A Pathway of Protein Translocation in Mitochondria Mediated by the AAA-ATPase Bcs1

Molecular Cell

Article A Pathway of Protein Translocation in Mitochondria Mediated by the AAA-ATPase Bcs1 Nikola Wagener,1,2 Markus Ackermann,1,2 Soledad Funes,2,3 and Walter Neupert1,2,* 1Max-Planck-Institut

fu¨r Biochemie, Am Klopferspitz 18, 82152 Martinsried, Germany fu¨r Physiologische Chemie, Universita¨t Mu¨nchen, Butenandtstrasse 5, 81377 Mu¨nchen, Germany 3Present address: Departamento de Gene ´ tica Molecular, Instituto de Fisiologı´a Celular, Circuito Exterior s/n, Ciudad Universitaria, Universidad Nacional Auto´noma de Me´xico, Me´xico, Distrito Federal, 04510, Me´xico *Correspondence: [email protected] DOI 10.1016/j.molcel.2011.07.036 2Institut

SUMMARY

The AAA+ family in eukaryotes has many members in various cellular compartments with a role in protein unfolding and degradation. We show that the mitochondrial AAA-ATPase Bcs1 has an unusual function in protein translocation. Bcs1 mediates topogenesis of the Rieske protein, Rip1, a component of respiratory chains in bacteria, mitochondria, and chloroplasts. The oligomeric AAA-ATPase Bcs1 is involved in export of the folded Fe-S domain of Rip1 across the inner membrane and insertion of its transmembrane segment into an assembly intermediate of the cytochrome bc1 complex, thus revealing an unexpected mechanistical concept of protein translocation across membranes. Furthermore, we describe structural elements of Rip1 required for recognition and export by as well as ATP-dependent lateral release from the AAA-ATPase. In bacteria and chloroplasts Rip1 uses the Tat machinery for topogenesis; however, mitochondria have lost this machinery during evolution and a member of the AAA-ATPase family has taken over its function.

INTRODUCTION AAA-proteins (ATPases associated with diverse cellular activities) show a wide distribution between all kingdoms of life. Proteins belonging to this family exhibit diverse functions such as unfolding and disaggregation of proteins and their degradation, but also protein extraction from membranes for their processing and maturation (Sauer et al., 2004; Truscott et al., 2010). They comprise one or two highly conserved ATP-binding domains that form a channel loop that is possibly involved in substrate transport through a central channel made up by six AAA-ATPase subunits (Zolkiewski, 2006). Within eukaryotic cells AAA-proteins are found in several cellular subcompartments. In mitochondria, members of the family are involved in crucial processes like organelle fusion and mitochondrial protein translation and degradation (Tatsuta and Langer, 2009). Another mitochondrial AAA-ATPase was identified to be required for stable

expression and assembly of the mitochondrial Rieske protein Rip1 (Nobrega et al., 1992; Cruciat et al., 1999). Rip1 is a key subunit of the ubiquinol-cytochrome c reductase, the bc1 complex (or complex III). It spans the inner mitochondrial membrane exposing its C-terminal domain to the intermembrane space and extends its N terminus into the matrix (Hunte et al., 2000). The C-terminal domain carries a 2Fe-2S center. The AAA-ATPase involved in its assembly was named Bcs1 for ubiquinol-cytochrome c reductase (bc1) synthesis. bcs1 null mutants lack bc1 complex activity and show reduced levels of Rip1. BCS1 genes are found in virtually all eukaryotic organisms from yeast to human and represent an outlying clade of the large family of AAA-proteins (Frickey and Lupas, 2004). The Bcs1 protein is anchored to the inner membrane with a single transmembrane domain located next to the short hydrophilic N-terminal segment that protrudes into the intermembrane space (IMS). The large C-terminal domain faces the matrix (Nobrega et al., 1992; Fo¨lsch et al., 1996). Various functions of Bcs1 were suggested, such as embedding of the Fe-S cluster into the Rip1 apoprotein, chaperoning the transfer of Rip1 into the bc1 precomplex, or involvement in the formation of the supercomplexes between the cytochrome bc1 and the cytochrome c oxidase complexes (Nobrega et al., 1992; Cruciat et al., 1999; Zara et al., 2007). Interestingly, mutations in the human BCS1L gene were reported to be linked to various neurological and metabolic disorders such as the Gracile syndrome (Fernandez-Vizarra et al., 2007; Bla´zquez et al., 2009; Kotarsky et al., 2010). Rip1 is synthesized as precursor with a classical mitochondrial matrix targeting signal. It enters the mitochondria with its N terminus, which is followed by a hydrophobic membrane segment that could act as a stop-transfer signal, in a process mediated by TOM and TIM23. However, in previous studies import in vitro of Rip1 was not stopped at the level of TIM23 (Hartl et al., 1986; van Loon and Schatz, 1987). The Fe-S cluster, or a precursor thereof, is added to the apoprotein in the mitochondrial matrix (Kispal et al., 1997; Kispal et al., 1999), apparently because biosynthesis of Fe-S clusters takes place there (for review, see Lill and Mu¨hlenhoff, 2006). These observations imply that the C-terminal domain is initially present in the matrix and then translocated with the bound Fe-S cluster back to the IMS. On its further assembly pathway, Rip1 is the penultimate subunit that is incorporated into the bc1 complex. A dimeric intermediate assembly complex (bc1 precomplex) was observed that contains

