disulfides to the proteins (<xref ref-type="bibr" rid="bib2">Chacinska et al., 2004</xref>; <xref ref-type="bibr" rid="bib25">Naoé et al., 2004</xref>; <xref ref-type="bibr" rid="bib34">Terziyska et al., 2005</xref>, <xref ref-type="bibr" rid="bib35">2007</xref>; <xref ref-type="bibr" rid="bib10">Grumbt et al., 2007</xref>; <xref ref-type="bibr" rid="bib23">Milenkovic et al., 2007</xref>; <xref ref-type="bibr" rid="bib24">Müller et al., 2008</xref>). <xref ref-type="bibr" rid="bib22">Mesecke et al. (2005)</xref> found that upon release of the oxidized substrate, reduced Mia40 was reoxidized by Erv1 (also containing three pairs of cysteines), thus permitting new rounds of precursor import. Here, we report that, contrary to the view that Erv1 acts only on substrate-free Mia40, we have identified a ternary complex of Erv1, Mia40, and precursor. This ternary complex assists in the transfer of disulfide bonds to IMS precursors by spatially connecting sulfhydryl oxidase, carrier protein, and substrate.</p></sec><sec><title>Results and discussion</title><sec><title>Disulfide-linked precursor–Mia40 intermediate in <italic>erv1</italic> mutant mitochondria</title><p>The import and assembly stages of the <sup>35</sup>S-labeled precursors of the small translocase of the inner mitochondrial membrane (Tim) proteins of the IMS were monitored by blue native electrophoresis. After disulfide-mediated interaction with Mia40, the oxidized proteins, which contain two disulfides each, assemble to the mature hexameric complexes (Tim9–Tim10 or Tim8–Tim13); Tim9 and Tim10 additionally assemble into the TIM22 complex of the inner membrane (<xref rid="fig1" ref-type="fig">Fig. 1 A</xref>; <xref ref-type="bibr" rid="bib2">Chacinska et al., 2004</xref>; <xref ref-type="bibr" rid="bib5">Curran et al., 2004</xref>; <xref ref-type="bibr" rid="bib23">Milenkovic et al., 2007</xref>; <xref ref-type="bibr" rid="bib32">Sideris and Tokatlidis, 2007</xref>; <xref ref-type="bibr" rid="bib24">Müller et al., 2008</xref>). Blue native analysis of a yeast mutant of Erv1 revealed that precursors were efficiently bound to Mia40 in the mutant mitochondria, whereas the subsequent assembly into mature complexes was inhibited (<xref ref-type="bibr" rid="bib29">Rissler et al., 2005</xref>; <xref ref-type="bibr" rid="bib24">Müller et al., 2008</xref>), shown here for Tim10 with three different <italic>erv1</italic> mutants (<xref rid="fig1" ref-type="fig">Fig. 1 A</xref>). These results questioned the prevailing model that Erv1 was only needed for reoxidation of Mia40 to activate it for substrate binding, and suggested that Erv1 may play a role in later steps of substrate maturation.</p><fig position="float" id="fig1"><label>Figure 1.</label><caption><p><bold>Accumulation of precursor proteins at Mia40.</bold> (A) Isolated mitochondria were incubated with <sup>35</sup>S-Tim10 and analyzed by native electrophoresis. WT, wild-type. (B) <sup>35</sup>S-Tim9 was imported into mitochondria (samples 1–10). Tim9-Mia40 was isolated by affinity purification from Mia40<sub>His</sub> mitochondria (right). The samples were analyzed by nonreducing SDS-PAGE. (C) <sup>35</sup>S-precursors were incubated with mitochondria and analyzed by native electrophoresis. Asterisk, nonspecific band. (D) Mia40 or Mia40<sub>His</sub> mitochondria were subjected to affinity purification, SDS-PAGE, and autoradiography (<sup>35</sup>S-Tim9) or immunoblotting. Load, 2.5%; eluate, 100%. (E) <sup>35</sup>S-Tim9 was imported and analyzed by nonreducing SDS-PAGE.</p></caption><graphic xlink:href="jcb1830195f01"></graphic></fig><p>To analyze the precursor–Mia40 conjugate under denaturing conditions, we used nonreducing SDS-PAGE (<xref ref-type="bibr" rid="bib23">Milenkovic et al., 2007</xref>). Formation of the conjugate was impaired in <italic>mia40-3</italic> mutant mitochondria, and its gel migration was faster (<xref rid="fig1" ref-type="fig">Fig. 1 B</xref>, lanes 4–6) because of a C-terminal truncation of Mia40-3 (<xref ref-type="bibr" rid="bib2">Chacinska et al., 2004</xref>). The conjugate was observed in similar amounts in <italic>erv1</italic> mutant mitochondria and wild-type mitochondria (<xref rid="fig1" ref-type="fig">Fig. 1 B</xref>, lanes 7–10), as well as after purification of tagged Mia40 from lysed mitochondria (<xref rid="fig1" ref-type="fig">Fig. 1 B</xref>, lanes 11–16). The total amount of imported precursor, determined by treatment of the mitochondria with protease, was decreased in <italic>erv1</italic> mutant mitochondria (<xref rid="fig1" ref-type="fig">Fig. 1 B</xref>, lanes 9 and 10), in agreement with the requirement of Erv1 for the Mia40-driven import reaction (<xref ref-type="bibr" rid="bib1">Allen et al., 2005</xref>; <xref ref-type="bibr" rid="bib22">Mesecke et al., 2005</xref>; <xref ref-type="bibr" rid="bib29">Rissler et al., 2005</xref>). We hypothesized that in <italic>erv1</italic> mutants, the MIA reaction cycle was impaired such that imported precursors remained bound to Mia40, preventing assembly of the proteins and further rounds of precursor import.