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A transcriptome screen in yeast identifies a novel assembly factor for the mitochondrial complex III
Mitochondrion 11 (2011) 391–396
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Mitochondrion j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m i t o
A transcriptome screen in yeast identifies a novel assembly factor for the mitochondrial complex III Lise Mathieu a,1, Sophie Marsy a,1, Yann Saint-Georges b, Claude Jacq b, Geneviève Dujardin a,⁎ a b
Centre de Génétique Moléculaire, CNRS FRE 3144, Avenue de la terrasse, Gif-sur-Yvette F-91190, France Laboratoire de Génétique Moléculaire, CNRS UMR 8541, Ecole Normale Supérieure, 46 rue d'Ulm, Paris F-75005, France
a r t i c l e
i n f o
Article history: Received 4 August 2010 Received in revised form 4 November 2010 Accepted 3 December 2010 Available online 16 December 2010 Keywords: Yeast Respiratory complex biogenesis Complex III assembly mRNA localization
a b s t r a c t Starting from a transcriptome based study of the spatio-temporal expression of yeast genes encoding mitochondrial proteins of unknown function, we have identified the gene BCA1 (YLR077W). A FISH analysis showed that the BCA1 mRNA co-localized with the mitochondrial network. Cellular fractionation revealed that Bca1 is bound to the mitochondrial inner-membrane and protrudes into the inter-membrane space. We show that Bca1 controls an early step in complex III assembly and that the supra-molecular organization of Bca1 is dependent upon the assembly level of complex III. Thus, Bca1 is a novel assembly factor for the respiratory complex III. © 2011 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
1. Introduction Mitochondrial respiratory enzymes are particularly fascinating examples of membrane complexes, because they are composed of numerous subunits of dual genetic origin, a feature that they share with the photosynthetic complexes in chloroplasts. A few core subunits of these complexes are encoded by the mitochondrial genome, translated on mitochondrial ribosomes and immediately inserted into the membrane, while most subunits are encoded by nuclear genes, translated within the cytoplasm and have to be imported into mitochondria. In addition, supra-molecular associations between different mitochondrial complexes have been reported (Cruciat et al., 2000; Schagger and Pfeiffer, 2000). The assembly of these large molecular machineries is a dynamic and complicated process that involves assembly factors or membrane chaperons that are extrinsic to the complexes. The complex III, also called complex bc1, occupies a key position in the respiratory chain and in the formation of the super-complexes. Its structure and function are highly conserved from lower to higher eukaryotes and several human diseases are related to complex III deficiencies (Benit et al., 2009). The complex operates as a homodimer
⁎ Corresponding author. Centre de Génétique Moléculaire, CNRS FRE 3144, Bâtiment 26, Avenue de la terrasse, Gif-sur-Yvette F-91190, France. Tel.: +33 1 69 82 31 69; fax: +33 1 69 82 31 50. E-mail address: [email protected] (G. Dujardin). 1 The first two authors contributed equally to this paper.
and in mammals, each monomeric unit consists of 11 different polypeptides. Three subunits, cytochrome b (Cytb), cytochrome c1 (Cyt1) and the Iron Sulphur Protein (ISP or Rip1) form the catalytic core and participate in the electron transfer process. The remaining subunits are thought to be involved in the stabilization and protection of the catalytic core. Cytb is the only mitochondrially-encoded subunit of complex III and it occupies the central position in the atomic structure with its eight transmembrane (TM) segments and constitutes the core scaffold of the complex (Hunte et al., 2000; Xia et al., 1997). The details of complex III assembly are still poorly understood. A modular mode of assembly has been proposed for the yeast Saccharomyces cerevisiae complex III by Zara et al., 2009a,b: two modules containing Cytb or Cyt1 and other non-catalytic subunits would first associate to form a 500 kDa pre-complex III (pre-III) which would subsequently be able to bind the remaining catalytic subunit Rip1 (Fig. 3D). In the absence of Rip1, the pre-complex III accumulates (Cruciat et al., 2000; Zara et al., 2009b). Complex III and complex IV show a similar level of complexity and more than 30 assembly-assisting factors or membrane chaperons are known to be involved in the assembly of complex IV (Fontanesi et al., 2008). However, only five complex III assembly factors have been identified until now: Cc1hl, Cbp3, Cbp4, Mzm1 and Bcs1. Cc1hl is a heme lyase which covalently binds heme to Cyt1 (Zollner et al., 1992). Cbp3 and Cbp4 interact and play a role in the biogenesis of the Cytb module (Kronekova and Rodel, 2005; Wu and Tzagoloff, 1989). Mzm1 influences a labile pool of mitochondrial zinc and its absence leads to a destabilized complex III (Atkinson et al., 2010). Bcs1 is involved in the binding of the catalytic subunit Rip1 (Cruciat et al., 1999; Nobrega et al., 1992; Nouet et al., 2009). The function of Bcs1 is conserved in higher
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eukaryotes and mutations in human Bcs1 are associated with mitochondrial diseases (e.g. de Lonlay et al., 2001; Fernandez-Vizarra et al., 2007). S. cerevisiae is able to rely exclusively on glucose fermentation for its energy requirements and most of the known assembly factors were discovered through the study of yeast mutants that were unable to grow on non-fermentable medium. These mutant screens were quite intensive and are probably close to saturation. Also mutations in some assembly factors may not lead to a clear respiratory phenotype. Thus, alternative screening methods are needed to isolate other complex III assembly factors. We have recently shown that there are differences in the spatiotemporal expression of yeast genes with a mitochondrial function: in synchronized cells the expression of genes encoding translation and assembly factors precedes the expression of genes encoding the respiratory complex components and the TCA cycle enzymes (Lelandais et al., 2009). In addition, these early-expressed genes appear to be translated in the vicinity of mitochondria and many present a conserved motif in their 3′end UTR that is known to be recognized by the RNA-binding protein Puf3 (Garcia et al., 2007; Saint-Georges et al., 2008). Based on these observations, we have proposed that new assembly factors may be encoded by genes of unknown function belonging to the subset of early expressed, Puf3-dependent genes. In this paper, we demonstrate that this transcriptome-based screen does allow the identification of a novel factor, Bca1 (bc1 assembly), controlling the assembly of the respiratory complex III. 2. Materials and methods 2.1. Yeast strains, media and genetic techniques All the strains were derived from W303-1B MATα ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 can1-100 (Table 1). CW252 is the wild-type strain (WT) and contains an intron-less mitochondrial genome (SaintGeorges et al., 2002). The series of mutants expressing the HA-tagged version of Bca1 were constructed by crossing each single mutant with the BCA1-HA strain (see below). Most media and genetic techniques were as described in Dujardin et al. (1980). The nonfermentable media contained either 2% glycerol or 2% lactate. The fermentable media contained either 2% glucose or 2% galactose and 0.1% glucose. The maintenance of the mitochondrial genome was tested by crossing the cells with the strain KL14-4A/60, which was devoid of the mitochondrial genome (MATa his1 trp2 rho°), and testing the growth of the resulting diploids on glycerol medium. 2.2. Epitope tagging and gene deletions Bca1p was tagged at the C-terminus with the 3HA epitope. We used the Schizosaccharomyces pombe his5 marker gene (which complements the S. cerevisiae his3 mutation) as described in Longtine et al. (1998). The PCR fragments were used to transform the CW252 strain to histidine prototrophy. Correct integration of the tag at the