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plex lacking the Rip1 fails to assemble into supercomplexes (Cruciat et al., 1999). Notably, in bacteria, the prokaryotic ancestors of mitochondria, as well as in chloroplasts, the Tat system functions as an export machinery for folded proteins, including the Rieske Fe-S protein, to the periplasmic space and to the intrathylakoid space, respectively (Molik et al., 2001; Aldridge et al., 2008). During evolution of mitochondria, this machinery was lost. This raises the intriguing question of how Rip1 reaches its final topology and which component would have taken over the function of the Tat system. In the present study, we have analyzed the biogenesis pathway of Rip1 as a model substrate of Bcs1 in S. cerevisiae with the aim of showing that Bcs1 is responsible for the export of the Fe-S domain. We report that the precursor of Rip1 is first completely imported into the mitochondrial matrix, where it becomes processed and the C-terminal domain folded. In a next step it associates with the Bcs1 protein to become translocated across the inner membrane to the IMS side. Surprisingly, Bcs1 recognizes Rip1 in its folded conformation, as mutants preventing folding are not exported. At this stage Rip1 is firmly associated with the inner membrane. In an ATPdependent reaction, it further assembles into the bc1 precomplex. We conclude that the AAA-ATPase Bcs1 is acting as a protein translocase and has apparently replaced the Tat system as an export machinery for folded mitochondrial Rieske Fe-S protein. We suggest that the pathway but not the exporting protein translocase has been conserved during the evolution of mitochondria. RESULTS

Figure 1. Cells Lacking Bcs1 Contain Soluble Unassembled Rip1 in the Mitochondrial Matrix (A) Mitochondria (150 mg protein) from wild-type (W303), Drip1, and Dbcs1 cells were solubilized with 3% digitonin, subjected to BNGE and immunoblotting using antibodies to cytochrome c1 (Cyt1) and Rip1. p-III: bc1 intermediate assembly complex; p-III2: p-III dimer; IV: cytochrome c oxidase (respiratory chain complex IV); IV2: cytochrome c oxidase dimer. (B) Mitochondria from wild-type and Dbcs1 cells were treated with carbonate (pH 10) for 30 min at 4 C. Soluble (S) and membrane (M) fractions were separated by sucrose flotation from a 1.6 M sucrose cushion through a 1.4 M sucrose to a 250 mM sucrose layer. Fractions were analyzed by SDS-PAGE and immunoblotting with antibodies to Rip1, Hep1 (matrix protein), and Oxa1 (inner membrane integrated). (C) Mitochondria from wild-type and Dbcs1 cells were incubated with increasing amounts of digitonin and Triton X-100 as indicated in the presence of proteinase K. Proteins were subjected to SDS-PAGE and immunoblotting using antibodies to Rip1, and the markers Hep1 and Tim50 (inner membrane protein exposed to the IMS). Dig.: digitonin.

all subunits, in particular cytochrome c1, cytochrome b, and the core proteins 1 and 2, except Rip1 and Qcr10 (Grivell, 1989; Tzagoloff, 1995; Cruciat et al., 1999; Zara et al., 2007). The precom-

Deletion of Bcs1 Leads to Accumulation of Rip1 in the Matrix Bcs1 has been identified as a component required for the assembly of Rip1 into the bc1 complex. We asked which of the various steps of the pathway of Rip1 would be affected by deletion of Bcs1 in yeast. First, we analyzed the complex by Blue native gel electrophoresis (BNGE) and immunodecoration with antibodies against cytochrome c1 (Cyt1) (Figure 1A). In wildtype mitochondria, Rip1 was present in the bc1 supercomplexes. In cells lacking Bcs1 (Dbcs1), the supercomplexes were missing and instead Cyt1 was found in the bc1 precomplex (p-III, p-III2). The same pattern was observed in mitochondria from a mutant in which the RIP1 gene was deleted (Figure 1A). In Dbcs1 mitochondria, in contrast, Rip1 was not detectable in a high molecular mass complex. Instead, Rip1 was detected in the lower molecular mass region, obviously an unassembled species of Rip1. This species was, on the other hand, not distinguishable in mitochondria from wild-type (data not shown). To characterize the submitochondrial localization of Rip1 present in Dbcs1 cells, mitochondria were subjected to alkaline treatment followed by flotation centrifugation. In wild-type mitochondria, Rip1 was virtually completely recovered in the membrane fraction; in contrast, in Dbcs1 mitochondria, the majority was found in the soluble fraction (Figure 1B). Apparently, the assembled Rip1 in wild-type is firmly embedded in the interior of the bc1 complex, whereas the intermediate species of

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To exclude the possibility that the observed assembly defect in the latter two mutant mitochondria was due to diminished import, Rip1 precursor was incubated with mitochondria from the different types of cells, either in the presence or absence of ATP and NADH (Figure 2B). Efficient import was apparent with all types of mitochondria in the presence of these added nucleotides, which supported formation of a membrane potential and maintenance of intramitochondrial ATP levels. In contrast, in their absence, import into mitochondria from the mutants was almost completely abolished. This is due to the diminished membrane potential of mitochondria from the mutant cells. In summary, we have worked out a system in which the complete process of assembly of Rip1 can be followed in vitro.

Figure 2. Rip1 Precursor Is Imported and Assembled into Supercomplexes in Isolated Mitochondria from Wild-Type but Not Dbcs1 or Dcyt1 Cells (A) Radiolabeled Rip1 was incubated with mitochondria (150 mg) from wildtype, Dbcs1, Drip1, and Dcyt1 cells for the indicated time periods. Samples were analyzed for assembled Rip1 by BNGE, western blotting, and autoradiography. (B) Radiolabeled Rip1 was incubated with mitochondria (100 mg) in the absence or presence of NADH and ATP; import was analyzed by SDS-PAGE and autoradiography. i: intermediate form of Rip1; m: mature Rip1.