</p></sec><sec><title>A ternary complex of Erv1, Mia40, and precursor protein</title><p>The hypothesis raised the possibility that Erv1 may associate with the substrate–Mia40 complex. To capture a putative ternary complex, we used the trap mutant form Mia40-4, which efficiently binds substrate but is impaired in its release (<xref ref-type="bibr" rid="bib2">Chacinska et al., 2004</xref>; <xref ref-type="bibr" rid="bib23">Milenkovic et al., 2007</xref>; <xref ref-type="bibr" rid="bib24">Müller et al., 2008</xref>). In <italic>mia40-4</italic> mitochondria, formation of the mature complexes (Tim9–Tim10, Tim8–13, and TIM22) is impaired, and precursor proteins accumulate at Mia40-4. We noticed an additional complex in the high molecular weight range of blue native gels upon import of <sup>35</sup>S-precursors (<xref rid="fig1" ref-type="fig">Fig. 1 C</xref>, Mia40–intermediate II). Mitochondria carrying His-tagged versions of Mia40 and Mia40-4 were lysed in digitonin and subjected to affinity purification. The amount of Erv1 bound to Mia40-4 was increased approximately twofold in the mutant (<xref rid="fig1" ref-type="fig">Fig. 1 D</xref>). The yield of copurification of the imported precursor of Tim9 with Mia40-4 was also enhanced (<xref rid="fig1" ref-type="fig">Fig. 1 D</xref>), and nonreducing SDS-PAGE revealed a precursor conjugate in the higher molecular weight range of <italic>mia40-4</italic> mitochondria (<xref rid="fig1" ref-type="fig">Fig. 1 E</xref>, lane 2). We conclude that both substrate and Erv1 were accumulated at the mutant protein Mia40-4.</p><p>In wild-type mitochondria, a ternary complex of Erv1, Mia40, and precursor would represent a short-lived intermediate in the reaction cycle for precursor oxidation. We searched for conditions to slow down the reaction and stabilize an intermediate complex with wild-type Mia40. Upon rupturing the outer membrane by swelling (mitoplasting), IMS components like the small Tim proteins were released, and the amount of Erv1 was reduced by approximately half (Erv1 was found in two fractions, soluble and peripherally membrane associated; <xref rid="fig2" ref-type="fig">Fig. 2 A</xref>, lanes 10 and 12; and Fig. S1, available at <ext-link ext-link-type="uri" xlink:href="http://www.jcb.org/cgi/content/full/jcb.200804095/DC1">http://www.jcb.org/cgi/content/full/jcb.200804095/DC1</ext-link>). Upon import of Tim9 into mitoplasts, the formation of mature TIM complexes is impaired, whereas precursors accumulate at the membrane-bound Mia40 (<xref ref-type="bibr" rid="bib23">Milenkovic et al., 2007</xref>). In addition to increased amounts of the Mia40–intermediate I, we observed the high molecular weight intermediate II (<xref rid="fig2" ref-type="fig">Fig. 2 A</xref>, lanes 5–8). Formation of the intermediate depended on the presence of precursors, as immunodecoration of the mature complexes did not reveal the high molecular weight complex (<xref rid="fig2" ref-type="fig">Fig. 2 A</xref>, lanes 9 and 10). Mia40–intermediate I disappeared with time, whereas the amount of intermediate II increased (<xref rid="fig2" ref-type="fig">Fig. 2 A</xref>, lanes 5–8), which indicates that the <sup>35</sup>S-precursor moved from intermediate I to intermediate II.</p><fig position="float" id="fig2"><label>Figure 2.</label><caption><p><bold>Ternary complex of Mia40, Erv1, and substrate.</bold> (A) <sup>35</sup>S-Tim9 was incubated with mitochondria or mitoplasts and analyzed by native electrophoresis (lanes 1–8). Immunoblotting of mitochondria and mitoplasts (lanes 9–12) is shown. Asterisk, nonspecific band. (B) After import of <sup>35</sup>S-precursors, mitoplasts were incubated with antisera and analyzed by native electrophoresis. Asterisk, nonspecific band. (C) <sup>35</sup>S-Tim9 was incubated with mitochondria or mitoplasts for 30 min. SDS-PAGE ± 50 mM DTT. (D) After import of <sup>35</sup>S-Tim9, Mia40, or Mia40<sub>His</sub>, mitochondria or mitoplasts were subjected to affinity purification, and eluates were analyzed by nonreducing SDS-PAGE. (E) After import of <sup>35</sup>S-Tim9, mitoplasts (WT and <italic>mia40-3</italic>) or mitochondria (<italic>mia40-4</italic>) were subjected to immunoprecipitation.