Table 1 List of strains. Name
Genotype
Origin
CW252 Δbca1 Δbca1 Δrip1 Bca1-HA Δcyt1 Bca1-HA Δcbp3 Bca1-HA Δrip1 Bca1-HA Δbcs1 Bca1-HA
Wild type bca1::G418 bca1::G418 rip1::LEU2 BCA1-3HA-HIS3 cyt1::LEU2 BCA1-3HA-HIS3 cbp3::G418 BCA1-3HA-HIS3 rip1::LEU2 BCA1-3HA-HIS3 bcs1::URA3 BCA1-3HA-HIS3
Saint-Georges et al., 2002 This work This work This work This work This work This work This work
BCA1 locus in the BCA1-HA strain was confirmed by PCR amplification and sequencing. The Δbca1 strain carrying the deleted allele bca1::kanMX4 was constructed by transformation of CW252 with a PCR fragment that contained the KanR gene flanked by 50 bp of the BCA1 5′ and 3′ UTR. The integration was confirmed by PCR analysis of the genomic DNA and the [G418R] phenotype was shown to segregate 2:2 and to correlate with the presence of the deleted version of BCA1. The other null mutants were constructed by similar PCR-based techniques. The double mutant Δbca1Δrip1 was constructed by crossing the two single mutants. 2.3. FISH analysis and mRNA localization WT cells were grown to mid-exponential phase and were treated as described in Garcia et al. (2007) and Saint-Georges et al. (2008). The combined hybridization of three to five antisense oligonucleotides probes was used to enhance the visualization of each mRNA. Typically, each probe was designed so that it contained 50 to 55 nucleotides, with five aminoallyl thymidines and a coherent Tm value. The probes were directly labeled with Cy3 or Cy5 fluorochromes and hybridized to fixed cells. 2.4. Cytochrome absorption spectra Cytochrome absorption spectra of whole cells grown on galactose medium were recorded at liquid nitrogen temperature after the addition of the reducing agent, sodium dithionite, using a Cary 400 (Varian, San Fernando, CA) spectrophotometer. Absorption maxima for the bands of cytochromes c, c1, b, and aa3 are expected at 546, 552, 558 and 602 nm, respectively. 2.5. Isolation of mitochondria, SDS-polyacrylamide gels (SDS-PAGE) Cells were grown in galactose medium, and mitochondria were isolated by differential centrifugation after digestion of cell walls with Zymolyase-100T (Lemaire and Dujardin, 2008). The protein concentration of the final mitochondrial suspension was determined using the Bio-Rad assay (Hercules, CA). Mitochondrial proteins were resolved on denaturating 12% SDS-PAGE. Protein markers were used to estimate protein molecular masses. 2.6. Separation of mitochondrial protein complexes in Blue Native non-denaturing gels (BN-PAGE) Mitochondria were solubilised with 2% digitonin (w/v) in the presence of antiprotease and DNase and the complexes were separated on 5–10% or 6–15% polyacrylamide gradient gels as described in Schagger and Pfeiffer (2000) and modified in Lemaire and Dujardin (2008). Protein markers (High Molecular Weight Calibration kit for native electrophoresis, Amersham Biosciences) were used to estimate the molecular weights. In some cases, the gel strips excised from the BN-PAGE were analyzed in a second dimension SDS-PAGE. The gel strips were incubated in a solution containing 60 mM Tris HCl (pH 6.8) for five minutes at room temperature. Each gel strip was then placed horizontally above an SDS PAGE separating gel (12% polyacrylamide and 0.1% SDS). The gel slice was then encased in the 5% polyacrylamide stacking gel. This second dimension SDS-PAGE allows the utilization of antibodies that do not recognize the native protein. 2.7. Immuno-precipitation experiments Mitochondria were isolated from the Bca1p-HA strain and solubilised in 1% digitonine or lauryl maltoside. The suspension was centrifuged for 15 min at 4 °C at 100,000 g. Polyclonal HA-antibodies
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coupled agarose beads were added to the supernatant. Samples were incubated under gentle shaking for 90 min at 4 °C. The beads were washed twice with the lysis buffer and the immuno-precipitate was analyzed by SDS-PAGE and western blotting experiments.
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co-localized with the mitochondrial network (15S rRNA) in 45% of cells analyzed (Fig. 1). Control mRNAs were previously identified as mitochondrially-localized (ATP3) and non-mitochondrially-localized (ATP16). Our results show that BCA1 mRNAs are strongly co-localized with the mitochondrial network.
2.8. Immunoblotting experiments of SDS-PAGE and BN-PAGE 3.2. Topology of the Bca1 protein within mitochondria SDS-PAGE and BN-PAGE were blotted on nitrocellulose (Schleicher & Schuell, Keene, NH). Polyclonal antibodies against cytochrome c1 (Cyt1) or cytochrome b (Cytb) were raised against a fusion proAapocytochrome c1 expressed in E. coli or a synthetic peptide of Cytb coupled to KLH (AGRO-BIO) respectively. Monoclonal antibodies against Cox2, Hsp60, and PGK were from Molecular Probes (Eugene, OR) and the anti-HA was from Santa Cruz Biotechnology. Other antibodies were gifts: Cyb2 (B. Guiard, Gif-sur-Yvette, France), Rip1 (C. Godinot, Lyon, France) and anti-Atp4 (J. Velours, Bordeaux, France). Bound antibodies were detected by horseradish-peroxidase-conjugated secondary antibodies (Promega, Madison, WI). Proteins were visualized using a chemiluminescent substrate (Pierce Chemical, Rockford, IL). The anti-Cyt1, anti-Atp2 and anti-Cox2 recognize the native protein within the complexes. 3. Results and discussion 3.1. BCA1 mRNA co-localize with the mitochondria According to the systematic transcriptome analysis (Lelandais et al., 2009), BCA1 mRNA (YLR077W) belongs to the regulon of earlyexpressed genes that were translated in the vicinity of mitochondria in a Puf3 dependent way. The 3′UTR of the BCA1 mRNA contains the characteristic-Puf3 binding motif: CAUGUAUAUA. To quantify BCA1 mRNA localization, we have performed an in situ FISH analysis in wildtype cells by labeling the BCA1 mRNAs and observed that BCA1 mRNAs
A large-scale analysis of the localization of S. cerevisiae proteins suggested that Bca1 is a mitochondrial protein and the PSORT program predict a mitochondrial location. To verify its subcellular localization, Bca1 was tagged at its C-terminus with three HA epitopes. The HA antibody revealed a protein of the expected size, ~70 kDa, that was recovered in the mitochondrial fraction, as was the mitochondrial protein Atp4, whereas the cytosolic phosphoglycerate kinase (PGK) was found mainly in the postmitochondrial supernatant (Fig. 2A, left panel). Bca1 is predicted to have a transmembrane domain between residues 83 and 105 and a large soluble C-terminal domain (Fig. 2B, right panel). To test whether Bca1 was associated with mitochondrial membranes, mitochondria purified from cells expressing Bca1-HA, were “alkali”-treated to extract the non-integral membrane proteins. As shown in Fig. 2A (right panel), Bca1-HA was found in the pellet fraction, as was the inner membrane protein Atp4. In addition, proteinase K treatment of mitochondria did not degrade the Bca1-HA protein or modify its mobility (Fig. 2B, lane 2) indicating that Bca1 did not span the outer membrane. Thus Bca1 is embedded in the inner membrane. To determine the topology of Bca1, we performed osmotic swelling experiments that disrupt the outer membrane of mitochondria while keeping the inner membrane intact. The proteinase K treatment of the swollen mitochondria led to the degradation of 90% of Bca1-HA while the matrix protein Hsp60 remained protected and the intermembrane space soluble protein cytochrome b2 (Cyb2) was completely degraded
Fig. 1. Localization of BCA1 mRNA by FISH analysis. BCA1, ATP3, ATP16 mRNAs were labeled with specific sets of fluorescent probes as described in Materials and methods (green in the Merge). Five fluorescent probes specific for the 15S mitochondrial ribosomal RNA delimited the mitochondrial compartment (red in the Merge). The nucleus was stained by DAPI (blue in the 3D reconstruction). Quantification of the mitochondria/mRNA colocalization rate was done using Corsen software and presented in the histogram (Jourdren et al., 2010).
