Tài liệu Báo cáo khóa học: Further insights into the assembly of the yeast cytochrome bc1 complex based on analysis of single and double deletion mutants lacking supernumerary subunits and cytochrome b pdf

Further insights into the assembly of the yeast cytochrome bc 1 complex based on analysis of single and double deletion mutants lacking supernumerary subunits and cytochrome b Vincenzo Zara 1 , Ilaria Palmisano 1 , Laura Conte 1 and Bernard L. Trumpower 2 1 Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Universita ` di Lecce, Italy; 2 Department of Biochemistry, Dartmouth Medical School, Hanover, NH, USA The cytochrome bc 1 complex of the yeast Saccharomyces cerevisiae is composed of 10 different subunits that are assembled as a symmetrical dimer in the inner mitochondrial membrane. Three of the subunits contain redox centers and participate in catalysis, whereas little is known about the function of the seven supernumerary subunits. To gain fur- ther insight into the function of the supernumerary subunits in the assembly process, we have examined the subunit composition of mitochondrial membranes isolated from yeast mutants in which the genes for supernumerary sub- units and cytochrome b were deleted and from yeast mutants containing double deletions of supernumerary subunits. Deletion of any one of the genes encoding cyto- chrome b, subunit 7 or subunit 8 caused the loss of the other two subunits. This is consistent with the crystal structure of the cytochrome bc 1 complex that shows that these three subunits comprise its core, around which the remaining subunits are assembled. Absence of the cytochrome b/sub- unit 7/subunit 8 core led to the loss of subunit 6, whereas cytochrome c 1 , iron–sulfur protein, core protein 1, core protein 2 and subunit 9 were still assembled in the mem- brane, although in reduced amounts. Parallel changes in the amounts of core protein 1 and core protein 2 in the mito- chondrial membranes of all of the deletion mutants suggest that these can be assembled as a subcomplex in the mito- chondrial membrane, independent of the presence of any other subunits. Likewise, evidence of interactions between subunit 6, subunit 9 and cytochrome c 1 suggests that a subcomplex between these two supernumerary subunits and the cytochrome might exist. Keywords: cytochrome bc 1 ; assembly; supernumerary sub- units; Saccharomyces cerevisiae. The cytochrome bc 1 complex is a multisubunit complex embedded in the inner membrane of mitochondria [1,2]. This respiratory enzyme catalyzes the transfer of electrons from ubiquinol to cytochrome c and couples the electron transfer to vectorial proton translocation across the inner mitochondrial membrane. The bc 1 complex has been crystallized and analyzed from bovine, chicken and yeast mitochondria [3–7]. In mitochondria of the yeast Saccharomyces cerevisiae, the cytochrome bc 1 complex is composed of 10 different subunits organized in the lipid bilayer as a homo-dimer as shown in Fig. 1A [8,9]. There are three catalytic subunits that contain redox prosthetic groups, cytochrome b,cyto- chrome c 1 and the Rieske iron–sulfur protein (ISP). In addition, there are seven supernumerary subunits that lack any cofactors. The supernumerarysubunits arecore protein 1 and core protein 2 [10,11], with apparent molecular masses of  44 and 40 kDa on SDS/PAGE, respectively, and five smaller proteins. The latter are Qcr6p [12], Qcr7p [13], Qcr8p [14], Qcr9p [15] and Qcr10p [8] with apparent molecular masses of about 17, 14, 11, 7.3 and 8.5 kDa, respectively. Although the supernumerary subunits of the mitochond- rial bc 1 complexes were discovered one to two decades ago [16], little is known about their function. It is also not known how these peripheral subunits are assembled around the catalytic core of the enzyme to arrive at the three dimen- sional organization revealed by the crystal structures (Fig. 1A). The supernumerary subunits and the catalytic subunits of the yeast cytochrome bc 1 complex show sequence similarities to those of the bc 1 complexes of higher eucaryotes [1,2,9]. In addition, the crystallographic analysis of the Saccharomyces cerevisiae cytochrome bc 1 complex has revealed an essentially identical overall structure of this complex and that of