</p></caption><graphic xlink:href="jcb1830195f02"></graphic></fig><p>To analyze the composition of the intermediate, we used antibody-shift blue native electrophoresis upon accumulation of <sup>35</sup>S-precursors in mitoplasts. Antibodies against Mia40 as well as antibodies against Erv1 shifted the intermediate II complex, whereas intermediate I was shifted only by anti-Mia40 (<xref rid="fig2" ref-type="fig">Fig. 2 B</xref>, lanes 1 and 5; Erv1-specific antibodies also depleted intermediate II formed in the <italic>mia40-4</italic> mutant; Fig. S1). To exclude that intermediate II was associated with the membrane translocases (translocase of the outer mitochondrial membrane [TOM] and TIM22), we used antibodies shifting those complexes (<xref ref-type="bibr" rid="bib28">Rehling et al., 2003</xref>; <xref ref-type="bibr" rid="bib40">Wiedemann et al., 2003</xref>). Neither anti-Tom22 nor anti-Tim54 shifted intermediate II. Anti-Tim54 shifted the TIM22 complex as expected, whereas neither anti-Mia40 nor anti-Erv1 shifted the TIM22 complex or the Tim9–Tim10 complex (<xref rid="fig2" ref-type="fig">Fig. 2 B</xref>). The dicarboxylate carrier (DIC), which is imported independently of Mia40/Erv1 (<xref ref-type="bibr" rid="bib28">Rehling et al., 2003</xref>; <xref ref-type="bibr" rid="bib2">Chacinska et al., 2004</xref>), was not shifted by the antibodies (<xref rid="fig2" ref-type="fig">Fig. 2 B</xref>). We conclude that Erv1, Mia40, and precursor are present in the intermediate II complex.</p><p>The precursor–Mia40 conjugates were dissociated by addition of the reductant DTT (<xref rid="fig2" ref-type="fig">Fig. 2 C</xref>), which indicates that they were linked by disulfide bonds. To compare the conjugates formed in mitoplasts and <italic>mia40-4</italic> mutants, we performed affinity purifications. Upon separation by nonreducing SDS-PAGE, the migration of the precursor–Mia40 conjugates was indistinguishable between mitochondria, mitoplasts, and <italic>mia40-4</italic> mutants (<xref rid="fig2" ref-type="fig">Fig. 2 D</xref>).</p><p>To obtain independent evidence for a ternary complex, we used coimmunoprecipitation. <sup>35</sup>S-Tim9 imported into mitoplasts was immunoprecipitated with antibodies against Mia40, as well as Erv1, but not control antibodies (<xref rid="fig2" ref-type="fig">Fig. 2 E</xref>, lanes 1–5). To distinguish if <sup>35</sup>S-Tim9 was directly coprecipitated with Erv1 or via binding to Mia40, we used the mutant form Mia40-3, which is impaired in precursor binding (<xref ref-type="bibr" rid="bib2">Chacinska et al., 2004</xref>; <xref ref-type="bibr" rid="bib24">Müller et al., 2008</xref>). The coprecipitation of <sup>35</sup>S-Tim9 with anti-Erv1 was impaired in the <italic>mia40-3</italic> mutant, like that with anti-Mia40 (<xref rid="fig2" ref-type="fig">Fig. 2 E</xref>, lanes 6 and 8). Because in mitoplasts the precursor would have direct access to Erv1 without a need for Mia40-dependent translocation across the outer membrane (<xref ref-type="bibr" rid="bib23">Milenkovic et al., 2007</xref>), we conclude that Mia40 is required for the interaction of Erv1 with the precursor. In <italic>mia40-4</italic> mitochondria, Tim9 was efficiently coprecipitated with anti-Erv1 as well as anti-Mia40 (<xref rid="fig2" ref-type="fig">Fig. 2 E</xref>), confirming the findings of the blue native analysis.</p><p>Collectively, we conclude that Erv1, Mia40, and precursor are present in a ternary complex, representing a transient intermediate stage during precursor oxidation, which is slowed down in mitoplasts and <italic>mia40-4</italic> mitochondria.