JCB: REPORT Y G O L O I B L L E C F O L A N R U O J E H T Mitochondrial protein import: precursor oxidation in a ternar y complex with disulﬁde carrier and sulfhydr yl oxidase Diana Stojanovski,1 Dusanka Milenkovic,1 Judith M. Müller,1 Kipros Gabriel,1 Agnes Schulze-Specking,1 Michael J. Baker,3 Michael T. Ryan,3 Bernard Guiard,4 Nikolaus Pfanner,1,2 and Agnieszka Chacinska1,2 1Institut für Biochemie und Molekularbiologie, Zentrum für Biochemie und Molekulare Zellforschung, and 2Centre for Biological Signalling Studies (bioss), Universität Freiburg, 79104 Freiburg, Germany 3Department of Biochemistry, La Trobe University, Melbourne 3086, Australia 4Centre de Génétique Moléculaire, Centre National de la Recherche Scientiﬁque, 91190 Gif-sur-Yvette, France T space proteins depends on speciﬁc machinery that he biogenesis of mitochondrial intermembrane transfers disulﬁde bonds to precursor proteins. The machinery shares features with protein relays for disulﬁde bond formation in the bacterial periplasm and endoplas- mic 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 bind- ing 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 precur- sor while both oxidase and precursor are associated with the disulﬁde carrier Mia40. Introduction Mitochondria use diverse mechanisms to import precursor pro- tein (Jensen and Johnson, 2001; Endo et al., 2003; Koehler, 2004; Oka and Mihara, 2005; Dolezal et al., 2006; Neupert and Herrmann, 2007). The mitochondrial intermembrane space (IMS) contains an import and assembly (MIA) machinery, which transfers disulfide bonds to precursors and promotes their im- port and maturation (Chacinska et al., 2004; Koehler, 2004; Mesecke et al., 2005; Tokatlidis, 2005). The MIA pathway was reported to operate as a protein relay for disulfide bond forma- tion like the disulfide relay systems of the bacterial periplasm (disulfide bond formation protein B [DsbB]–DsbA) and the ER (Ero1–protein disulfide isomerase [PDI]; Collet and Bardwell, 2002; Kadokura et al., 2004; Tu and Weissman, 2004; Sevier D. Stojanovski and D. Milenkovic contributed equally to this paper. Correspondence to Nikolaus Pfanner: nikolaus.pfanner@biochemie.uni-freiburg. de; or Agnieszka Chacinska: agnieszka.chacinska@biochemie.uni-freiburg.de K. Gabriel’s present address is Department of Genetics, University of Melbourne, Parkville 3010, Australia 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 in- ner mitochondrial membrane. The online version of this article contains supplemental material. The Rockefeller University Press $30.00 J. Cell Biol. Vol. 183 No. 2 195–202 www.jcb.org/cgi/doi/10.1083/jcb.200804095 and Kaiser, 2006; Herrmann and Köhl, 2007). 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 pro- tein arriving in the periplasm, ER, or IMS. Mixed disulfides are generated between oxidase and dis- ulfide 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; Collet and Bardwell, 2002; Kadokura et al., 2004; Tu and Weissman, 2004; Mesecke et al., 2005; Sevier and Kaiser, 2006; Herrmann and Köhl, 2007). 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 © 2008 Stojanovski et al. This article is distributed under the terms of an Attribution– Noncommercial–Share Alike–No Mirror Sites license for the ﬁrst six months after the publica- tion date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/). JCB 195