chicken and beef [6]. In yeast and higher eukaryotes, cytochrome b is encoded by mito- chondrial DNA, while the remaining subunits of the bc 1 complex are encoded in the nucleus, synthesized by cytosolic polysomes, and then imported into mitochondria, thereby reaching their final location in the inner membrane [17]. The similarities of the yeast bc 1 complex to the bc 1 complexes of higher eukaryotes suggest that the yeast enzyme may serve as a paradigm to understand how this oligomeric protein complex is assembled into the inner mitochondrial membrane. Correspondence to V. Zara, Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Universita ` di Lecce, Via Prov.le Lecce- Monteroni, I-73100 Lecce, Italy. Fax: + 39 0832 298626, Tel.: + 39 0832 298705, E-mail: vincenzo.zara@unile.it Abbreviations: DFP, diisopropyl fluorophosphate; ISP, Rieske iron–sulfur protein. (Received 8 January 2004, revised 23 January 2004, accepted 6 February 2004) Eur. J. Biochem. 271, 1209–1218 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04024.x In this study, we have investigated the role of the supernumerary subunits in the assembly of the bc 1 complex in S. cerevisiae mitochondria. To this end we have prepared single and double deletion yeast mutants in which one or two nuclear genes encoding the supernumerary subunits Qcr6p, Qcr7p, Qcr8p, Qcr9p and Qcr10p have been deleted and analyzed the bc 1 subunits present in mitochondrial membranes using antibodies directed against the various subunits. Yeast mutant strains containing single deletions of genes for supernumerary subunits were described previously [8,15,18–20], even though an exhaustive analysis of cyto- chrome bc 1 subunit composition in these yeast strains has not been reported. We have also created two yeast strains in which the mitochondrial gene encoding cytochrome b has been deleted or truncated and examined the subunit composition of membranes in which the catalytic and structural core of the enzyme is absent. Experimental procedures Materials Yeast extract and bacto-peptone were purchased from Difco. Yeast nitrogen base without amino acids, Coomas- sie Brilliant Blue, phenylmethylsulfonyl fluoride, glass beads, acrylamide, bis-acrylamide, N,N,N¢N¢-tetramethyl- ethylenediamine, ammonium persulfate, diisopropyl fluoro- phosphate (DFP), glucose and glycerol were from Sigma. Anti-mouse and anti-rabbit IgG, coupled to peroxidase, were from Bio-Rad. The ECL detection system for Western blotting was from Amersham. Nitrocellulose was from Pall Life Sciences, New York, NY, USA 1 . Polyclonal and monoclonal antibodies against the various subunits of the yeast cytochrome bc 1 complex were prepared in the Trumpower laboratory. The anti-Tom40 Igs were a gift of N. Pfanner 2 (Institute for Biochemistry and Molecular Biology, Freiburg, Germany). All other reagents were of analytical grade. Yeast strains, media and genetic methods The S. cerevisiae strains used in this study are listed in Table 1. The construction of the QCR7 deletion strain (VZ1) was performed following the procedure of homolog- ous recombination as described previously [21]. A DNA fragment prepared by PCR and carrying the coding region for the selectable TRP1 marker, plus the flanking sequences of the QCR7 open-reading frame at the 5¢-and3¢-regions, was used to transform yeast cells by treatment with lithium acetate [22]. The transformants were then selected for tryptophan prototrophy. The double deletion strains were constructed as follows. The haploid strains VZ1 (D7) and MES8 (D6), VZ2 (D7) and LLD9 (D8), JDP1 (D9) and LLD9 (D8), JDP2 (D9) and UBL2 (D10), were mated and the resulting diploids were sporulated to obtain the double deletion strains VZ4 (D6/D7), VZ6 (D7/D8), VZ14 (D8/D9) and VZ9 (D9/D10), respectively. The selectable markers exhibited a 2 : 2 segre- gation pattern, and some spores were prototrophic for both markers. Haploid spores of VZ4, VZ6, VZ14 and VZ9 were then selected for Trp + and Leu + ,Trp + and His + ,His + and Ura + ,orHis + and Leu + prototrophy, respectively. Other yeast genetic methods used were as described in [23]. The expected absence of the corresponding protein pro- ducts in mitochondrial membranes from the deletion strains was assessed by Western blot analysis (Results). The