</p></sec><sec><title>Erv1 is required for resolving the ternary complex</title><p>In <italic>erv1</italic> mutant mitochondria, the Mia40–intermediate II formed with an efficiency close to that of wild-type mitochondria (<xref rid="fig3" ref-type="fig">Fig. 3 A</xref>). Although the original <italic>erv1-1</italic> mutant showed only a minor effect on assembly of small Tim proteins, as described previously, the other <italic>erv1</italic> mutants were strongly impaired in the formation of the mature Tim9–Tim10 and TIM22 complexes (<xref rid="fig1" ref-type="fig">Figs. 1 A</xref> and <xref rid="fig3" ref-type="fig">3 A</xref>; <xref ref-type="bibr" rid="bib1">Allen et al., 2005</xref>; <xref ref-type="bibr" rid="bib29">Rissler et al., 2005</xref>; <xref ref-type="bibr" rid="bib23">Milenkovic et al., 2007</xref>; <xref ref-type="bibr" rid="bib24">Müller et al., 2008</xref>). We reasoned that Erv1 may be rate-limiting for the turnover of intermediate II. When Erv1 was overexpressed, the amount of intermediate II was indeed decreased (<xref rid="fig3" ref-type="fig">Fig. 3 B</xref>). Moreover, overexpression of Erv1 in <italic>mia40-4</italic> cells restored growth at elevated temperatures (<xref rid="fig3" ref-type="fig">Fig. 3 C</xref>) and decreased the amount of intermediate II (<xref rid="fig3" ref-type="fig">Fig. 3 D</xref>). To directly test if Erv1 was needed for dissociation of intermediate II, we generated the intermediate in mitoplasts and added recombinant Erv1. Thereby, the amount of intermediate II was significantly decreased (<xref rid="fig3" ref-type="fig">Fig. 3 E</xref>), and released precursor was found in the supernatant fraction (<xref rid="fig3" ref-type="fig">Fig. 3 F</xref>). Mutant forms of Erv1 (<xref ref-type="bibr" rid="bib12">Hofhaus et al., 2003</xref>) did not change the amount of intermediate II (<xref rid="fig3" ref-type="fig">Fig. 3 G</xref>). We conclude that Erv1 promotes a dissociation of the ternary complex.</p><fig position="float" id="fig3"><label>Figure 3.</label><caption><p><bold>Influence of Erv1 on the ternary complex.</bold> (A) <sup>35</sup>S-Tim9 was incubated with mitochondria and mitoplasts for 30 min and analyzed by native electrophoresis. (B) Mitoplasts were incubated with <sup>35</sup>S-Tim9. (C) Growth of <italic>mia40-4</italic> cells overproducing Erv1. (D) <sup>35</sup>S-Tim9 was incubated with mitochondria and analyzed by nonreducing SDS-PAGE. (E–G) Mitoplasts were incubated with recombinant Erv1 forms. Pellets (E and G) and supernatants (F) were analyzed by nonreducing SDS-PAGE.</p></caption><graphic xlink:href="jcb1830195f03"></graphic></fig></sec><sec><title>Disulfide transfer in the ternary complex</title><p>Each small Tim protein contains two CysX<sub>3</sub>Cys motifs, forming two intramolecular disulfide bonds in the mature protein (<xref ref-type="bibr" rid="bib4">Curran et al., 2002</xref>; <xref ref-type="bibr" rid="bib19">Koehler, 2004</xref>; <xref ref-type="bibr" rid="bib39">Webb et al., 2006</xref>). The fully oxidized proteins are released from Mia40 and assemble to hexameric complexes (<xref ref-type="bibr" rid="bib21">Lu et al., 2004</xref>; <xref ref-type="bibr" rid="bib24">Müller et al., 2008</xref>). To determine why Erv1 was needed for precursor release, we asked if it promoted disulfide formation on a Mia40-bound precursor. We monitored the oxidation of single cysteines of Tim9 by modification with the alkylating agent 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (AMS; 500 D). Mutant precursors with a replacement of one to four cysteines were used as standards (<xref rid="fig4" ref-type="fig">Fig. 4 A</xref>, lanes 1–5). By indirect thiol trapping, the number of oxidized cysteines after import into mitochondria was determined: first, free thiol groups were blocked by iodoacetamide, then the mitochondria were lysed and all disulfides were reduced, followed by modification of the originally oxidized cysteines by AMS. At a low temperature (15°C), a sequential oxidation of the cysteines was observed (<xref rid="fig4" ref-type="fig">Fig. 4 A</xref>, lanes 6–10). At physiological temperature (30°C), three species were dissected: reduced Tim9, Tim9 with one oxidized cysteine, and fully oxidized Tim9 (<xref rid="fig4" ref-type="fig">Fig. 4 B</xref>). In <italic>erv1</italic> mutant mitochondria, oxidation of the first cysteine was only mildly affected, but formation of the fully oxidized form was significantly inhibited (<xref rid="fig4" ref-type="fig">Fig. 4 B</xref>).</p><fig position="float" id="fig4"><label>Figure 4.</label><caption><p><bold>Erv1 promotes disulfide transfer to Mia40-bound precursors.