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3.3. Bca1 controls the assembly of complex III
Fig. 2. Mitochondrial localization and topology of Bca1. Mitochondria were purified from cells expressing Bca1-HA. Proteins were analyzed by SDS-PAGE and immunoblotting with different antibodies. (A) Left: Mitochondrial (M) and postmitochondrial supernatant (PS) protein fractions were separated on SDS-PAGE. Right: Mitochondria were alkali treated at pH 11.5 according to Lemaire and Dujardin (2008) to separate the soluble proteins (S) from the integral membrane proteins that remained in the pellet (P). (B) Left: Mitochondrial proteins (lane 1) were treated with 100 mg/ml of proteinase K before (lane 2) or after osmotic swelling (lane 3) according to Funes et al. (2004). Right: Topology of the Bca1 protein. TM: transmembrane segment; OM: outer membrane; IM: inner membrane.
(Fig. 2B, lane 3). Altogether, our results indicated that Bca1 is bound to the mitochondrial inner membrane and exposes its large soluble C-terminal domain to the intermembrane space (Fig. 2B, right panel).
To investigate the role of Bca1, a Δbca1 null allele was constructed (see Section 2.3). The Δbca1 exhibited a respiratory growth defect on a non-fermentable medium containing lactate as carbon source but not on glycerol medium (Fig. 3A). The effect of the absence of Bca1 on respiratory complex biogenesis was first studied by recording the cytochrome absorption spectra. The Δbca1 cells showed a wild-type level of cytochromes a + a3 and a clear decrease in the cytochrome b content (Fig. 3B). Next, the steady-state levels of the three complex III catalytic subunits, Cytb, Cyt1 and Rip1 as well as that of Cox2 and Atp4 that are representative of the respiratory complexes IV and V respectively, were analyzed by western blot. The steady state level of Rip1 and Cytb diminished, 10% for Cytb and 70% for Rip1, while Cyt1, Cox2, Atp4 and the control Hsp60 were not affected (Fig. 3C). Finally, the assembly level of complex III was analyzed by Blue Native Poly Acrylamide Gel Electrophoresis (BN-PAGE) of mitochondria solubilised with the mild detergent digitonin that preserves the interactions between complexes. As expected, the anti-Cyt1 antibody revealed the super-complexes III2 + IV, III2 + IV2 and the homo-dimer, III2 in the wild-type strain. In the Δbca1 mutant, the intensity of the three bands was strongly reduced by at least 70% (Fig. 3E). No lowmolecular-weight band that might correspond to the accumulation of assembly intermediates was revealed even after a long exposure. We have also verified that the assembly of complex V was not disturbed in the Δbca1 mutant (data not shown). Finally, we have constructed a double mutant Δrip1Δbca1 and compare the assembly of complex III of the double mutant to that of the single mutant Δrip1. In the absence of Rip1, a pre-complex III of about 500 kDa resulting from the association of the cytochrome b and c1 modules is known to accumulate (Cruciat et al., 2000; Schagger and Pfeiffer, 2000; Fig. 3D). In the double mutant Δrip1Δbca1, this pre-complex III is detected but its steady state level was reduced by about 60%. The fact that the absence of the inter-membrane space protein Bca1 affects the amount of cytochrome b as well as the steady state levels of precomplex III and complex III suggests that Bca1 controls the assembly of complex III at an early stage, before the insertion of Rip1 into the
Fig. 3. Effect of Δbca1 mutation on the assembly of the respiratory complex III. (A) Wild-type (WT) and Δbca1 were spotted onto fermentable glucose (glu) and respiratory glycerol (gly) or lactate (lac) media. Photographs were taken after 1 day (glucose) or 3 days (glycerol, lactate) of incubation at 28 °C. (B) Low temperature cytochrome absorption spectra of the wild-type and Δbca1 of cells grown on galactose medium were recorded. Cytochromes c1 and b are part of complex III and cytochromes aa3 of complex IV. Abs: Absorbance. (C) Mitochondrial proteins from wild-type and Δbca1 were analyzed by SDS-PAGE and immunoblotting with different antibodies. (D) Model for the assembly of complex III (see Introduction and this work for Bca1). (E) Mitochondrial proteins from wild type, Δbca1, Δrip1, Δbca1 Δrip1 mutants were analyzed by BN-PAGE (5–10%) and immunoblotting with antibodies against Cyt1. Positions of pre-complex III, dimers III2, supercomplexes III2IV and III2IV2, are indicated.
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pre-complex III, the strong effect on the level of Rip1 being the consequence of this upstream effect in the assembly process (Fig. 3C and D). 3.4. Complex III assembly affects the supra-molecular organization of Bca1 BLAST searches reveal that Bca1-like proteins are only found in closely related yeasts such as Kluyveromyces lactis or Candida glabrata (about 40% identity) and some filamentous fungi such as Neurospora crassa (about 20% identity). In addition, the C-terminal domain of Bca1 presents RCC1/BLIPII motifs (Regulator of Chromosome Condensation, Renault et al., 1998; β-lactamase inhibitor protein-II, Lim et al., 2001, see Fig. S1). RCC1/BLIP-II motifs are structural motifs forming a β-propeller fold that can bind protein ligands of very different types in eukaryotic cells, suggesting that Bca1 might interact with protein partners. In order to study the interactions between Bca1 and complex III, we have compared the effect of three mutations affecting the various modules involved in complex III assembly, Δcyt1, Δcbp3 and Δrip1 on Bca1-HA accumulation. As shown in Fig. 4A, none of these mutants modified the steady-state level of Bca1. Next, proteins of wild type mitochondria were solubilised with 1% digitonin and subjected to
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co-immuno-precipitation using agarose-bound antibodies against HA. Bca1 was clearly detected in the immuno-precipitate while none of the three catalytic subunits of complex III or representative subunits of complexes IV and V immuno-precipitated with Bca1 (Fig. 4B). A similar result was obtained with laurylmaltoside solubilisation and with mitochondria from a Δrip1 strain. Thus, if there are any interaction between Bca1 and the catalytic subunits of complex III, they are probably labile or transitory. We have also compared the supra-molecular organization of Bca1 in the wild type and mutants by combining the first dimension BN-PAGE (Fig. 4C) with a second-dimension SDS-PAGE (Fig. 4D). In the Δrip1 and Δbcs1 mutants, Bca1 accumulated in a high molecular weight complex of about 600 kDa that was not detected in the wild type or in Δcyt1 and Δcbp3 mutants. Thus, this 600 kDa Bca1containing complex only accumulates in the two mutants that also accumulate the 500 kDa pre-complex III (Fig. 4B). The second dimension gel reveals that in the wild type strain, Bca1-HA was indeed present in a continuous streak of low molecular weight complexes from about 140 to 440 kDa (Fig. 4C). Thus the supra molecular organization of the Bca1 protein clearly differs in the wild type and in the mutants affecting the assembly of the Rip1 subunit, showing that its supramolecular organization was dependent of the assembly state of complex III. The presence of Bca1 in a continuous
Fig. 4. Supramolecular organization of Bca1 and complex III assembly. Mitochondrial proteins were purified from wild-type (WT), Δbca1 and four other mutants affecting different steps of the assembly of complex III: Δcyt1, Δrip1, Δcbp3, Δbcs1 and expressing Bca1-HA. (A) Mitochondrial proteins were analyzed by SDS-PAGE and immunoblotting with antibodies against HA, the three catalytic complex III subunits and Hsp60. (B) Mitochondrial proteins from wild-type strain expressing Bca1-HA were solubilised with 1% digitonin and incubated with antibodies against HA coupled to agarose beads. The different samples were subjected to SDS-PAGE and immunoblotting with antibodies. (M) mitochondrial lysate; (S) supernatant of beads; (W) washing; (IP) immuno-precipitate. (C) Mitochondrial proteins were analyzed by BN-PAGE (6–15%) and immunoblotting with anti-Cyt1 and anti-HA. Positions of pre-complex III (pre-III) and of the 600 kDa Bca1-containing complex are indicated. (D) Mitochondrial proteins of wild-type and Δrip1 cells were analyzed by BN-PAGE (6–15%) and then in a second dimension by SDS-PAGE (12%) and immunoblotting with anti-HA and anti-Cyt1. Positions of size markers are indicated.