Rip1 in Dbcs1 mitochondria is not membrane-integrated. The double band of Rip1 that can be sometimes observed corresponds to the intermediate and the mature form of the protein. Incomplete processing is observed under a variety of conditions, such as import in vitro and episomal expression of Rip1. This, however, is not relevant for correct targeting of Rip1, since even complete block of the second processing does not affect correct assembly (Nett et al., 1998). We then asked in which subcompartment of Dbcs1 mitochondria Rip1 was located. The mitochondrial membranes were opened in a stepwise fashion by incubation with increasing amounts of digitonin in the presence of proteinase K. Rip1 remained stable when the outer membrane was opened due to its integration into the larger bc1 complex, but, in contrast to wild-type, Rip1 was degraded as soon as the inner membrane was opened (Figure 1C, compare lanes 6 and 12). We conclude that in Dbcs1 mitochondria, Rip1 is present in the matrix, either freely soluble or attached to the inner membrane. An In Vitro System to Analyze Assembly of Rip1 from Import into Mitochondria to the Formation of Cytochrome bc1 Supercomplexes In order to follow the individual steps of the assembly pathway, it was necessary to develop an in vitro system starting with the precursor form of Rip1 and isolated mitochondria. Radiolabeled Rip1 precursor was incubated with mitochondria from wild-type as well as Dbcs1, Drip1, and Dcyt1 cells in the presence of ATP, NADH, and an ATP-regenerating system (Glick, 1995). With wildtype and Drip1 mitochondria, imported Rip1 was recovered in complexes of the same size as authentic bc1 supercomplexes. In contrast, in Dbcs1 and Dcyt1 mitochondria, no complexes containing imported Rip1 were observed (Figure 2A).

Export of Rip1 from the Matrix Depends on the Presence of Bcs1 but Not of the Cytochrome bc1 Precomplex In order to study the steps of Rip1 topogenesis after import into the matrix, we analyzed its submitochondrial location in cytochrome c1 deficient cells. Dcyt1 cells lack the bc1 precomplex, therefore Rip1 cannot take the step of integration into this intermediate complex. Radiolabeled Rip1 was imported into Dcyt1 and, as a control, into wild-type and Dbcs1 mitochondria. Then, they were treated at alkaline pH, and extracted material was separated by centrifugation from sedimentable material (Figure 3A). Imported Rip1 in Dbcs1 mitochondria was found in the extract fraction. In wild-type, a considerable part was present in the pellet, consistent with our previous observation with the observed assembly into supercomplexes (see Figure 2A). In contrast, in Dcyt1 mitochondria, Rip1 was found in the soluble fraction upon alkaline treatment. We also localized the Rip1 intermediate accumulating in Dcyt1 in vivo by titration of isolated mitochondria with digitonin in the presence of proteinase K. At concentrations of digitonin sufficient to open the outer membrane, Rip1 was degraded (Figure 3B). This was in contrast to the results with Dbcs1 mitochondria, in which Rip1 remained protected against proteolysis until the inner membrane was opened (see Figure 1C). We conclude that in the presence of Bcs1, Rip1 is translocated out of the matrix across the inner membrane, or at least parts of it. Furthermore, this translocation process is not dependent on the presence of the bc1 precomplex. Is the exported species translocated completely into the intermembrane space, or does it remain associated with the inner membrane? We ruptured mitochondria from wild-type, Dbcs1, and Dcyt1 cells by repeated freezing and thawing in buffer containing between 0 and 250 mM KCl and then separated the soluble and membrane fraction by centrifugation. In mitochondria from wild-type and Dcyt1 cells, Rip1 was recovered completely in the membrane fraction. In contrast, in Dbcs1 cells, there was an equal distribution between soluble and membrane fraction, presumably due to hydrophobic interactions at all KCl concentrations applied (Figure 3C). In summary, in the presence of Bcs1 but absence of cytochrome c1 when the receiving bc1 precomplex is missing, Rip1 is exported from the matrix and is arrested in tight association with the inner membrane. We then asked whether Rip1 in the matrix of Dbcs1 mitochondria was present in a folded or unfolded state with regard to the domain that binds the Fe-S cluster and has to be translocated across

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the inner membrane. Mitochondria from wild-type, Dcyt1, and Dbcs1 cells were opened by sonication and the resulting fractions treated with protease in the presence or absence of SDS. In wild-type, Rip1 was not degraded by proteinase K, and only when SDS was present was a 15 kDa fragment produced. In the Dbcs1 samples, Rip1 was degraded even in the absence of SDS, but a 15 kDa fragment remained that interacted with the Rip1 antibody directed against a peptide segment of the C-terminal domain (residues 135–149). The formation of this folded fragment upon protease treatment was described previously by Li et al., 1981. In the samples from the Dcyt1 mitochondria, much less of Rip1 intermediate was present, but also this intermediate was degraded to yield a 15 kDa C-terminal fragment (Figure 3D). In summary, we conclude that the Fe-S domain of Rip1 acquires a folded state already in the matrix. Whether the domain contained an Fe-S cluster or was stably folded in its absence is not known. However, since Rip1 was shown to become assembled in vivo in the absence of the Fe-S cluster (Graham and Trumpower, 1991), an answer to this question appears not to be relevant in the context of the analysis of the role of Bcs1 in the topogenesis pathway of Rip1.

Figure 3. In Dbcs1 Mitochondria Folded Rip1 Is Present in the Matrix, Whereas in Dcyt1 Mitochondria It Is Exposed to the IMS Side (A) Radiolabeled Rip1 was imported into 100 mg mitochondria from wild-type, Dcyt1, and Dbcs1 cells. Subsequently, mitochondria were treated with carbonate (pH 10), and membrane-bound proteins (P1), aggregates (P2), and soluble proteins (S) were separated by stepwise centrifugation and analyzed by SDS-PAGE and autoradiography. (B) Mitochondria from Dcyt1 cells were treated and analyzed as in Figure 1C. (C) Mitochondria were ruptured by repeated freezing and thawing at different salt concentrations. Membrane-bound (M) and soluble (S) proteins were separated by centrifugation and subjected to SDS-PAGE and immunodecoration of Rip1 and the markers aconitase (Aco1, soluble), fumarase (Fum1, loosely membrane-associated) and Oxa1 (membrane integrated). (D) Mitochondria from the indicated strains were sonicated and treated with proteinase K in the absence or presence of 0.1% or 0.5% SDS. Formation of