</bold> (A) <sup>35</sup>S-Tim9 precursors were treated with AMS (left) or imported into mitochondria at 15°C, and subjected to indirect thiol trapping (right). Samples were analyzed by Tricine–SDS-PAGE. (B) <sup>35</sup>S-Tim9 was imported into mitochondria at 30°C and treated as described in A. (C) <sup>35</sup>S-Tim9 precursors were imported into WT mitochondria. (D) <sup>35</sup>S-Tim9 precursors were imported into Mia40<sub>His</sub> mitochondria and subjected to affinity purification and indirect thiol trapping. (E) <sup>35</sup>S-Tim9<sub>C55S</sub> was imported into mitochondria for the indicated times or for 24 min in the presence of iodoacetamide (IA). Samples were treated as described in A. (F) <sup>35</sup>S-Tim9<sub>C55S</sub> with one or three oxidized cysteines were quantified; error bars indicate SEM (<italic>n</italic> = 5). (G) <sup>35</sup>S-Tim9<sub>C55S</sub> was imported into Mia40<sub>His</sub> mitochondria and analyzed as in D.</p></caption><graphic xlink:href="jcb1830195f04"></graphic></fig><p>The first cysteine residue of Tim9 is required for binding to Mia40 (<xref ref-type="bibr" rid="bib23">Milenkovic et al., 2007</xref>; <xref ref-type="bibr" rid="bib32">Sideris and Tokatlidis, 2007</xref>). We imported mutant precursors lacking three, two, or one of the other cysteines and observed the generation of one, two, and three oxidized cysteines, respectively (<xref rid="fig4" ref-type="fig">Fig. 4 C</xref>). We hypothesized that species with an odd number of oxidized cysteines may represent Mia40-linked precursors. After affinity purification of His-tagged Mia40, bound precursors were eluted, and the number of originally oxidized cysteines was determined. The precursors lacking three or two cysteines contained one oxidized cysteine in the Mia40-bound form (<xref rid="fig4" ref-type="fig">Fig. 4 D</xref>), which reflects the mixed disulfide with Mia40. Of particular importance was the precursor Tim9<sub>C55S</sub>. Here, two forms, one with a single oxidized cysteine and one with all three cysteines oxidized, were bound to Mia40 (<xref rid="fig4" ref-type="fig">Fig. 4 D</xref>). Thus, in addition to the mixed disulfide with Mia40, a further disulfide bond was transferred to the precursor bound to Mia40. In <italic>erv1</italic> mutant mitochondria, the oxidation of Tim9<sub>C55S</sub> was impaired with a particular defect in generation of the third oxidized cysteine (<xref rid="fig4" ref-type="fig">Fig. 4, E and F</xref>). We determined the number of oxidized cysteines in the Mia40-bound precursor of wild-type and <italic>erv1</italic> mutant mitochondria. Full oxidation of the precursor strongly depended on the activity of Erv1, whereas oxidation of the first cysteine (mixed disulfide with Mia40) was only moderately affected in the <italic>erv1</italic> mutant (<xref rid="fig4" ref-type="fig">Fig. 4 G</xref>). Thus, active Erv1 is critical for the efficient transfer of a further disulfide bond to the precursor, which is bound to Mia40 via a mixed disulfide, demonstrating the function of Erv1 in the ternary complex. We conclude that the association of Erv1 with the precursor–Mia40 complex permits an efficient transfer of disulfides in a ternary complex.</p></sec><sec><title>Conclusions</title><p>We report that the interactions of Mia40 with Erv1 and precursor are not mutually exclusive, but all three components associate in a ternary complex. This intermediate complex is a transient step in precursor oxidation and thus has escaped experimental detection in previous studies. We found two means to trap precursors in a ternary complex. First, a trap mutant form of Mia40 accumulated precursors and Erv1. Second, mitochondria were swollen, leading to a release of IMS components, including approximately half of Erv1, whereas the amount of membrane-bound Mia40 was unchanged. Erv1 was rate-limiting for dissociation of the precursor–Mia40 complex because overexpression as well as addition of purified Erv1 promoted substrate release from Mia40. The ternary complex thus accumulated in mitoplasts containing limiting amounts of Erv1.</p><p>Two possibilities are conceivable for the organization of the ternary complex. Erv1 may bind to the precursor or to Mia40. Our findings support the scenario that Erv1 does not directly bind the precursor but acts on Mia40 because the coprecipitation of precursor with Erv1 requires the presence of functional Mia40, and is enhanced by the Mia40-trap form. We suggest that Erv1 promotes maturation (oxidation) of Mia40-bound substrate in the ternary complex through the transfer of disulfide bonds to precursors via Mia40, and this serves as a prerequisite for substrate release.