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streak of low molecular weight complexes in BN-PAGE suggests that it is involved in dynamic and thus probably transient interactions. The arrest of the assembly process of complex III at the step of Rip1 insertion might modify these transitory interactions leading to the stable accumulation of Bca1 in the high molecular weight oligomeric complex of 600 kDa. 4. Conclusions In this paper, we validate our transcriptome-based screen by demonstrating that the early expressed, Puf3 dependent BCA1 gene encodes a novel assembly factor for the yeast respiratory complex III. Bca1 appears to act upstream of Rip1 insertion in the assembly of complex III. According to the modular model of assembly, Bca1 could control either the formation/stabilization of the cytochrome b module or the interactions between the cytochrome b and cytochrome c1 modules before Rip1 insertion. It is very likely that the study of the function of other genes that are part of the same regulon should lead to the discovery of more assembly factors of respiratory complexes. The further characterization of Bca1 and other similarly regulated assembly factors will allow a better understanding of the assembly pathway of the mitochondrial respiratory complexes. In human cells, it would be also very interesting to test if a similar subset of genes encoding translation and assembly factors are translated at the vicinity of mitochondria. If this is the case, such transcriptome-based screens should lead to the identification of new nuclear encoded factors that are required for the biogenesis of the human respiratory complexes. Mutations in the genes encoding these factors could be responsible for pathologies for which the genetic determinism is still unknown. Supplementary materials related to this article can be found online at doi:10.1016/j.mito.2010.12.002. Acknowledgements We thank CJ. Herbert, B. Guiard, B. Meunier and C. Panozzo for critical reading of the manuscript and CJ Herbert for checking the English language. This work was supported by a grant from the Agence Nationale pour la Recherche (LAN06-0234). References Atkinson, A., Khalimonchuk, O., Smith, P., Sabic, H., Eide, D., Winge, D.R., 2010. Mzm1 influences a labile pool of mitochondrial zinc important for respiratory function. J. Biol. Chem. 285, 19450–19459. Benit, P., Lebon, S., Rustin, P., 2009. Respiratory-chain diseases related to complex III deficiency. Biochim. Biophys. Acta 1793, 181–185. Cruciat, C.M., Hell, K., Folsch, H., Neupert, W., 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. Cruciat, C.M., Brunner, S., Baumann, F., Neupert, W., Stuart, R.A., 2000. The cytochrome bc1 and cytochrome c oxidase complexes associate to form a single supracomplex in yeast mitochondria. J. Biol. Chem. 275, 18093–18098. de Lonlay, P., Valnot, I., Barrientos, A., Gorbatyuk, M., Tzagoloff, A., Taanman, J.W., Benayoun, E., Chretien, D., Kadhom, N., Lombes, A., de Baulny, H.O., Niaudet, P., Munnich, A., Rustin, P., Rotig, A., 2001. A mutant mitochondrial respiratory chain assembly protein causes complex III deficiency in patients with tubulopathy, encephalopathy and liver failure. Nat. Genet. 29, 57–60.
Dujardin, G., Pajot, P., Groudinsky, O., Slonimski, P.P., 1980. Long range control circuits within mitochondria and between nucleus and mitochondria. I. Methodology and phenomenology of suppressors. Mol. Gen. Genet. 179, 469–482. Fernandez-Vizarra, E., Bugiani, M., Goffrini, P., Carrara, F., Farina, L., Procopio, E., Donati, A., Uziel, G., Ferrero, I., Zeviani, M., 2007. Impaired complex III assembly associated with BCS1L gene mutations in isolated mitochondrial encephalopathy. Hum. Mol. Genet. 16, 1241–1252. Fontanesi, F., Soto, I.C., Barrientos, A., 2008. Cytochrome c oxidase biogenesis: new levels of regulation. IUBMB Life 60, 557–568. Funes, S., Nargang, F.E., Neupert, W., Herrmann, J.M., 2004. The Oxa2 protein of Neurospora crassa plays a critical role in the biogenesis of cytochrome oxidase and defines a ubiquitous subbranch of the Oxa1/YidC/Alb3 protein family. Mol. Biol. Cell 15, 1853–1861. Garcia, M., Darzacq, X., Delaveau, T., Jourdren, L., Singer, R.H., Jacq, C., 2007. Mitochondria-associated yeast mRNAs and the biogenesis of molecular complexes. Mol. Biol. Cell 18, 362–368. Hunte, C., Koepke, J., Lange, C., Rossmanith, T., Michel, H., 2000. Structure at 2.3 A resolution of the cytochrome bc(1) complex from the yeast Saccharomyces cerevisiae co-crystallized with an antibody Fv fragment. Structure 8, 669–684. Jourdren, L., Delaveau, T., Marquenet, E., Jacq, C., Garcia, M., 2010. CORSEN, a new software dedicated to microscope-based 3D distance measurements: mRNA-mitochondria distance, from single-cell to population analyses. RNA 16, 1301–1307. Kronekova, Z., Rodel, G., 2005. Organization of assembly factors Cbp3p and Cbp4p and their effect on bc(1) complex assembly in Saccharomyces cerevisiae. Curr. Genet. 47, 203–212. Lelandais, G., Saint-Georges, Y., Geneix, C., Al-Shikhley, L., Dujardin, G., Jacq, C., 2009. Spatio-temporal dynamics of yeast mitochondrial biogenesis: transcriptional and post-transcriptional mRNA oscillatory modules. PLoS Comput. Biol. 5, e1000409. Lemaire, C., Dujardin, G., 2008. Preparation of respiratory chain complexes from Saccharomyces cerevisiae wild-type and mutant mitochondria: activity measurement and subunit composition analysis. Meth. Mol. Biol. 432, 65–81. Lim, D., Park, H.U., De Castro, L., Kang, S.G., Lee, H.S., Jensen, S., Lee, K.J., Strynadka, N.C., 2001. Crystal structure and kinetic analysis of beta-lactamase inhibitor protein-II in complex with TEM-1 beta-lactamase. Nat. Struct. Biol. 8, 848–852. Longtine, M.S., McKenzie 3rd, A., Demarini, D.J., Shah, N.G., Wach, A., Brachat, A., Philippsen, P., Pringle, J.R., 1998. Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14, 953–961. Nobrega, F.G., Nobrega, M.P., Tzagoloff, A., 1992. BCS1, a novel gene required for the expression of functional Rieske iron-sulfur protein in Saccharomyces cerevisiae. EMBO J. 11, 3821–3829. Nouet, C., Truan, G., Mathieu, L., Dujardin, G., 2009. Functional analysis of yeast bcs1 mutants highlights the role of Bcs1p-specific amino acids in the AAA domain. J. Mol. Biol. 388, 252–261. Renault, L., Nassar, N., Vetter, I., Becker, J., Klebe, C., Roth, M., Wittinghofer, A., 1998. The 1.7A crystal structure of the regulator of chromosome condensation (RCC1) reveals a seven-bladed propeller. Nature 392, 97–101. Saint-Georges, Y., Bonnefoy, N., di Rago, J.P., Chiron, S., Dujardin, G., 2002. A pathogenic cytochrome b mutation reveals new interactions between subunits of the mitochondrial bc1 complex. J. Biol. Chem. 277, 49397–49402. Saint-Georges, Y., Garcia, M., Delaveau, T., Jourdren, L., Le Crom, S., Lemoine, S., Tanty, V., Devaux, F., Jacq, C., 2008. Yeast mitochondrial biogenesis: a role for the PUF RNA-binding protein Puf3p in mRNA localization. PLoS ONE 3, e2293. Schagger, H., Pfeiffer, K., 2000. Supercomplexes in the respiratory chains of yeast and mammalian mitochondria. EMBO J. 19, 1777–1783. Wu, M., Tzagoloff, A., 1989. Identification and characterization of a new gene (CBP3) required for the expression of yeast coenzyme QH2-cytochrome c reductase. J. Biol. Chem. 264, 11122–11130. Xia, D., Yu, C.A., Kim, H., Xia, J.Z., Kachurin, A.M., Zhang, L., Yu, L., Deisenhofer, J., 1997. Crystal structure of the cytochrome bc1 complex from bovine heart mitochondria. Science 277, 60–66. Zara, V., Conte, L., Trumpower, B.L., 2009a. Biogenesis of the yeast cytochrome bc1 complex. Biochim. Biophys. Acta 1793, 89–96. Zara, V., Conte, L., Trumpower, B.L., 2009b. Evidence that the assembly of the yeast cytochrome bc1 complex involves the formation of a large core structure in the inner mitochondrial membrane. FEBS J. 276, 1900–1914. Zollner, A., Rodel, G., Haid, A., 1992. Molecular cloning and characterization of the Saccharomyces cerevisiae CYT2 gene encoding cytochrome-c1-heme lyase. Eur. J. Biochem. 207, 1093–1100.
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Mitochondrion j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m i t o
A transcriptome screen in yeast identifies a novel assembly factor for the mitochondrial complex III Lise Mathieu a,1, Sophie Marsy a,1, Yann Saint-Georges b, Claude Jacq b, Geneviève Dujardin a,⁎ a b
Centre de Génétique Moléculaire, CNRS FRE 3144, Avenue de la terrasse, Gif-sur-Yvette F-91190, France Laboratoire de Génétique Moléculaire, CNRS UMR 8541, Ecole Normale Supérieure, 46 rue d'Ulm, Paris F-75005, France
a r t i c l e
i n f o
Article history: Received 4 August 2010 Received in revised form 4 November 2010 Accepted 3 December 2010 Available online 16 December 2010 Keywords: Yeast Respiratory complex biogenesis Complex III assembly mRNA localization
a b s t r a c t Starting from a transcriptome based study of the spatio-temporal expression of yeast genes encoding mitochondrial proteins of unknown function, we have identified the gene BCA1 (YLR077W). A FISH analysis showed that the BCA1 mRNA co-localized with the mitochondrial network. Cellular fractionation revealed that Bca1 is bound to the mitochondrial inner-membrane and protrudes into the inter-membrane space. We show that Bca1 controls an early step in complex III assembly and that the supra-molecular organization of Bca1 is dependent upon the assembly level of complex III. Thus, Bca1 is a novel assembly factor for the respiratory complex III. © 2011 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
1. Introduction Mitochondrial respiratory enzymes are particularly fascinating examples of membrane complexes, because they are composed of numerous subunits of dual genetic origin, a feature that they share with the photosynthetic complexes in chloroplasts. A few core subunits of these complexes are encoded by the mitochondrial genome, translated on mitochondrial ribosomes and immediately inserted into the membrane, while most subunits are encoded by nuclear genes, translated within the cytoplasm and have to be imported into mitochondria. In addition, supra-molecular associations between different mitochondrial complexes have been reported (Cruciat et al., 2000; Schagger and Pfeiffer, 2000). The assembly of these large molecular machineries is a dynamic and complicated process that involves assembly factors or membrane chaperons that are extrinsic to the complexes. The complex III, also called complex bc1, occupies a key position in the respiratory chain and in the formation of the super-complexes. Its structure and function are highly conserved from lower to higher eukaryotes and several human diseases are related to complex III deficiencies (Benit et al., 2009). The complex operates as a homodimer
⁎ Corresponding author. Centre de Génétique Moléculaire, CNRS FRE 3144, Bâtiment 26, Avenue de la terrasse, Gif-sur-Yvette F-91190, France. Tel.: +33 1 69 82 31 69; fax: +33 1 69 82 31 50. E-mail address: [email protected] (G. Dujardin). 1 The first two authors contributed equally to this paper.