Newly Imported Rip1 in the Matrix Interacts with Bcs1 in an ATP-Dependent Manner and Is an On-Pathway Intermediate Since Bcs1 turned out to be essential for translocation of Rip1, we looked for a direct interaction of these proteins. In wildtype cells, no Rip1 was detectable in association with Bcs1. This was not surprising, since in wild-type cells all Rip1 can assemble into the bc1 complex. We therefore imported radiolabeled Rip1 into isolated mitochondria from cells that contained either N-terminally His-tagged or wild-type Bcs1. Coprecipitation using NiNTA beads followed by BNGE showed a radioactive band at the position at which the native BCS1 complex migrated in the case of the mitochondria from the His-tagged cells, but not in case of the wild-type control (Figure 4A). Thus, a complex between Rip1 and Bcs1 is detectable in mitochondria containing newly imported Rip1. Interestingly, the amount of bound Rip1 depended on the temperature of the import reaction. At lower temperature more Rip1 in relation to total material imported was associated with Bcs1 (Figure 4B). This suggests that the Rip1 intermediate is partially arrested at Bcs1 at lower temperatures. In order to investigate the role of nucleotides in this interaction, radiolabeled precursor was imported into mitochondria containing His-tagged Bcs1, which were then further incubated in the presence or absence of ATP, ADP, or AMP-PNP. The amounts of Rip1 associated with Bcs1 were relatively low in the absence of any nucleotide and in the presence of ATP or ADP. In the presence of the slowly hydrolyzable AMP-PNP, a much higher proportion of imported Rip1 was found in association with Bcs1 (Figure 4C). This suggested that the interaction of Bcs1 with Rip1 is adenine nucleotide-dependent and critical for the

the Rip1 fragment representing the Fe-S-binding domain was analyzed by SDS-PAGE, western blot, and immunodecoration.

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assembly process of Rip1. To confirm this, we first imported Rip1 into mitochondria at a lower temperature (15 C) until import came to completion, and then continued incubation in the presence or absence of ATP at 25 C. Indeed, in the absence of ATP, assembly was drastically reduced, comparable to mitochondria from Dbcs1 cells that were analyzed in parallel as a control (Figure 4D). These observations suggest that in order to allow Bcs1 to function and release the Rip1 intermediate, hydrolysis of ATP is an essential step in the overall assembly process of Rip1. To strengthen this conclusion, we performed chase experiments, in which we followed the release of Rip1 from Bcs1 and its appearance in the cytochrome bc1 complex. Indeed, during the chase, Rip1 disappeared from Bcs1 and appeared in the supercomplexes (Figure 4E). This supports the notion that the Bcs1-Rip1 complex represents a true intermediate on the assembly pathway of Rip1. We then asked whether the release of Rip1 from Bcs1 is dependent on the presence of the bc1 precomplex. We performed NiNTA coprecipitation with mitochondria from cells that contained His-tagged Bcs1 and were either wild-type or lacked Cyt1. In neither case was endogenous Rip1 found in association with Bcs1. This was true whether the isolated mitochondria were preincubated with ATP or with AMP-PNP (Figure 4F). In addition, we performed the NiNTA coprecipitation with newly imported Rip1 and could chase Rip1 from Bcs1 in the absence of the bc1 precomplex, as in wild-type (data not shown). From this, we conclude that the release of Rip1 does not require the bc1 precomplex. Notably, the total levels of Rip1 in the Dcyt1 cells were greatly reduced as compared to wild-type (Figure 4G; see also Figure 3B). Unassembled Rip1 might be prone to degradation, in contrast to assembled Rip1. In conclusion, the Rip1 intermediate in the matrix interacts tightly and in an adenine nucleotide-dependent fashion with Bcs1, whose function does not depend on the availability of the accepting bc1 precomplex. Sequence Elements of Rip1 Required for Interaction with Bcs1 To investigate the nature of the interaction of Bcs1 and Rip1, we used several approaches. In a first approach we generated truncation mutants lacking an increasing number of amino acid residues at the very C terminus. The tight embedding of the C terminus in a b sheet conformation has most likely a critical role for stable folding of the Fe-S domain, and folding of Rip1 might be prerequisite for its recognition by Bcs1 and, therefore, its translocation. We asked whether the truncation mutants were able to complement the function of full-length Rip1(residues 1–215) in a Drip1 mutant. Interestingly, mutants lacking more than five residues could not rescue growth on a nonfermentable carbon source. A construct expressing Rip1(1–211) under the endogenous promoter was the shortest one with this capacity. Rip1(1–214) displayed reduced growth at 37 C (data not shown). The altered C terminus in these mutants apparently leads to a destabilized Fe-S domain at higher temperatures. Analysis of the supercomplexes revealed a close correlation between the level of assembled Rip1 and complementation of the growth defect (Figure 5A, left panel). Rip1(1–211), Rip1(1– 214), and full-length Rip1 were assembled into supercomplexes,