</p><p>Most disulfide relay components of mitochondria, bacteria, and ER share only limited homology on the primary structure level; however, basic functional mechanisms of disulfide shuttling may be related (<xref ref-type="bibr" rid="bib8">Gerber et al., 2001</xref>; <xref ref-type="bibr" rid="bib31">Sevier et al., 2001</xref>; <xref ref-type="bibr" rid="bib9">Gross et al., 2002</xref>; <xref ref-type="bibr" rid="bib18">Kadokura et al., 2004</xref>; <xref ref-type="bibr" rid="bib38">Tu and Weissman, 2004</xref>; <xref ref-type="bibr" rid="bib14">Inaba et al., 2006</xref>; <xref ref-type="bibr" rid="bib30">Sevier and Kaiser, 2006</xref>). By splitting bacterial DsbB into two domains, disulfide transfer in a complex of three polypeptides, DsbA with the two DsbB domains was shown (<xref ref-type="bibr" rid="bib17">Kadokura and Beckwith, 2002</xref>). Here, the central polypeptide contained one redox-active pair of cysteines; in the ternary complex, each cysteine was bound to a different partner polypeptide. Mia40 may function in a similar manner with one redox-active pair of cysteines (<xref ref-type="bibr" rid="bib10">Grumbt et al., 2007</xref>). Alternatively, more than one cysteine residue of precursor and Mia40 may be involved in forming transient disulfides in the ternary complex. Several components of the ER and bacterial systems contain more than one redox-active pair of cysteines (<xref ref-type="bibr" rid="bib37">Tsai and Rapoport, 2002</xref>; <xref ref-type="bibr" rid="bib18">Kadokura et al., 2004</xref>; <xref ref-type="bibr" rid="bib20">Kulp et al., 2006</xref>; <xref ref-type="bibr" rid="bib30">Sevier and Kaiser, 2006</xref>). Moreover, more than one molecule of Mia40 may participate in the reaction, as human Mia40 forms a homo-oligomer linked by noncovalent interactions (<xref ref-type="bibr" rid="bib13">Hofmann et al., 2005</xref>). In addition to disulfide-mediated interactions, noncovalent interactions and conformational changes likely contribute to the cooperation of the partners in the ternary complex.</p><p>The findings presented here change the view that the disulfide carrier (Mia40) strictly alternates between binding to Erv1 and precursor in a sequential relay system (<xref ref-type="bibr" rid="bib22">Mesecke et al., 2005</xref>; <xref ref-type="bibr" rid="bib11">Herrmann and Köhl, 2007</xref>). The identification of a ternary complex suggests that the flow of electrons between oxidase, carrier, and substrate does not require sequential association and dissociation of the components, but rather their concerted action within a single complex. Thus, two distinct redox reactions are spatially coupled, which results in a channeling mechanism for disulfide transfer. Moreover, for substrates that receive more than one disulfide, the binding of Erv1 to the Mia40–precursor complex may permit an efficient transfer of multiple disulfides in a ternary complex. We speculate that the concept of mitochondrial disulfide channeling, comprising oxidase, disulfide carrier, and substrate, provides mechanistic implications for the ER and bacterial systems.</p></sec></sec><sec sec-type="materials|methods"><title>Materials and methods</title><sec><title>Strains</title><p>Strains generated and used in this study are derivatives of the wild-type YPH499 strain of yeast <italic>Saccharomyces cerevisiae</italic> (<italic>MAT</italic>a, <italic>ade2-101</italic>, <italic>his3-Δ200</italic>, <italic>leu2-Δ1</italic>, <italic>ura3-52</italic>, <italic>trp1-Δ63</italic>, and <italic>lys2-801</italic>). The temperature-sensitive <italic>erv1-1</italic> (YPH-BG-Erv1ts-F124S), <italic>erv1-2</italic> (YBG-0702b), <italic>erv1-3</italic> (YBG-0701a), and <italic>erv1-5</italic> (YPH-BGErv1ts-C159S) mutants have been described previously (<xref ref-type="bibr" rid="bib29">Rissler et al., 2005</xref>; <xref ref-type="bibr" rid="bib23">Milenkovic et al., 2007</xref>; <xref ref-type="bibr" rid="bib24">Müller et al., 2008</xref>). The mutant strain <italic>erv1-6</italic> was generated by site-directed mutagenesis followed by plasmid shuffling in the background of the genomic deletion of <italic>ERV1</italic> (YPH-BG-Erv1ts-C176S; <italic>MAT</italic>a, <italic>ade2-101</italic>, <italic>his3-Δ200</italic>, <italic>leu2-Δ1</italic>, <italic>ura3-52</italic>, <italic>trp1-Δ63</italic>, <italic>lys2-801</italic>, and <italic>erv1∷ADE2</italic> [pFL39-<italic>erv1-</italic>C176S]). The <italic>erv1-6</italic> mutant possesses the amino acid substitution Cys176Ser (<xref ref-type="bibr" rid="bib12">Hofhaus et al., 2003</xref>).