and in mammals, each monomeric unit consists of 11 different polypeptides. Three subunits, cytochrome b (Cytb), cytochrome c1 (Cyt1) and the Iron Sulphur Protein (ISP or Rip1) form the catalytic core and participate in the electron transfer process. The remaining subunits are thought to be involved in the stabilization and protection of the catalytic core. Cytb is the only mitochondrially-encoded subunit of complex III and it occupies the central position in the atomic structure with its eight transmembrane (TM) segments and constitutes the core scaffold of the complex (Hunte et al., 2000; Xia et al., 1997). The details of complex III assembly are still poorly understood. A modular mode of assembly has been proposed for the yeast Saccharomyces cerevisiae complex III by Zara et al., 2009a,b: two modules containing Cytb or Cyt1 and other non-catalytic subunits would first associate to form a 500 kDa pre-complex III (pre-III) which would subsequently be able to bind the remaining catalytic subunit Rip1 (Fig. 3D). In the absence of Rip1, the pre-complex III accumulates (Cruciat et al., 2000; Zara et al., 2009b). Complex III and complex IV show a similar level of complexity and more than 30 assembly-assisting factors or membrane chaperons are known to be involved in the assembly of complex IV (Fontanesi et al., 2008). However, only five complex III assembly factors have been identified until now: Cc1hl, Cbp3, Cbp4, Mzm1 and Bcs1. Cc1hl is a heme lyase which covalently binds heme to Cyt1 (Zollner et al., 1992). Cbp3 and Cbp4 interact and play a role in the biogenesis of the Cytb module (Kronekova and Rodel, 2005; Wu and Tzagoloff, 1989). Mzm1 influences a labile pool of mitochondrial zinc and its absence leads to a destabilized complex III (Atkinson et al., 2010). Bcs1 is involved in the binding of the catalytic subunit Rip1 (Cruciat et al., 1999; Nobrega et al., 1992; Nouet et al., 2009). The function of Bcs1 is conserved in higher
1567-7249/$ – see front matter © 2011 Elsevier B.V. and Mitochondria Research Society. All rights reserved. doi:10.1016/j.mito.2010.12.002
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eukaryotes and mutations in human Bcs1 are associated with mitochondrial diseases (e.g. de Lonlay et al., 2001; Fernandez-Vizarra et al., 2007). S. cerevisiae is able to rely exclusively on glucose fermentation for its energy requirements and most of the known assembly factors were discovered through the study of yeast mutants that were unable to grow on non-fermentable medium. These mutant screens were quite intensive and are probably close to saturation. Also mutations in some assembly factors may not lead to a clear respiratory phenotype. Thus, alternative screening methods are needed to isolate other complex III assembly factors. We have recently shown that there are differences in the spatiotemporal expression of yeast genes with a mitochondrial function: in synchronized cells the expression of genes encoding translation and assembly factors precedes the expression of genes encoding the respiratory complex components and the TCA cycle enzymes (Lelandais et al., 2009). In addition, these early-expressed genes appear to be translated in the vicinity of mitochondria and many present a conserved motif in their 3′end UTR that is known to be recognized by the RNA-binding protein Puf3 (Garcia et al., 2007; Saint-Georges et al., 2008). Based on these observations, we have proposed that new assembly factors may be encoded by genes of unknown function belonging to the subset of early expressed, Puf3-dependent genes. In this paper, we demonstrate that this transcriptome-based screen does allow the identification of a novel factor, Bca1 (bc1 assembly), controlling the assembly of the respiratory complex III. 2. Materials and methods 2.1. Yeast strains, media and genetic techniques All the strains were derived from W303-1B MATα ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 can1-100 (Table 1). CW252 is the wild-type strain (WT) and contains an intron-less mitochondrial genome (SaintGeorges et al., 2002). The series of mutants expressing the HA-tagged version of Bca1 were constructed by crossing each single mutant with the BCA1-HA strain (see below). Most media and genetic techniques were as described in Dujardin et al. (1980). The nonfermentable media contained either 2% glycerol or 2% lactate. The fermentable media contained either 2% glucose or 2% galactose and 0.1% glucose. The maintenance of the mitochondrial genome was tested by crossing the cells with the strain KL14-4A/60, which was devoid of the mitochondrial genome (MATa his1 trp2 rho°), and testing the growth of the resulting diploids on glycerol medium. 2.2. Epitope tagging and gene deletions Bca1p was tagged at the C-terminus with the 3HA epitope. We used the Schizosaccharomyces pombe his5 marker gene (which complements the S. cerevisiae his3 mutation) as described in Longtine et al. (1998). The PCR fragments were used to transform the CW252 strain to histidine prototrophy. Correct integration of the tag at the
Table 1 List of strains. Name
Genotype
Origin
CW252 Δbca1 Δbca1 Δrip1 Bca1-HA Δcyt1 Bca1-HA Δcbp3 Bca1-HA Δrip1 Bca1-HA Δbcs1 Bca1-HA
Wild type bca1::G418 bca1::G418 rip1::LEU2 BCA1-3HA-HIS3 cyt1::LEU2 BCA1-3HA-HIS3 cbp3::G418 BCA1-3HA-HIS3 rip1::LEU2 BCA1-3HA-HIS3 bcs1::URA3 BCA1-3HA-HIS3
Saint-Georges et al., 2002 This work This work This work This work This work This work This work
BCA1 locus in the BCA1-HA strain was confirmed by PCR amplification and sequencing. The Δbca1 strain carrying the deleted allele bca1::kanMX4 was constructed by transformation of CW252 with a PCR fragment that contained the KanR gene flanked by 50 bp of the BCA1 5′ and 3′ UTR. The integration was confirmed by PCR analysis of the genomic DNA and the [G418R] phenotype was shown to segregate 2:2 and to correlate with the presence of the deleted version of BCA1. The other null mutants were constructed by similar PCR-based techniques. The double mutant Δbca1Δrip1 was constructed by crossing the two single mutants. 2.3. FISH analysis and mRNA localization WT cells were grown to mid-exponential phase and were treated as described in Garcia et al. (2007) and Saint-Georges et al. (2008). The combined hybridization of three to five antisense oligonucleotides probes was used to enhance the visualization of each mRNA. Typically, each probe was designed so that it contained 50 to 55 nucleotides, with five aminoallyl thymidines and a coherent Tm value. The probes were directly labeled with Cy3 or Cy5 fluorochromes and hybridized to fixed cells. 2.4. Cytochrome absorption spectra Cytochrome absorption spectra of whole cells grown on galactose medium were recorded at liquid nitrogen temperature after the addition of the reducing agent, sodium dithionite, using a Cary 400 (Varian, San Fernando, CA) spectrophotometer. Absorption maxima for the bands of cytochromes c, c1, b, and aa3 are expected at 546, 552, 558 and 602 nm, respectively. 2.5. Isolation of mitochondria, SDS-polyacrylamide gels (SDS-PAGE) Cells were grown in galactose medium, and mitochondria were isolated by differential centrifugation after digestion of cell walls with Zymolyase-100T (Lemaire and Dujardin, 2008). The protein concentration of the final mitochondrial suspension was determined using the Bio-Rad assay (Hercules, CA). Mitochondrial proteins were resolved on denaturating 12% SDS-PAGE. Protein markers were used to estimate protein molecular masses. 2.6. Separation of mitochondrial protein complexes in Blue Native non-denaturing gels (BN-PAGE) Mitochondria were solubilised with 2% digitonin (w/v) in the presence of antiprotease and DNase and the complexes were separated on 5–10% or 6–15% polyacrylamide gradient gels as described in Schagger and Pfeiffer (2000) and modified in Lemaire and Dujardin (2008). Protein markers (High Molecular Weight Calibration kit for native electrophoresis, Amersham Biosciences) were used to estimate the molecular weights. In some cases, the gel strips excised from the BN-PAGE were analyzed in a second dimension SDS-PAGE. The gel strips were incubated in a solution containing 60 mM Tris HCl (pH 6.8) for five minutes at room temperature. Each gel strip was then placed horizontally above an SDS PAGE separating gel (12% polyacrylamide and 0.1% SDS). The gel slice was then encased in the 5% polyacrylamide stacking gel. This second dimension SDS-PAGE allows the utilization of antibodies that do not recognize the native protein. 2.7. Immuno-precipitation experiments Mitochondria were isolated from the Bca1p-HA strain and solubilised in 1% digitonine or lauryl maltoside. The suspension was centrifuged for 15 min at 4 °C at 100,000 g. Polyclonal HA-antibodies
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coupled agarose beads were added to the supernatant. Samples were incubated under gentle shaking for 90 min at 4 °C. The beads were washed twice with the lysis buffer and the immuno-precipitate was analyzed by SDS-PAGE and western blotting experiments.
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co-localized with the mitochondrial network (15S rRNA) in 45% of cells analyzed (Fig. 1). Control mRNAs were previously identified as mitochondrially-localized (ATP3) and non-mitochondrially-localized (ATP16). Our results show that BCA1 mRNAs are strongly co-localized with the mitochondrial network.