in contrast to the shorter forms. We also studied the assembly of the Rip1 variants in our in vitro assay (Figure 5A, right panel). Surprisingly, compared to full-length Rip1, only minor amounts of Rip1(1–214) were assembled and only traces of Rip1(1–211), indicating that their assembly kinetics were strongly reduced compared to wild-type Rip1. In agreement with these in vitro data, growth rates of the truncation mutants were decreased accordingly on nonfermentable carbon sources (Figure 5B). To investigate whether the C terminus of Rip1 is required for the interaction with Bcs1, we performed coprecipitation experiments with different radiolabeled Rip1 proteins upon import into isolated mitochondria containing His-tagged Bcs1 and determined complex formation between Bcs1 and Rip1. Indeed, Rip1(1–209) and Rip1(1–205) were not bound to Bcs1 when analyzed by BNGE (Figure 5C) or by SDS-PAGE (Figure 5D). We conclude that the lack of the very C terminus of Rip1 impairs assembly because interaction with Bcs1 is defective. We expressed mutant and wild-type Rip1 in Drip1 background and checked the mitochondria for presence of folded Rip1, by protease treatment. The Rip1 variant that lacked the C-terminal ten residues did not yield a protease-resistant 15 kDa fragment, in contrast to wild-type Rip1 (Figure 5E). Because of the increased expression of the Rip1 variants from the plasmid used, these variants were subject to aggregation in the matrix to a high degree. Therefore, the yield of the 15 kDa fragment was rather low, even in wild-type. We conclude from these experiments that interaction of Rip1 in the matrix with Bcs1 is correlated with the presence of a folded C-terminal domain in Rip1. Rip1 Can Bind to Bcs1 In Vitro To study the interaction of Rip1 with Bcs1 in more detail, we used a cell- and organelle-free system. First, we lysed mitochondria with digitonin, incubated the lysate with different nucleotides, and subjected it to BNGE and immunoblotting with antibodies against Bcs1. Upon addition of ATP, the band of Bcs1 was migrating at a slightly lower apparent molecular mass, as compared to addition of AMP-PNP (Figure 6A, left panel). BCS1 apparently undergoes conformational changes, as demonstrated for other AAA-ATPases, such as Hsp104 (Wendler et al., 2009), depending on the type of adenine nucleotide bound. We also studied the effect of addition of the recombinant Rip1 protein on the electrophoretic mobility of Bcs1. A massive shift of about 150–200 kDa apparent molecular mass of the BCS1 oligomer resulted when mature or C-terminally truncated Rip1 was added in the absence of nucleotides. In contrast, in the presence of ATP and Rip1, the electrophoretic mobility of Bcs1 was similar to that when both ATP and Rip1 were absent. To check the specificity of the effect of Rip1, we added recombinant Su9DHFR, a precursor protein used for studying mitochondrial import. No change of the electrophoretic mobility was observed in comparison to that seen in the presence of Rip1 (Figure 6A, right panel). We then performed the same experiments in a system in which Bcs1 was first isolated from cells containing His-tagged Bcs1. In this case, after BNGE, the proteins were stained with Coomassie Brilliant Blue (Figure 6B). The same behavior as described in Figure 6A was observed. In addition, reduced carboxymethylated

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Figure 4. Rip1 Imported into Isolated Mitochondria Associates with Bcs1 and Is an On-Pathway Intermediate in the Assembly of the bc1 Complex (A) Radiolabeled Rip1 was incubated with mitochondria from wild-type cells and cells containing His6-tagged Bcs1. Samples were analyzed for Bcs1-bound Rip1 by BNGE, western blotting, and autoradiography. (B) Radiolabeled Rip1 was imported into mitochondria from cells containing His6-Bcs1 at the indicated temperatures and analyzed for imported and Bcs1-bound Rip1 by NiNTA coprecipitation, SDS-PAGE, western blotting, autoradiography, and quantification.

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lactalbumin (RCMLA), a natively unfolded protein, was used as a control. The presence of Rip1 in the complex that was formed when no nucleotide was added was verified by immunoblotting (data not shown). Surprisingly, the interaction between BCS1 and Rip1 was independent of the presence of the C-terminal eight residues of Rip1 (Figures 6A and 6B). To further analyze the ATP dependence of this reaction, mitochondria were lysed as described in Figure 6A and incubated for 20 min in the presence of isolated Rip1 to allow its binding to BCS1. Aliquots were then incubated for a further 20 min, either in the absence of nucleotide or in the presence of ATP, AMPPNP, or ADP (Figure 6C, left panel). Dissociation of Rip1 from BCS1 occurred after addition of ATP, but not of AMP-PNP or ADP or in absence of any nucleotide. We further analyzed a Bcs1 mutant in which the Walker B Box contains the point mutation E319Q that is blocked in ATP hydrolysis. Bcs1 from this mutant was able to bind Rip1, but not to release it upon addition of ATP. Interestingly, this mutant BCS1 did not undergo a shift to an apparent higher molecular mass upon binding of Rip1 (Figure 6C, right panel). These findings demonstrate that Rip1 can tightly bind to BCS1 in the absence of ATP in vitro. The interaction causes a massive conformational change of BCS1, resulting in a highly altered mobility in BNGE that cannot only be caused by the binding of the small substrate. In contrast, ATP hydrolysis leads to release of Rip1 from BCS1 that moves again faster in BNGE. In a further approach, we used a peptide library of 15-mers comprising full-length Rip1 staggered by four residues to screen for binding of purified BCS1 complex (Figure 6D). One series of peptides was highly prominent in its efficiency of binding. The overlapping region comprised residues 165–187. Another, weaker, signal was observed covering the stretch of residues 145–167. These residues are presented on the surface of the Fe-S domain (Hunte et al., 2000) and may reflect the requirement of a correctly folded Fe-S domain for binding of Rip1 to Bcs1 and be responsible for recognition of folded Rip1 in the matrix. In order to search for further elements in Rip1 that contribute to the interaction with Bcs1, we generated a number of recombinant His-tagged variants of Rip1. Wild-type Rip1(31–215) (the numbering refers to the precursor form of Rip1) and the following variants were analyzed: N-terminal truncation mutants consisting of residues 61–215 or 91–215; a C-terminal truncation mutant lacking the complete FeS cluster-binding domain (31–123); and a mutant that was generated based on our results obtained from the peptide library, in which the highly conserved stretch comprising residues 172–179 was replaced by an Ala8 stretch. These recombinant Rip1 proteins were incubated with either