</p><p>To overproduce Erv1, the yeast YPH499 strain and <italic>mia40-4</italic> mutant strain were transformed with the plasmid p9014-8 harboring the <italic>ERV1</italic> gene under the control of its endogenous promoter and terminator in the multicopy Yep352 yeast vector. The suppression by p9014-8 was analyzed on YPD plates (1% [wt/vol] yeast extract, 2% [wt/vol] bactopeptone, and 2% [wt/vol] glucose). The C-terminal His<sub>10</sub>-tagged versions of Mia40 were generated by genomic integration in a wild-type background, resulting in Mia40<sub>His</sub> (yAC53; <italic>MAT</italic>a, <italic>ade2-101</italic>, <italic>his3-Δ200</italic>, <italic>leu2-Δ1</italic>, <italic>ura3-52</italic>, <italic>trp1-Δ63</italic>, <italic>lys2-801</italic>, and <italic>MIA40<sub>His10</sub></italic>; <xref ref-type="bibr" rid="bib23">Milenkovic et al., 2007</xref>), and in the <italic>erv1-2</italic> mutant background, resulting in <italic>erv1-2</italic> Mia40<sub>His</sub> (yAC55-81; <italic>MAT</italic>a, <italic>ade2-101</italic>, <italic>his3-Δ200</italic>, <italic>leu2-Δ1</italic>, <italic>ura3-52</italic>, <italic>trp1-Δ63</italic>, <italic>lys2-801</italic>, <italic>MIA40<sub>His10</sub></italic>, and <italic>erv1∷ADE2</italic> [pFL39-<italic>erv1-2</italic>]). The Mia40-4int strain (BG-fomp2-7-int-4-12; <italic>MAT</italic>a, <italic>ade2-101</italic>, <italic>his3-Δ200</italic>, <italic>leu2-Δ1</italic>, <italic>ura3-52</italic>, <italic>trp1-Δ63</italic>, <italic>lys2-801</italic>, and <italic>mia40-4</italic>) was constructed by integration of the <italic>mia40-4</italic> allele (amino acids substitutions T313A, I316M, and F342L according to original open reading frame assignment in the <italic>Saccharomyces</italic> Genome Database [<xref ref-type="bibr" rid="bib2">Chacinska et al., 2004</xref>]; T289A, I292M, and F318L according to the current open reading frame assignment) into the genome of YPH499. Subsequently, a His<sub>10</sub> tag was introduced at the 3′ end of the <italic>mia40-4</italic> genomic coding sequence, giving rise to Mia40-4<sub>His</sub> (yAC64-1; <italic>MAT</italic>a, <italic>ade2-101</italic>, <italic>his3-Δ200</italic>, <italic>leu2-Δ1</italic>, <italic>ura3-52</italic>, <italic>trp1-Δ63</italic>, <italic>lys2-801</italic>, and <italic>mia40-4<sub>His10</sub></italic>).</p></sec><sec><title>Mitochondrial procedures</title><p>Mitochondria were isolated from <italic>S. cerevisiae</italic> strains grown on YPG medium (1% [wt/vol] yeast extract, 2% [wt/vol] bactopeptone, and 3% [wt/vol] glycerol) at 19, 24, or 30°C by differential centrifugation, as described previously, and mitoplasts were generated by hypoosmotic swelling (<xref ref-type="bibr" rid="bib33">Stojanovski et al., 2007</xref>). Mitochondrial precursor proteins were synthesized in rabbit reticulocyte lysate in the presence of [<sup>35</sup>S]methionine and subjected to in vitro import assays (<xref ref-type="bibr" rid="bib33">Stojanovski et al., 2007</xref>). Import reactions were performed at 25°C (dicarboxylate carrier and Tim9), 30°C (Tim8, Tim9, and Tim13), or 35°C (Tim10 and Tim22). Samples analyzed by nonreducing SDS-PAGE were solubilized in Laemmli buffer (minus reducing agent) containing 50 mM iodoacetamide. Carbonate extractions were performed as described previously (<xref ref-type="bibr" rid="bib33">Stojanovski et al., 2007</xref>).</p><p>For coimmunoprecipitation experiments, <sup>35</sup>S-labeled Tim9 was imported into mitochondria or mitoplasts, and the reaction was stopped by the addition of 50 mM iodoacetamide. Mitochondria were isolated and solubilized in digitonin-containing buffer (1% digitonin, 20 mM Tris-Cl, pH 7.4, 50 mM NaCl, 10% [wt/vol] glycerol, and 50 mM iodoacetimide) for 20 min on ice. After clarification of the solubilized material, supernatants were isolated and incubated with IgGs coupled to protein–A Sepharose for 1 h at 4°C. Antibody-bound material was washed three times in digitonin-containing buffer, upon which Laemmli buffer was added to the samples. Samples were heated at 95°C for 5 min and analyzed by SDS-PAGE.