respiratory capacity of the yeast strains was checked on nonfermentable solid medium containing 1% (w/v) yeast extract, 2% (w/v) bacto-peptone, 2% (w/v) agar, 3% (v/v) glycerol and 2% (v/v) ethanol (YPEG). Viability of the strains on fermentable medium was confirmed on 1% (w/v) yeast extract, 2% (w/v) bacto-peptone, 2% (w/v) agar and Fig. 1. The yeast cytochrome bc 1 complex. (A) The structure of the dimeric yeast bc 1 complex with the redox subunits, cytochrome b,cyto- chrome c 1 , and the Rieske ISP colored blue, red and yellow, respectively. The supernumerary subunits are colored gray. The structure is oriented as it would appear in the inner mitochondrial membrane, with the mitochondrial matrix at the bottom. (B) The structure of cytochrome b and supernumerary subunits 7 and 8 in one monomer (the Ôcytochrome b, subunit 7, subunit 8 coreÕ). Cytochrome b is colored blue, subunit 7 is colored pink, and subunit 8 is colored green. The arrow labeled (a) points to the N-terminus of cytochrome b where it is enveloped by subunit 7. The arrows labeled (b) and (c) point to the areas of interaction between the transmembrane helix of subunit 8 and helices G and H1 of cytochrome b and between the N-terminus of subunit 8 and helix a of cytochrome b. The figure was constructed from the crystal structure of the yeast bc 1 complex [6]. 1210 V. Zara et al. (Eur. J. Biochem. 271) Ó FEBS 2004 2% (w/v) glucose (YPD). For the isolation of mitochondrial membranes, the yeast strains were grown in liquid YPD medium containing 1% (w/v) yeast extract, 2% (w/v) bacto- peptone and 2% (w/v) glucose, pH 5.0. Isolation of mitochondrial membranes Mitochondrial membranes were isolated from the various yeast strains by a modification of a previously described method [24]. Yeast cells were grown overnight at 30 °C, unless otherwise specified, in 800 mL of YPD until expo- nential growth phase was reached (D 600 3 of 1–2). Cells were recovered by centrifugation at 3200 g for 15 min and then washed once with distilled water. The pellet was resuspended in 25 mL of MTE buffer (400 m M mannitol, 50 m M Tris/ HCl, 2 m M EDTA, pH 7.4). Acid-washed glass beads were added up to a final volume of 30 mL to the mixture kept at 4 °Cand1m M DFP was then added. Afterwards, the cells were mixed with a vortex mixer at maximum speed for 10 min at 4 °C. After the further addition of MTE buffer to a final volume of 50 mL, the mixture was centrifuged at 1000 g for 10 min at 4 °C. The supernatant was then centrifuged at 18 500 g for 30 min at 4 °C in order to pellet the mitochondrial membranes. The pellet was washed with 20–30 mL of MTE and re-isolated by centrifugation as described above. The mitochondrial membranes were then resuspended in 1 mL of MTE buffer, divided in aliquots of 50 lL each, and stored at )80 °C for subsequent analysis by SDS/PAGE and Western blotting. SDS/PAGE and Western blotting Mitochondrial membranes were analyzed by standard SDS/PAGE with 15% (w/v) acrylamide and an acryl- amide/bis-acrylamide ratio of 30 : 0.8 (w/w) [25]. The proteins were then stained with Coomassie Blue or transferred to nitrocellulose membranes. Immunodetection of the yeast mitochondrial proteins was carried out with monoclonal and polyclonal antibodies by chemilumines- cence. The stained polyacrylamide gels and the fluoro- graphs containing the immunodetected proteins were scanned and quantified using an Imaging Densitometer GS-700 from Bio-Rad. Other methods Protein concentrations were determined by the Bradford method [26] or the modified Lowry method [27]. Electro- phoretic analysis of DNA on agarose gels, restriction endonuclease analysis, ligation of DNA fragments, Table 1. Yeast strains used in this study. Strain Genotype Reference W303–1 A (WT) MATa, ade2–1, his3–11,15, trp1–1, leu2–3,112, ura3–1, can1–100 Gift from A. Tzagoloff, Columbia University, New York W303–1B (WT) MATa, ade2–1, his3–11,15, trp1–1, leu2–3,112, ura3–1, can1–100 Gift from A. Tzagoloff, Columbia University, New York MES8 (D6) MATa, ade2–1, his3–11,15, trp1–1, leu2–3,112, ura3–1, can1–100, qcr6D::LEU2 [37] VZ1 (D7) MATa, ade2–1, his3–11,15, trp1–1, leu2–3,112, ura3–1, can1–100, qcr7D::TRP1 This study VZ2 (D7) MATa, ade2–1, his3–11,15, trp1–1, leu2–3,112, ura3–1, can1–100, qcr7D::TRP1 