Summary The biogenesis of mitochondria requires the import of a large number of proteins from the cytosol [1, 2]. While numerous studies have defined the proteinaceous machineries that mediate mitochondrial protein sorting, little is known about the role of lipids in mitochondrial protein import. Cardiolipin, the signature phospholipid of the mitochondrial inner membrane [3–5], affects the stability of many inner membrane protein complexes [6–12]. Perturbation of cardiolipin metabolism leads to the X-linked cardioskeletal myopathy, Barth syndrome [13–18]. We report that cardiolipin affects the preprotein translocases of the mitochondrial outer membrane. Cardiolipin mutants genetically interact with mutants of outer membrane translocases. Mitochondria from cardiolipin yeast mutants, as well as Barth syndrome patients, are impaired in the biogenesis of outer membrane proteins. Our findings reveal a new role for cardiolipin in protein sorting at the mitochondrial outer membrane and bear implications for the pathogenesis of Barth syndrome.

The intermembrane space of mitochondria contains the specific mitochondrial intermembrane space assembly (MIA) machinery that operates in the biogenesis pathway of precursor proteins destined to this compartment. The Mia40 component of the MIA pathway functions as a receptor and binds incoming precursors, forming an essential early intermediate in the biogenesis of intermembrane space proteins. The elements that are crucial for the association of the intermembrane space precursors with Mia40 have not been determined. In this study, we found that a region within the Tim9 and Tim10 precursors, consisting of only nine amino acid residues, functions as a signal for the engagement of substrate proteins with the Mia40 receptor. Furthermore, the signal contains sufficient information to facilitate the transfer of proteins across the outer membrane to the intermembrane space. Thus, here we have identified the mitochondrial intermembrane space sorting signal required for delivery of proteins to the mitochondrial intermembrane space. INTRODUCTIONMitochondria pose a great challenge for the proper delivery of proteins because of their complex architecture. Mitochondrial precursors must find their way to one of the four mitochondrial subcompartments: the outer membrane, intermembrane space, inner membrane, or matrix. As a direct consequence of this complexity, several machineries for the translocation and sorting of mitochondrial precursors have evolved. Interplay between these machineries and specific signals present in the precursors drive different protein targeting pathways (Schatz and Dobberstein, 1996;Emanuelsson and von Heijne, 2001;Jensen and Johnson, 2001;Endo et al., 2003;Koehler, 2004;Oka and Mihara, 2005;Dolezal et al., 2006;Neupert and Herrmann, 2007;Bolender et al., 2008). Initially, mitochondrial precursors are recognized in a signal-dependent manner by specific receptors and are transferred across the barrier of outer mitochondrial membrane by using the translocase of the outer membrane (TOM) complex. On the trans-side of the outer mitochondrial membrane, sorting machineries decode specific signals in precursors, and this results in the branching of protein import pathways. The most well characterized is the presequence pathway across the inner membrane driven by a cleavable and positively charged signal sequence, called a presequence, and the translocase of the inner membrane (TIM) 23 complex Endo et al., 2003;Oka and Mihara, 2005;Neupert and Herrmann, 2007;Bolender et al., 2008). The presequence is cleaved off by a specific protease liberating the mature protein. However, other mitochondrial signals are not proteolytically removed and remain as part of the native mitochondrial protein. One example is the recently identified ␤-signal that is recognized by the sorting and assembly machinery (SAM) complex to sort ␤-barrel proteins to the mitochondrial outer membrane . In other mitochondrial membrane proteins, the membrane domains, anchors, and their surrounding regions are used to some extent for selection of ...

Abbreviations used in this paper: AAC, ADP/ATP carrier; Crd1, cardiolipin synthase; DIC, dicarboxylate carrier; LCMS, liquid chromatography/mass spectrometry; Mmp37, mitochondrial matrix protein 37; PA, phosphatidic acid; PAM, presequence translocase-associated motor; PG, phosphatidylglycerol; PGP, phosphatidylglycerophosphate; Pgs1, PGP synthase; Tam41, translocator assembly and maintenance protein 41; TIM, translocase of the inner membrane.

The mitochondrial inner membrane harbors the complexes of the respiratory chain and translocase complexes for precursor proteins. We have identified a further subunit of the carrier translocase (TIM22 complex) that surprisingly is identical to subunit 3 of respiratory complex II, succinate dehydrogenase (Sdh3). The membrane-integral protein Sdh3 plays specific functions in electron transfer in complex II. We show by genetic and biochemical approaches that Sdh3 also plays specific functions in the TIM22 complex. Sdh3 forms a subcomplex with Tim18 and is involved in biogenesis and assembly of the membrane-integral subunits of the TIM22 complex. We conclude that the assembly of Sdh3 with different partner proteins, Sdh4 and Tim18, recruits it to two different mitochondrial membrane complexes with functions in bioenergetics and protein biogenesis, respectively.

The mitochondrial inner membrane contains preprotein translocases that mediate insertion of hydrophobic proteins. Little is known about how the individual components of these inner membrane preprotein translocases combine to form multisubunit complexes. We have analyzed the assembly pathway of the three membrane-integral subunits Tim18, Tim22, and Tim54 of the twin-pore carrier translocase. Tim54 displayed the most complex pathway involving four preprotein translocases. The precursor is translocated across the intermembrane space in a supercomplex of outer and inner membrane translocases. The TIM10 complex, which translocates the precursor of Tim22 through the intermembrane space, functions in a new posttranslocational manner: in case of Tim54, it is required for the integration of Tim54 into the carrier translocase. Tim18, the function of which has been unknown so far, stimulates integration of Tim54 into the carrier translocase. We show that the carrier translocase is built via a modular process and that each subunit follows a different assembly route. Membrane insertion and assembly into the oligomeric complex are uncoupled for each precursor protein. We propose that the mitochondrial assembly machinery has adapted to the needs of each membrane-integral subunit and that the uncoupling of translocation and oligomerization is an important principle to ensure continuous import and assembly of protein complexes in a highly active membrane.The majority of mitochondrial proteins are nucleus encoded and imported into mitochondria through protein translocase complexes (6,7,17,25,29,33,42). The translocase of the outer membrane (TOM complex) is the general entry gate for mitochondrial precursor proteins. Two translocases of the inner membrane (TIM), the presequence translocase (TIM23 complex) and the twin-pore carrier translocase (TIM22 complex), mediate signal-selective transport of precursor proteins. While the TIM23 complex translocates the majority of substrates into the matrix and inserts only a limited number of substrates into the inner membrane (7,10,13,17,25,26,40), the TIM22 complex is dedicated to the insertion of multispanning hydrophobic proteins into the inner membrane, including a large number of metabolite carriers (7,17,25,27,29,46). The TIM22 complex is a voltage-dependent 300-kDa complex with three membrane-integral subunits, Tim18, Tim22, and Tim54. Tim22 forms the voltage-sensitive channels of the twin-pore translocase (21, 30). Tim54 was shown to play a role in the assembly of a protease complex (Yme1) of the inner membrane, yet the molecular mechanism of its action has not been elucidated (12). Thus, the molecular functions of Tim54 and Tim18 in the TIM22 complex are unknown (6,7,17,25,29).The precursors of metabolite carriers are not directly transferred from the TOM complex to the TIM22 complex, but the TIM10 translocase complex of the intermembrane space binds to the precursors and functions in a chaperone-like manner to guide them through the aqueous space between outer and inner membranes. ...