lysed mitochondria or with purified BCS1 complex from yeast mitochondria in the presence of AMP-PNP and subjected to BNGE to determine conformational alterations of BCS1 (Figure 6E, left and right panel, respectively). N-terminally truncated Rip1(61–215) and Rip1(91–215) did not induce the conformational shift caused by the mature full-length protein (31–215), while neither lack of the cluster-binding domain nor replacement by the Ala8 stretch did affect the shift. Furthermore, radiolabeled Rip1(A-stretch) and N- and C-terminal truncation mutants were imported into mitochondria containing His6-Bcs1. Neither the Rip1(A-stretch) mutant nor Rip1(31–123) were coisolated with BCS1 (Figure 6F). In contrast, the N-terminally truncated Rip1 was still able to interact under these conditions, apparently, since it contains a folded C terminus. In contrast to wild-type, however, Rip1(61–215) and Rip1(91–215) could not be chased efficiently from Bcs1 (Figure 6F). In addition, all mutants failed to assemble into the bc1 complex (data not shown).These data suggest that the N-terminal part comprising residues 31–60 (residues 1–30 of mature Rip1) is required for the release of Rip1 from Bcs1. This step is dependent on ATP and leads to a conformational change of Bcs1. The polypeptide segment identified by use of the peptide library that is not needed in vitro, but in organello, for the interaction between Rip1 with BCS1 is then a reflection of the requirement for a folded Fe-S domain of Rip1 for recognition and binding to Bcs1 in the matrix. With the solubilized and isolated proteins, binding of Rip1 could occur from both sides of the BCS1 complex and might, therefore, not depend on the proper folding of Rip1. These data suggest different roles of the different parts of Rip1 for binding to and release from Bcs1 when studied with the isolated components. On the one hand, a C-terminal domain is not required for binding to isolated Bcs1, for the triggering of a conformational change, and for ATP-dependent release and reverse change of Bcs1 conformation. On the other hand, the N-terminal 30 residues are required for triggering the conformational change in isolated Bcs1. This seems to be in contrast to the behavior observed in organello. We conclude that this difference is due to the presence of two interaction sites on Bcs1 for Rip1. One is occupied selectively from the matrix side. The other one, present on the intermembrane space side, is occupied after translocation of the C-terminal domain of Rip1. BCS1 in the isolated state can be reached by Rip1 from both sides. DISCUSSION Here, we describe an unusual and unexpected role of a AAAATPase in translocation of a folded substrate protein. In the

(C) Radiolabeled Rip1 was imported into mitochondria from His6-Bcs1 cells for 20 min at 15 C. A 20 min chase was performed in the presence of ATP, ADP, AMPPNP, or in the absence of any nucleotide. Imported (T) and Bcs1-bound (B) Rip1 were analyzed by SDS-PAGE and autoradiography (left panel) and quantified as in (B) (right panel). (D) Radiolabeled Rip1 was imported into mitochondria from wild-type and Dbcs1 cells in the presence or absence of ATP for the indicated time periods; the samples were analyzed for assembled Rip1 by BNGE, western blotting, and autoradiography. (E) Radiolabeled Rip1 was imported into mitochondria from cells containing His6-Bcs1 for 20 min at 15 C followed by a 20 min chase in the presence of ATP at 25 C. Imported and Bcs1-bound Rip1 were analyzed by SDS-PAGE (left panel); assembled Rip1 was visualized by BNGE (right panel) and autoradiography. (F) Bcs1 was purified from cells expressing His6-Bcs1 in wild-type and in Dcyt1 background in the presence of AMP-PNP or ATP. Analysis was done by SDSPAGE and immunodecoration. (G) Endogenous levels of Rip1 in wild-type, Dbcs1, and Dcyt1 mitochondria were analyzed by SDS-PAGE, western blotting, and immunodecoration. All error bars represent standard deviations of three independent experiments.

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Figure 5. Folding of Rip1 Is Important for Its Interaction with Bcs1 In Vivo (A) Mitochondria from cells expressing C-terminally truncated Rip1 variants were subjected to BNGE and SDS-PAGE and decorated for Rip1 (left panel). Radiolabeled, truncated Rip1 variants were imported into wild-type mitochondria for 90 min. Assembly of Rip1 was analyzed by BNGE and autoradiography (right panel). (B) The indicated truncation mutants were grown in synthetic medium containing 2% galactose to log phase and then shifted to YP medium containing 2% glycerol. While keeping them in log phase, OD600 was recorded. (C) Radiolabeled full-length and truncated forms of Rip1 were imported into mitochondria from wild-type and His6-Bcs1-expressing cells. Imported Rip1 and Bcs1-bound Rip1 were analyzed by coprecipitation, BNGE, and autoradiography. (D) Same as (C), with the exception that analysis was by SDS-PAGE and autoradiography (left panel) and quantification (right panel). Three independent experiments were quantified and the value for full-length Rip1 was set as 100%. (E) Mitochondria from Drip1 cells expressing the indicated constructs from a plasmid were sonicated and treated with proteinase K in the presence or absence of SDS. Formation of a 15 kDa fragment representing the Fe-S domain of Rip1 was analyzed by SDS-PAGE, western blot, and immunodecoration of Rip1 with an antibody directed against a C-terminal peptide (135SALKDPQTDADRVKD149). All error bars represent standard deviations of three independent experiments.