This study LLD9 (D8) MATa, ade2–1, his3–11,15, trp1–1, leu2–3,112, ura3–1, can1–100, qcr8D::HIS3 Daniels and Trumpower, unpublished data JDP1 (D9) MATa, ade2–1, his3–11,15, trp1–1, leu2–3,112, ura 3–1, can1–100, qcr9D1::URA3 [15] JDP2 (D9) MATa, leu2–3,112, his3, can 1–11, qcr9D2::HIS3 [15] UBL2 (D10) MATa, ade2–1, his3–11,15, leu2–3,112, ura3–1, can1–100, qcr10D2::LEU2 [8] VZ4 (D6/D7) MATa, ade2–1, his3–11,15, trp1–1, leu2–3,112, ura3–1, can1–100, qcr6D::LEU2, qcr7D::TRP1 This study VZ6 (D7/D8) MATa, ade2–1, his3–11,15, trp1–1, leu2–3,112, ura3–1, can1–100, qcr7D::TRP1, qcr8D::HIS3 This study VZ14 (D8/D9) MATa, ade2–1, his3–11,15, trp1–1, leu2–3,112, ura3–1, can1–100, qcr8D::HIS3, qcr9D1::URA3 This study VZ9 (D9/D10) MATa, ade2–1, leu2–3,112, qcr9D2::HIS3, qcr10D2::LEU2 This study SUY 106-a MATa, his3-D200, leu2-D, qcr6D1::LEU2, qcr10D1::HIS3 [8] W303–1B q° MATa, ade2–1, his3-, trp1–1, leu2–3,112, ura3–1 (no mtDNA) Gift from B. Meunier, UCL CKWT MATa, leu1, kar1–1 (WT mtDNA, intronless) Gift from B. Meunier, UCL CKL57 MATa, leu1, kar1–1 (intronless mtDNA, point mutation in cytochrome b gene) Gift from B. Meunier, UCL Ó FEBS 2004 Assembly of the yeast cytochrome bc 1 complex (Eur. J. Biochem. 271) 1211 transformation of Escherichia coli and isolation of plasmid DNA from bacterial cells were carried out by standard procedures [28]. Results Growth phenotype of single and double deletion mutants The growth phenotype of the yeast strains with deletions of genes encoding various subunits of the bc 1 complex was determined by plating the cells on solid media containing fermentable or nonfermentable carbon sources and then incubating at 30 °C. The results are summarized in Table 2. Among the single deletion mutants, only the subunit 6 (MES8) and subunit 10 (UBL2) deletion strains were able to grow on nonfermentable carbon source at a rate compar- able to the wild-type strain (W303). Under the same conditions, the strain JDP1, in which the nuclear gene encoding subunit 9 had been deleted, exhibited a reduced growth rate with respect to the wild-type strain as reported previously [15,29]. The yeast mutants with deletions for the genes encoding subunit 7 (VZ1) or subunit 8 (LLD9) failed to grow on the nonfermentable YPEG medium. Among the double deletion mutants, the strain with the genes encoding subunits 9 and 10 deleted (VZ9) and that with the genes encoding subunits 6 and 10 deleted (SUY 106-a) grew on nonfermentable medium, although at a reduced rate compared to the wild-type strain. In the case of the VZ9 strain, this was to be expected, based on the reduced growth rate of the single deletion strain lacking subunit 9. The remaining double deletion mutants, VZ4 (D6/D7), VZ6 (D7/D8) and VZ14 (D8/D9), were unable to grow on the same medium. Cytochrome bc 1 subunit analysis of single deletion mutants We sought to determine how the absence of individual supernumerary subunits affected the composition of bc 1 subunits in the mitochondrial membranes. For this purpose, mitochondrial membranes were isolated from the single deletion strains grown at 30 °C in YPD, then transferred to nitrocellulose and probed with an antiserum against Tom40p, an outer membrane protein belonging to the import machinery of yeast mitochondria (data not shown). In this way we adjusted the amount of mitochondrial membranes in order to use comparable amounts of protein for the subsequent immunoblot experiments. The blot in Fig. 2 shows the cytochrome bc 1 subunits in the mitochondrial membranes from the mutants in which genes for subunit 6 (MES8), 7 (VZ1), 8 (LLD9), 9 (JDP1) or 10 (UBL2) were deleted. Relative amounts of the subunits determined by densitometry scanning of the stained gels are tabulated in Table 3. The relative amounts of cytochrome b and the mature forms of both cytochrome c 1 and Rieske ISP decreased to 52, 64 and 68%, respectively, in the subunit 6 deletion strain compared to the wild-type strain. Table 2. Growth phenotype of single and double deletion mutants. All the strains were first grown in liquid YPD medium to the same original density and subsequently plated on solid media containing ferment- able (YPD) or nonfermentable carbon sources (YPEG). Normal growth, +; reduced growth rate (+); no growth, ). Strain Lacking subunit(s) Growth YPD YPEG W303 – + + MES8 Qcr6p + + VZ1 Qcr7p + – LLD9 Qcr8p + – JDP1 Qcr9p (+) (+) UBL2 Qcr10p + + VZ4 Qcr6p/Qcr7p + – VZ6 Qcr7p/Qcr8p + – VZ14 Qcr8p/Qcr9p + – VZ9 Qcr9p/Qcr10p + (+) SUY 106-a Qcr6p/Qcr10p (+) (+) Fig. 2. Subunit composition of mitochondrial membranes from yeast mutants with single deletions of genes for each of the nuclear encoded supernumerary subunits. Yeast strains were grown on YPD medium and mitochondrial membranes were analyzed by SDS/PAGE and Western blotting with antibodies to the subunits of the yeast bc 1 complex indicated on the left side of the blots. 