2.8. Immunoblotting experiments of SDS-PAGE and BN-PAGE 3.2. Topology of the Bca1 protein within mitochondria SDS-PAGE and BN-PAGE were blotted on nitrocellulose (Schleicher & Schuell, Keene, NH). Polyclonal antibodies against cytochrome c1 (Cyt1) or cytochrome b (Cytb) were raised against a fusion proAapocytochrome c1 expressed in E. coli or a synthetic peptide of Cytb coupled to KLH (AGRO-BIO) respectively. Monoclonal antibodies against Cox2, Hsp60, and PGK were from Molecular Probes (Eugene, OR) and the anti-HA was from Santa Cruz Biotechnology. Other antibodies were gifts: Cyb2 (B. Guiard, Gif-sur-Yvette, France), Rip1 (C. Godinot, Lyon, France) and anti-Atp4 (J. Velours, Bordeaux, France). Bound antibodies were detected by horseradish-peroxidase-conjugated secondary antibodies (Promega, Madison, WI). Proteins were visualized using a chemiluminescent substrate (Pierce Chemical, Rockford, IL). The anti-Cyt1, anti-Atp2 and anti-Cox2 recognize the native protein within the complexes. 3. Results and discussion 3.1. BCA1 mRNA co-localize with the mitochondria According to the systematic transcriptome analysis (Lelandais et al., 2009), BCA1 mRNA (YLR077W) belongs to the regulon of earlyexpressed genes that were translated in the vicinity of mitochondria in a Puf3 dependent way. The 3′UTR of the BCA1 mRNA contains the characteristic-Puf3 binding motif: CAUGUAUAUA. To quantify BCA1 mRNA localization, we have performed an in situ FISH analysis in wildtype cells by labeling the BCA1 mRNAs and observed that BCA1 mRNAs
A large-scale analysis of the localization of S. cerevisiae proteins suggested that Bca1 is a mitochondrial protein and the PSORT program predict a mitochondrial location. To verify its subcellular localization, Bca1 was tagged at its C-terminus with three HA epitopes. The HA antibody revealed a protein of the expected size, ~70 kDa, that was recovered in the mitochondrial fraction, as was the mitochondrial protein Atp4, whereas the cytosolic phosphoglycerate kinase (PGK) was found mainly in the postmitochondrial supernatant (Fig. 2A, left panel). Bca1 is predicted to have a transmembrane domain between residues 83 and 105 and a large soluble C-terminal domain (Fig. 2B, right panel). To test whether Bca1 was associated with mitochondrial membranes, mitochondria purified from cells expressing Bca1-HA, were “alkali”-treated to extract the non-integral membrane proteins. As shown in Fig. 2A (right panel), Bca1-HA was found in the pellet fraction, as was the inner membrane protein Atp4. In addition, proteinase K treatment of mitochondria did not degrade the Bca1-HA protein or modify its mobility (Fig. 2B, lane 2) indicating that Bca1 did not span the outer membrane. Thus Bca1 is embedded in the inner membrane. To determine the topology of Bca1, we performed osmotic swelling experiments that disrupt the outer membrane of mitochondria while keeping the inner membrane intact. The proteinase K treatment of the swollen mitochondria led to the degradation of 90% of Bca1-HA while the matrix protein Hsp60 remained protected and the intermembrane space soluble protein cytochrome b2 (Cyb2) was completely degraded
Fig. 1. Localization of BCA1 mRNA by FISH analysis. BCA1, ATP3, ATP16 mRNAs were labeled with specific sets of fluorescent probes as described in Materials and methods (green in the Merge). Five fluorescent probes specific for the 15S mitochondrial ribosomal RNA delimited the mitochondrial compartment (red in the Merge). The nucleus was stained by DAPI (blue in the 3D reconstruction). Quantification of the mitochondria/mRNA colocalization rate was done using Corsen software and presented in the histogram (Jourdren et al., 2010).
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3.3. Bca1 controls the assembly of complex III
Fig. 2. Mitochondrial localization and topology of Bca1. Mitochondria were purified from cells expressing Bca1-HA. Proteins were analyzed by SDS-PAGE and immunoblotting with different antibodies. (A) Left: Mitochondrial (M) and postmitochondrial supernatant (PS) protein fractions were separated on SDS-PAGE. Right: Mitochondria were alkali treated at pH 11.5 according to Lemaire and Dujardin (2008) to separate the soluble proteins (S) from the integral membrane proteins that remained in the pellet (P). (B) Left: Mitochondrial proteins (lane 1) were treated with 100 mg/ml of proteinase K before (lane 2) or after osmotic swelling (lane 3) according to Funes et al. (2004). Right: Topology of the Bca1 protein. TM: transmembrane segment; OM: outer membrane; IM: inner membrane.
(Fig. 2B, lane 3). Altogether, our results indicated that Bca1 is bound to the mitochondrial inner membrane and exposes its large soluble C-terminal domain to the intermembrane space (Fig. 2B, right panel).
To investigate the role of Bca1, a Δbca1 null allele was constructed (see Section 2.3). The Δbca1 exhibited a respiratory growth defect on a non-fermentable medium containing lactate as carbon source but not on glycerol medium (Fig. 3A). The effect of the absence of Bca1 on respiratory complex biogenesis was first studied by recording the cytochrome absorption spectra. The Δbca1 cells showed a wild-type level of cytochromes a + a3 and a clear decrease in the cytochrome b content (Fig. 3B). Next, the steady-state levels of the three complex III catalytic subunits, Cytb, Cyt1 and Rip1 as well as that of Cox2 and Atp4 that are representative of the respiratory complexes IV and V respectively, were analyzed by western blot. The steady state level of Rip1 and Cytb diminished, 10% for Cytb and 70% for Rip1, while Cyt1, Cox2, Atp4 and the control Hsp60 were not affected (Fig. 3C). Finally, the assembly level of complex III was analyzed by Blue Native Poly Acrylamide Gel Electrophoresis (BN-PAGE) of mitochondria solubilised with the mild detergent digitonin that preserves the interactions between complexes. As expected, the anti-Cyt1 antibody revealed the super-complexes III2 + IV, III2 + IV2 and the homo-dimer, III2 in the wild-type strain. In the Δbca1 mutant, the intensity of the three bands was strongly reduced by at least 70% (Fig. 3E). No lowmolecular-weight band that might correspond to the accumulation of assembly intermediates was revealed even after a long exposure. We have also verified that the assembly of complex V was not disturbed in the Δbca1 mutant (data not shown). Finally, we have constructed a double mutant Δrip1Δbca1 and compare the assembly of complex III of the double mutant to that of the single mutant Δrip1. In the absence of Rip1, a pre-complex III of about 500 kDa resulting from the association of the cytochrome b and c1 modules is known to accumulate (Cruciat et al., 2000; Schagger and Pfeiffer, 2000; Fig. 3D). In the double mutant Δrip1Δbca1, this pre-complex III is detected but its steady state level was reduced by about 60%. The fact that the absence of the inter-membrane space protein Bca1 affects the amount of cytochrome b as well as the steady state levels of precomplex III and complex III suggests that Bca1 controls the assembly of complex III at an early stage, before the insertion of Rip1 into the
Fig. 3. Effect of Δbca1 mutation on the assembly of the respiratory complex III. (A) Wild-type (WT) and Δbca1 were spotted onto fermentable glucose (glu) and respiratory glycerol (gly) or lactate (lac) media. Photographs were taken after 1 day (glucose) or 3 days (glycerol, lactate) of incubation at 28 °C. (B) Low temperature cytochrome absorption spectra of the wild-type and Δbca1 of cells grown on galactose medium were recorded. Cytochromes c1 and b are part of complex III and cytochromes aa3 of complex IV. Abs: Absorbance. (C) Mitochondrial proteins from wild-type and Δbca1 were analyzed by SDS-PAGE and immunoblotting with different antibodies. (D) Model for the assembly of complex III (see Introduction and this work for Bca1). (E) Mitochondrial proteins from wild type, Δbca1, Δrip1, Δbca1 Δrip1 mutants were analyzed by BN-PAGE (5–10%) and immunoblotting with antibodies against Cyt1. Positions of pre-complex III, dimers III2, supercomplexes III2IV and III2IV2, are indicated.