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following, we propose a model for the function of the mitochondrial AAA-ATPase Bcs1 in the biogenesis pathway of the Rip1 protein (Figure 7). Upon synthesis on cytosolic ribosomes, the precursor of Rip1 is imported into the mitochondrial matrix, where it acquires its 2Fe-2S cluster. The Fe-S cluster domain of Rip1 adopts a folded conformation before translocation from the matrix across the inner membrane. The Rip1 intermediate then interacts with the oligomeric BCS1 complex. BCS1 is anchored to the inner membrane by the transmembrane sequences of its monomers and exposes its ATPase domains into the matrix. The model suggests that the recognition of the Rip1 intermediate occurs by more than one signal. The first one is located in the Fe-S-containing domain and the second one in the N-terminal part. Rip1 translocation starts with the Fe-S cluster domain. The BCS1 complex interacts with the precomplex. We propose that the hydrophobic segment of Rip1, when it has reached the transmembrane part of BCS1, is laterally released in an ATP-dependent fashion. It switches over to the precomplex to be integrated into the protein core of the bc1 complex, followed by formation of supercomplexes. The role of the BCS1 AAA-ATPase in translocation of a folded protein domain extends the concept of the functions of AAA-ATPases. A major role described for AAA-ATPases is the translocation of essentially unfolded proteins through an interior channel formed by hexameric rings (Hoskins et al., 2001; Jarosch et al., 2002; Sauer et al., 2004; White and Lauring, 2007; Mogk et al., 2008). A remarkable relation exists also to the AAA-proteases (Tatsuta and Langer, 2009). These hybrids of chaperones and proteases are likely to encage transmembrane segments of integral membrane proteins that are subject to degradation. They are taking up transmembrane segments of membrane proteins into the interior of their hexameric ring to extract them from the membrane in an ATP-dependent manner. With their proximal chaperone domains, they move them further to reach the distal protease domains. We speculate that, in the case of Bcs1, the sequence of events occurs in the reverse direction. In agreement with such a mechanism are our following findings: The structural integrity of the Fe-S cluster domain of Rip1 is required for the interaction with Bcs1, but not sufficient to induce a nucleotidedependent conformational change that leads to the lateral release of Rip1 upon hydrolysis of the nucleotide. After translocation, an N-terminally located segment close to the transmembrane segment but not the C-terminal domain is required to induce the observed conformational change. The proposed mechanism of translocation of the Rieske protein implies the capacity of BCS1 to transport a folded domain of about 120 amino acid residues, corresponding to a diameter of about 2.5–3 nm. This implication is all but trivial, since so far AAA-ATPases were believed to unfold proteins to present them to associated proteases, and, therefore, only unfolded proteins may pass through the central channel. However, recent studies have shown that loops of polypeptides can be accommodated in the channel (Glynn et al., 2009). Furthermore, the central channel of the NBD1 ring of Hsp104 in certain conformational states can assume a diameter of up to 7.8 nm, depending on the added nucleotide (Wendler et al., 2009). In fact, preliminary experiments with isolated BCS1 upon negative staining show central stain-filled grooves or holes

of several nm in diameter (P. Wendler, personal communication). These findings are in very good agreement with the biochemical data presented here and support the model of translocation of the Fe-S domain in a folded state. Since it has been shown that in vivo Rip1 can be expressed and correctly assembled even in the absence of the Fe-S cluster (Graham and Trumpower, 1991), we have not followed the details of Fe-S cluster formation and its addition to the Rip1 protein in this study. It is currently not clear whether the complete cluster is assembled on the precursor or modifications are made which, in a later step, will lead to cluster formation. A number of interesting questions arise from our model. A major question is why Rip1 has first to be imported into the matrix and why a AAA-ATPase then is required to reach its final topology. First, Rip1 has to pick up its Fe-S cluster in the matrix, the site of its synthesis. Second, there is a conserved protein translocase in the mitochondria that inserts proteins from the matrix side into the inner membrane, the OXA1 translocase. However, this translocase is not required to export Rip1 from the matrix, since in mitochondria in which OXA1 was deleted, Rip1 was found to be translocated across the inner membrane (data not shown). These findings imply a number of evolutionary considerations. The homologs of Rip1 in bacteria and in chloroplasts are transported by the Tat system across the bacterial plasma membrane and the thylakoid membrane, respectively (Molik et al., 2001; Aldridge et al., 2008). The Tat system transports proteins in a folded conformation, often, but not always, carrying a cofactor (Weiner et al., 1998, reviewed in Robinson and Bolhuis, 2004; Palmer et al., 2005; Mu¨ller and Klo¨sgen, 2005). The mitochondria of virtually no organism analyzed contain the homologous components of the Tat system. One exception is Reclinomonas americana, which carries a mitochondrial DNA that encodes still a large number of proteins, among them components of the Tat system, TatA and TatC (Gray et al., 2004). Therefore, we suggest that in mitochondria the BCS1 system replaces the Tat system. This represents an intriguing evolutionary development, since thereby the basic mechanism of the formation and topogenesis of the Rip1 protein has been conserved, yet not the component(s) mediating a critical step in this pathway, the translocation of a folded cofactor-containing domain across the inner membrane of mitochondria. EXPERIMENTAL PROCEDURES Plasmids and Yeast Strains DNA manipulations were carried out according to standard procedures. All yeast strains were isogenic to W303-Mata wild-type cells. For expression of Bcs1 derivatives carrying an N-terminal histidinyl tag (His6-Bcs1), the sequence of BCS1 was amplified by PCR, digested with BamHI and HindIII and cloned into the yeast expression plasmid pYep51vk that was transformed into Dbcs1 cells. Import and Assembly Assays in Isolated Mitochondria Radiolabeled precursor proteins were synthesized in the presence of [35S] methionine in reticulocyte lysate according to the protocol of the manufacturer (Promega). Import into isolated yeast mitochondria (150 mg) was carried out in 0.6 M sorbitol, 0.5 mg/ml BSA, 2 mM K phosphate, 75 mM KCl, 10 mM Mg acetate, 2 mM EDTA, 2.5 mM MnCl2, 2 mM ATP, 2 mM NADH, 10 mM creatine phosphate, 0.1 mg/ml creatine kinase, 2.5 mM malate, 2.5 mM succinate, and 50 mM HEPES-KOH (pH 7.2) at 25 C, if not indicated otherwise. Import was