1212 V. Zara et al. (Eur. J. Biochem. 271) Ó FEBS 2004 Interestingly, the absence of subunit 6 also resulted in an increase in the ratio of intermediate to mature cyto- chrome c 1 and a disappearance of the intermediate form of the Rieske protein. At the same time, the levels of subunits 7, 8 and 9 significantly decreased in this mutant strain. However, the amounts of core protein 1 and core protein 2 were relatively unaffected. Therefore, the absence of subunit 6 appeared to alter the rates of processing of two of the redox subunits and caused minor changes in amounts of the small supernumerary subunits, but it did not cause dramatic changes in the cytochrome bc 1 composition. Accordingly, this yeast strain was respiratory-competent. Deletion of the gene encoding either subunit 7 or subunit 8 resulted in a more severe phenotype, and the changes in bc 1 subunit composition of the membranes were compar- able in these two deletion strains, as can be seen from the blot in Fig. 2. In addition, the absence of subunit 7 caused a strong decrease in subunit 8 and vice versa, suggesting a correlation between these two subunits. In both strains, cytochrome b and the Rieske protein were almost unde- tectable, while the amounts of cytochrome c 1 were similar to that found in the wild-type strain. Subunit 9 decreased to 36% of the wild-type level in both mutant strains, and the twocoreproteinsdecreasedinparallelinbothmutants,with lower amounts found in the subunit 8 deletion strain. The only difference between the two strains was that subunit 6 was present in small amounts in the subunit 7 deletion strain but completely absent in the subunit 8 deletion strain. In mitochondrial membranes from the strain JDP1, in which the gene encoding subunit 9 had been deleted, there was a significant decrease in cytochrome c 1 (45% of the wild-type content), a barely detectable amount of Rieske protein and low levels of cytochrome b (12% of the wild- type content). Core protein 1 and core protein 2 decreased significantly to about 40% of the wild-type levels. Subunit 8 decreased to the same extent as the core proteins, whereas a greater decrease was seen in the case of both subunits 6 and 7. Interestingly, a higher amount of cytochrome b,almost equivalent to that of wild-type cells, was detected in the JDP1 (D9) mitochondrial membranes when this mutant strain was grown at 25 °C instead of 30 °C (results not shown). This effect of temperature on cytochrome b content was not observed in the case of the other single deletion mutants. Among the single deletion strains tested, UBL2, in which the gene for subunit 10 was deleted, was the only one showing wild-type levels of all of the bc 1 subunits (Fig. 2 and Table 3). It is also worth noting that the mitochondrial membranes from these mutant cells also showed the same ratio of intermediate to mature form of ISP when compared to the wild-type membranes (Fig. 2). Accordingly, deletion of QCR10 did not affect mitochondrial respiration, even though bc 1 activity was significantly reduced [8]. This is due tothefactthatactivityofthebc 1 complex in wild-type yeast is significantly greater than what is required to support normal rates of respiration. Cytochrome bc 1 subunit analysis of double deletion mutants Mitochondrial membranes were isolated from the double deletion strains and processed by SDS/PAGE and Western blotting using Tom40p to normalize the protein load in the same manner as for the single deletion strains. The immuno- detection of bc 1 subunits in the mitochondrial membranes isolated from the double deletion mutants is shown in Fig. 3, and the corresponding quantifications are reported in Table 