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pre-complex III, the strong effect on the level of Rip1 being the consequence of this upstream effect in the assembly process (Fig. 3C and D). 3.4. Complex III assembly affects the supra-molecular organization of Bca1 BLAST searches reveal that Bca1-like proteins are only found in closely related yeasts such as Kluyveromyces lactis or Candida glabrata (about 40% identity) and some filamentous fungi such as Neurospora crassa (about 20% identity). In addition, the C-terminal domain of Bca1 presents RCC1/BLIPII motifs (Regulator of Chromosome Condensation, Renault et al., 1998; β-lactamase inhibitor protein-II, Lim et al., 2001, see Fig. S1). RCC1/BLIP-II motifs are structural motifs forming a β-propeller fold that can bind protein ligands of very different types in eukaryotic cells, suggesting that Bca1 might interact with protein partners. In order to study the interactions between Bca1 and complex III, we have compared the effect of three mutations affecting the various modules involved in complex III assembly, Δcyt1, Δcbp3 and Δrip1 on Bca1-HA accumulation. As shown in Fig. 4A, none of these mutants modified the steady-state level of Bca1. Next, proteins of wild type mitochondria were solubilised with 1% digitonin and subjected to
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co-immuno-precipitation using agarose-bound antibodies against HA. Bca1 was clearly detected in the immuno-precipitate while none of the three catalytic subunits of complex III or representative subunits of complexes IV and V immuno-precipitated with Bca1 (Fig. 4B). A similar result was obtained with laurylmaltoside solubilisation and with mitochondria from a Δrip1 strain. Thus, if there are any interaction between Bca1 and the catalytic subunits of complex III, they are probably labile or transitory. We have also compared the supra-molecular organization of Bca1 in the wild type and mutants by combining the first dimension BN-PAGE (Fig. 4C) with a second-dimension SDS-PAGE (Fig. 4D). In the Δrip1 and Δbcs1 mutants, Bca1 accumulated in a high molecular weight complex of about 600 kDa that was not detected in the wild type or in Δcyt1 and Δcbp3 mutants. Thus, this 600 kDa Bca1containing complex only accumulates in the two mutants that also accumulate the 500 kDa pre-complex III (Fig. 4B). The second dimension gel reveals that in the wild type strain, Bca1-HA was indeed present in a continuous streak of low molecular weight complexes from about 140 to 440 kDa (Fig. 4C). Thus the supra molecular organization of the Bca1 protein clearly differs in the wild type and in the mutants affecting the assembly of the Rip1 subunit, showing that its supramolecular organization was dependent of the assembly state of complex III. The presence of Bca1 in a continuous
Fig. 4. Supramolecular organization of Bca1 and complex III assembly. Mitochondrial proteins were purified from wild-type (WT), Δbca1 and four other mutants affecting different steps of the assembly of complex III: Δcyt1, Δrip1, Δcbp3, Δbcs1 and expressing Bca1-HA. (A) Mitochondrial proteins were analyzed by SDS-PAGE and immunoblotting with antibodies against HA, the three catalytic complex III subunits and Hsp60. (B) Mitochondrial proteins from wild-type strain expressing Bca1-HA were solubilised with 1% digitonin and incubated with antibodies against HA coupled to agarose beads. The different samples were subjected to SDS-PAGE and immunoblotting with antibodies. (M) mitochondrial lysate; (S) supernatant of beads; (W) washing; (IP) immuno-precipitate. (C) Mitochondrial proteins were analyzed by BN-PAGE (6–15%) and immunoblotting with anti-Cyt1 and anti-HA. Positions of pre-complex III (pre-III) and of the 600 kDa Bca1-containing complex are indicated. (D) Mitochondrial proteins of wild-type and Δrip1 cells were analyzed by BN-PAGE (6–15%) and then in a second dimension by SDS-PAGE (12%) and immunoblotting with anti-HA and anti-Cyt1. Positions of size markers are indicated.
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streak of low molecular weight complexes in BN-PAGE suggests that it is involved in dynamic and thus probably transient interactions. The arrest of the assembly process of complex III at the step of Rip1 insertion might modify these transitory interactions leading to the stable accumulation of Bca1 in the high molecular weight oligomeric complex of 600 kDa. 4. Conclusions In this paper, we validate our transcriptome-based screen by demonstrating that the early expressed, Puf3 dependent BCA1 gene encodes a novel assembly factor for the yeast respiratory complex III. Bca1 appears to act upstream of Rip1 insertion in the assembly of complex III. According to the modular model of assembly, Bca1 could control either the formation/stabilization of the cytochrome b module or the interactions between the cytochrome b and cytochrome c1 modules before Rip1 insertion. It is very likely that the study of the function of other genes that are part of the same regulon should lead to the discovery of more assembly factors of respiratory complexes. The further characterization of Bca1 and other similarly regulated assembly factors will allow a better understanding of the assembly pathway of the mitochondrial respiratory complexes. In human cells, it would be also very interesting to test if a similar subset of genes encoding translation and assembly factors are translated at the vicinity of mitochondria. If this is the case, such transcriptome-based screens should lead to the identification of new nuclear encoded factors that are required for the biogenesis of the human respiratory complexes. Mutations in the genes encoding these factors could be responsible for pathologies for which the genetic determinism is still unknown. Supplementary materials related to this article can be found online at doi:10.1016/j.mito.2010.12.002. Acknowledgements We thank CJ. Herbert, B. Guiard, B. Meunier and C. Panozzo for critical reading of the manuscript and CJ Herbert for checking the English language. This work was supported by a grant from the Agence Nationale pour la Recherche (LAN06-0234). References Atkinson, A., Khalimonchuk, O., Smith, P., Sabic, H., Eide, D., Winge, D.R., 2010. Mzm1 influences a labile pool of mitochondrial zinc important for respiratory function. J. Biol. Chem. 285, 19450–19459. Benit, P., Lebon, S., Rustin, P., 2009. Respiratory-chain diseases related to complex III deficiency. Biochim. Biophys. Acta 1793, 181–185. Cruciat, C.M., Hell, K., Folsch, H., Neupert, W., 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. Cruciat, C.M., Brunner, S., Baumann, F., Neupert, W., Stuart, R.A., 2000. The cytochrome bc1 and cytochrome c oxidase complexes associate to form a single supracomplex in yeast mitochondria. J. Biol. Chem. 275, 18093–18098. de Lonlay, P., Valnot, I., Barrientos, A., Gorbatyuk, M., Tzagoloff, A., Taanman, J.W., Benayoun, E., Chretien, D., Kadhom, N., Lombes, A., de Baulny, H.O., Niaudet, P., Munnich, A., Rustin, P., Rotig, A., 2001. A mutant mitochondrial respiratory chain assembly protein causes complex III deficiency in patients with tubulopathy, encephalopathy and liver failure. Nat. Genet. 29, 57–60.
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