</p><p>Recombinant Erv1<sub>His</sub> and its mutant versions (C130S and C133S) were purified via nickel–nitrilotriacetic acid–agarose as described previously (<xref ref-type="bibr" rid="bib12">Hofhaus et al., 2003</xref>; <xref ref-type="bibr" rid="bib23">Milenkovic et al., 2007</xref>). For release assays, recombinant Erv1 (1 and 2 μg per 50-μl reaction volume) was added, and samples were incubated at 30°C for 20 min. The reaction was stopped by the addition of 50 mM iodoacetamide. Analysis of protein complexes by blue native electrophoresis and antibody-shift/depletion analysis was performed as described previously (<xref ref-type="bibr" rid="bib40">Wiedemann et al., 2003</xref>; <xref ref-type="bibr" rid="bib2">Chacinska et al., 2004</xref>; <xref ref-type="bibr" rid="bib33">Stojanovski et al., 2007</xref>). Affinity purification of Mia40<sub>His</sub> and Mia40-4<sub>His</sub> was performed as described previously (<xref ref-type="bibr" rid="bib29">Rissler et al., 2005</xref>; <xref ref-type="bibr" rid="bib23">Milenkovic et al., 2007</xref>). Radiolabeled proteins were detected by digital autoradiography (Storm imaging system; GE Healthcare) and analyzed by ImageQuant software (GE Healthcare). Western blot analysis was performed using enhanced chemiluminescence.</p></sec><sec><title>Thiol-trapping experiments</title><p>Thiol modifications were performed as described previously (<xref ref-type="bibr" rid="bib15">Jakob et al., 1999</xref>; <xref ref-type="bibr" rid="bib4">Curran et al., 2002</xref>). Oxidation reactions of <sup>35</sup>S-precursors during their import into isolated mitochondria were blocked by modification of their free sulfhydryl groups with 50 mM iodoacetamide. After reisolation, mitochondria were solubilized in digitonin-containing buffer (1% digitonin, 20 mM Tris-Cl, pH 7.4, 50 mM NaCl, and 10% [wt/vol] glycerol) supplemented with 10 mM of the reducing agent Tris(2-carboxyethyl) phosphine hydrochloride (TCEP) at 30°C for 15 min to reduce existing disulfide bonds. After protein precipitation, samples were dissolved in Laemmli buffer containing 15 mM AMS at 25°C for 30 min to alkylate liberated thiols, and were subsequently denatured at 60°C for 15 min. For affinity purification, <sup>35</sup>S-precursors were imported into Mia40<sub>His</sub> mitochondria, and free thiols were subsequently blocked with 50 mM iodoacetamide. After reisolation, mitochondria were solubilized in digitonin-containing buffer (1% digitonin, 20 mM Tris-Cl, pH 7.4, 50 mM NaCl, 10% [wt/vol] glycerol, and 50 mM iodoacetamide) and subjected to nickel–nitrilotriacetic acid–agarose affinity chromatography. Elution fractions were treated with 10 mM TCEP at 30°C. After precipitation, samples were dissolved in Laemmli buffer containing 15 mM AMS at 25°C for 30 min followed by incubation at 60°C for 15 min. In some figures, nonrelevant gel lanes were excised digitally, and the loading intensities of the precursors were used for standardization. Proteins were separated by Tricine–SDS-PAGE.</p></sec><sec><title>Online supplemental material</title><p>Fig. S1 demonstrates conditions to form the ternary complex. Online supplemental material is available at <ext-link ext-link-type="uri" xlink:href="http://www.jcb.org/cgi/content/full/jcb.200804095/DC1">http://www.jcb.org/cgi/content/full/jcb.200804095/DC1</ext-link>.</p></sec></sec><sec sec-type="supplementary-material"><title>Supplementary Material</title><supplementary-material id="PMC_1" content-type="local-data"><caption><title>[Supplemental Material Index]</title></caption><media mimetype="text" mime-subtype="html" xlink:href="jcb.200804095_index.html"></media><media xlink:role="associated-file" mimetype="text" mime-subtype="html" xlink:href="jcb.200804095_1.html"></media><media xlink:role="associated-file" mimetype="image" mime-subtype="jpeg" xlink:href="jcb.200804095_JCB_200804095_FS1.jpg"></media><media xlink:role="associated-file" mimetype="image" mime-subtype="jpeg" xlink:href="jcb.200804095_JCB_200804095_FS1-01.jpg"></media></supplementary-material></sec></body><back><ack><p>We thank P. Rehling and N. Wiedemann for discussion.</p><p>This work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 746, the Excellence Initiative of the German Federal and State Governments (EXC 294), the Gottfried Wilhelm Leibniz Program, and the Fonds der Chemischen Industrie. D. Stojanovski and K. Gabriel were supported by Alexander von Humboldt research fellowships.</p></ack><ref-list><ref id="bib1"><citation citation-type="journal">Allen, S., V. Balabanidou, D.P. Sideris, T. Lisowsky, and K. Tokatlidis. <year>2005</year>. Erv1 mediates the Mia40-dependent protein import pathway and provides a functional link to the respiratory chain by shuttling electrons to cytochrome <italic>c</italic>. <source>J. Mol. Biol.</source> <volume>353</volume>:<fpage>937</fpage>–944.<pub-id pub-id-type="pmid">16185707</pub-id></citation></ref><ref id="bib2"><citation citation-type="journal">Chacinska, A., S. Pfannschmidt, N. Wiedemann, V. Kozjak, L.K. Sanjuán Szklarz, A. Schulze-Specking, K.N. 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