4. A comparison of the immunoblots in Figs 3 and 2 reveals that the double deletions of genes encoding bc 1 subunits had more marked effects on the composition of bc 1 subunits in the mitochondrial membranes than was observed with the single deletion mutants. The membranes from the D6/D7 double deletion strain (VZ4) exhibited the strongest defect in the assembly of the catalytic subunits of cytochrome bc 1 complex. This strain showed only 18% and 6% of the wild-type levels of iron– sulfur protein and cytochrome b, respectively, while mature cytochrome c 1 disappeared completely and only a small amount of the intermediate form was visible. Subunits 8 and 9 were reduced to about one third of the original levels. However, the core proteins were only slightly diminished. The most notable difference between this double deletion strain and the others (see below) was the complete absence of mature cytochrome c 1 . The mitochondrial membranes from the D7/D8 double deletion strain (VZ6) showed no cytochrome b and only a negligible amount of ISP, as expected on the basis of the results obtained with the single deletion strains. The relative amount of cytochrome c 1 decreased by 50%, as did both core proteins. There was also a strong reduction in the amounts of both subunit 6 and subunit 9 in this strain. The highest amount of cytochrome c 1 , approximately 80% of the normal amount, was found in the mitochondrial membranes from the D8/D9 double deletion strain (VZ14). However, there was a strong defect in both cytochrome b and ISP in this strain, similar to what was observed in the other double deletion strains. Core proteins 1 and 2 were reduced to approximately half of the wild-type levels, while subunits 6 and 7 were present only in small amounts (18% and 8%, respectively). The mitochondrial membranes from the D9/D10 double deletion strain (VZ9), which is one of the two respiratory Table 3. Cytochrome bc 1 subunit analysis of single deletion mutants. The values represent the percentages of the amounts of the individual subunits present in the yeast mutant strains with respect to the amounts present in the wild-type strain W303, which were set to 100%. The numbers are the averages of at least three independent experi- ments. Subunits Yeast mutant strains MES8 (D6) VZ1 (D7) LLD9 (D8) JDP1 (D9) ULB2 (D10) Cytochrome b 52 5 2 12 109 Cytochrome c 1 64 90 90 45 106 ISP 68 10 9 3 113 Core 1 103 57 30 38 101 Core 2 104 58 28 45 116 Qcr6p – 18 – 10 120 Qcr7p 64 – 5 28 111 Qcr8p 43 12 – 40 113 Qcr9p 36 36 36 – 100 Ó FEBS 2004 Assembly of the yeast cytochrome bc 1 complex (Eur. J. Biochem. 271) 1213 competent double deletion strains characterized here, showed decreased levels of all three catalytic subunits, cytochrome b, ISP and cytochrome c 1 . In addition, core proteins 1 and 2 and subunit 6 were reduced to about half of their original levels, while subunits 7 and 8 were reduced to about one quarter of their original levels. The deletion of both genes encoding subunit 6 and 10 in the strain SUY 106-a caused significant changes in the amount of catalytic subunits not observed previously with the single deletion strains lacking either subunit 6 or subunit 10. In fact, cytochrome b and ISP were reduced to about 12% and 27% of the original levels. Cytochrome c 1 and core proteins 1 and 2 decreased by about 50%, whereas a greater decrease was found in the case of subunits 7, 8 and 9. Cytochrome bc 1 subunit analysis of cytochrome b deletion mutants Crystal structures of the bc 1 complexes indicate that cytochrome b is the organizing component of the bc 1 complex, providing eight transmembrane helices that form the central core of the complex [6]. This central core is surrounded by four additional transmembranes helices contributed by cytochrome c 1 , the Rieske protein, and subunits 8 and 9. It is therefore clear that cytochrome b plays a fundamental role in organizing and stabilizing the structure of the entire complex in the inner mitochondrial membrane. For this reason, we investigated the composition of cytochrome bc 1 complex subunits in mitochondrial membranes from yeast strains in which the gene encoding cytochrome b had been deleted or truncated. To this end, we used the yeast strain W303–1B q°,devoid of mitochondrial