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Figure 6. Rip1 Interacts with Bcs1 In Vitro and Its N Terminus Induces a Conformational Shift in the BCS1 Complex (A) Lysed, wild-type mitochondria were incubated with ATP or AMP-PNP (left panel) and with recombinant full-length or truncated Rip1 or Su9-DHFR in the absence or presence of ATP for 20 min at 10 C (right panel). Analysis by BNGE, western blot, and decoration with antibodies to Bcs1. (B) Purified BCS1 complex was incubated with ATP or AMP-PNP (left panel) and recombinant full-length or truncated forms of Rip1, Su9-DHFR, or reduced carboxymethylated lactalbumin (RCMLA) in the absence or presence of ATP (right panel). Analysis by BNGE and Coomassie blue staining. (C) Wild-type and Bcs1(E319Q)-expressing mitochondria were lysed and incubated in the absence (lane 1) or presence (lanes 2–6) of recombinant Rip1 for 20 min at 10 C. The samples incubated with Rip1 were analyzed either without further incubation (lane 2) or incubated for 20 more min at 10 C in the presence of either no nucleotide, ATP, AMP-PNP, or ADP (lanes 3–6). Then all samples were subjected either to BNGE and decorated for Bcs1, or to SDS-PAGE and decorated for Rip1. (D) A peptide library of Rip1 was incubated with purified BCS1 complex. Bound BCS1 complex was transferred onto a PVDF membrane and visualized by decoration with antibodies to Bcs1.

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Figure 7. Model of the Role of Bcs1 in the Topogenesis of the Mitochondrial Rieske Protein The Rip1 precursor is synthesized in the cytosol and first imported into the matrix of mitochondria via TOM and TIM23 complexes. Rip1 is processed twice: by the mitochondrial-processing peptidase and the intermediate peptidase. It acquires its 2Fe-2S cluster in the matrix and adopts a folded conformation. The Rip1 intermediate then interacts with the BCS1 complex, first by a recognition site located on the surface of the folded Fe-Sbinding domain, and then is translocated with the C-terminal domain ahead through the central channel of BCS1. Lateral release of Rip1 then depends on a second recognition site located next to the N-terminal transmembrane segment and occurs in an ATP-dependent fashion when the hydrophobic segment of Rip1 has reached the transmembrane ring of the BCS1 complex. As the BCS1 complex interacts with the bc1 precomplex, Rip1 can be directly transferred to its final location. The completed bc1 complex then forms dimers and supercomplexes with cytochrome c oxidase.

stopped by diluting the reaction tenfold in ice-cold 0.6 M sorbitol, 20 mM HEPES-KOH (pH 7.2) with or without 50 mg/ml proteinase K. Samples were subjected to either SDS-PAGE or BNGE, according to Wittig et al., 2006. Radiolabeled proteins were detected by autoradiography.

Peptide Library Analysis A PepSpot library was purchased from JPT Peptide Technologies containing 15-mer peptides covering the complete Rip1 sequence. Each peptide overlapped by 11 amino acid residues with the previous peptide. The membrane containing the peptides was incubated with purified native His6-Bcs1. Bound Bcs1 was transferred to the PVDF membrane by western blotting, which was finally decorated with antibodies generated against Bcs1.

Expression of Recombinant Rip1 Proteins Bacteria containing the respective expression plasmids were cultured at 37 C in LB medium with 100 mg/l ampicillin. Protein expression was induced at an OD600 of 0.5 by addition of 1 mM IPTG. The cells were lysed in 7 M urea, 20 mM imidazole, 1 mM PMSF, 0.5% Triton X-100, 50 mM Na phosphate (pH 8.0), sonicated on ice and applied to NiNTA beads (Novagen) to allow binding of His-tagged proteins. The affinity matrix was washed extensively with 7 M urea, 20 mM imidazole, 50 mM Na phosphate (pH 8.0). Rip1 and variant proteins were eluted by incubation with 300 mM imidazole, 50 mM Na phosphate (pH 8.0).

Miscellaneous Antibodies against Bcs1 were generated by injecting rabbits with purified His6-Bcs1. For BNGE, mitochondria were solubilized with lysis buffer containing

3% digitonin. The resulting lysates were layered onto 4%–13% polyacrylamide gradient gels (Wittig et al., 2006). ACKNOWLEDGMENTS We thank Kai Hell for the yeast strain expressing His6-tagged Bcs1, Sandra Esser and Christiane Kotthoff for technical assistance, and the Fonds der Chemischen Industrie (M.A.) and the Deutsche Forschungsgemeinschaft for financial support (SFB 594 Teilprojekt B3). Received: December 3, 2010 Revised: May 31, 2011 Accepted: July 15, 2011 Published: October 20, 2011 REFERENCES Aldridge, C., Spence, E., Kirkilionis, M.A., Frigerio, L., and Robinson, C. (2008). Tat-dependent targeting of Rieske iron-sulphur proteins to both the plasma and thylakoid membranes in the cyanobacterium Synechocystis PCC6803. Mol. Microbiol. 70, 140–150. Bla´zquez, A., Gil-Borlado, M.C., Mora´n, M., Verdu´, A., Cazorla-Calleja, M.R., Martı´n, M.A., Arenas, J., and Ugalde, C. (2009). Infantile mitochondrial encephalomyopathy with unusual phenotype caused by a novel BCS1L mutation in an isolated complex III-deficient patient. Neuromuscul. Disord. 19, 143–146. Cruciat, C.M., Hell, K., Fo¨lsch, H., Neupert, W., and Stuart, R.A. (1999). Bcs1p, an AAA-family member, is a chaperone for the assembly of the cytochrome bc(1) complex. EMBO J. 18, 5226–5233.

(E) Recombinant full-length, truncated, or mutated Rip1 proteins (5 mg) (scaled maps are provided) were incubated with detergent-lysed mitochondria (left panel) or purified BCS1 complex (right panel) in the presence of AMP-PNP. Analysis was performed as in (A). (F) Radiolabeled Rip1 variants were imported and analyzed as described in Figure 4E.

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