DNA, and therefore without the gene encoding cytochrome b. We performed similar experiments with the strain CKL57 that contains a point mutation (L263-STOP) in the cytochrome b gene that results in a nonfunctional, truncated protein (Table 1). Both of these yeast strains were respiratory-deficient. Figure 4 shows the subunit composition of the mito- chondrial membranes from the W303–1B q° and CKL57 strains and from the corresponding wild-type cells grown in YPD at 30 °C. In general, the pattern of subunits present in the mitochondrial membranes was identical for these two mutant strains, although the decrease in amounts of the subunits was more severe in the q° strain. As expected, cytochrome b was absent from the W303–1B q° strain. Likewise, no cytochrome b protein was detectable in the CKL57 strain. We do not know whether the lack of immunoreactivity in the latter strain was due to the inability of the truncated protein to insert into and be stable in the inner mitochondrial membrane or lack of detection of the truncated protein by the antibodies. In the W303–1B q° strain the amounts of the other two catalytic subunits, cytochrome c 1 and the ISP, were reduced by about 70–80% (Fig. 4A,C). In the case of the strain Table 4. Cytochrome bc 1 subunit analysis of double deletion mutants. The values represent the percentages of the amounts of individual subunits present in the yeast mutant strains with respect to the amounts present in the wild-type strain W303, which were set to 100%. The numbers are the averages of at least three independent experi- ments. Subunits Yeast mutant strains VZ4 (D6/D7) VZ6 (D7/D8) VZ14 (D8/D9) VZ9 (D9/D10) SUY 106-a (D6/D10) Cytochrome b 6 – 7 26 12 Cytochrome c 1 –507940 50 ISP 18 15 15 33 27 Core 1 74 42 42 59 45 Core 2 83 54 49 53 45 Qcr6p – 23 18 51 – Qcr7p – – 8 26 23 Qcr8p 28 – – 27 33 Qcr9p 30 31 – – 40 Fig. 3. Subunit composition of mitochondrial membranes from yeast mutants with double deletions of genes for nuclear encoded super- numerary subunits. Yeast strains were grown on YPD medium and mitochondrial membranes were analyzed by SDS/PAGE and Western blotting with antibodies to the subunits of the yeast bc 1 complex indicated on the left side of the blots. 1214 V. Zara et al. (Eur. J. Biochem. 271) Ó FEBS 2004 CKL57 the amount of ISP was 40%, while the cyto- chrome c 1 content was almost unaffected (Fig. 4B,D). Core 1 and core 2 proteins were significantly reduced in both mutant strains, being 33 and 29%, respectively, in the W303–1B q° strain (Fig. 4A,C) and 57 and 53%, respect- ively, in the CKL57 strain (Fig. 4B,D). Interestingly, the small subunits 6, 7 and 8 were totally absent in both mutant strains. Only a small amount (22%) of subunit 9 was present in the W303–1B q° strain, Fig. 4A,C, while essentially normal amounts of this subunit were present in the CKL57 strain. When the cytochrome b mutant strains were grown in YPD at 25 °C instead of 30 °C, the defects in subunit composition appeared less evident, especially in the case of the W303–1B q° strain (results not shown). In the mito- chondrial membranes from this strain, the content of cytochrome c 1 increased from 29 to 73%, and the amounts of core proteins 1 and 2 increased from 33 to 74% and 29 to 81%, respectively. Likewise, the relative amount of sub- unit 9 increased from 22 to 48%. The amount of ISP changed only slightly at the lower growth temperature, from 23 to 32%. In the CKL57 mutant strain, the amounts of all subunits increased by about 10–20%. Subunit 9, as already seen at 30 °C, was present in wild-type amounts. Subunit 6 was present in only small amounts in the W303–1B q° strain (22%) and in considerably greater amounts (80%) in the CKL57 strain. Interestingly, subunits 7 and 8 remained undetectable, even at the lower growth temperature, in both cytochrome b mutants. Discussion We have analyzed the composition of cytochrome bc 1 subunits in mitochondrial membranes of yeast mutants in which genes for individual and pairs of bc 1 subunits have been deleted. As far as we know, this is the first time that such a large collection of single and double deletion mutants of the yeast cytochrome bc 1 complex has been characterized simultaneously. Our results add to and extend previous work on the assembly of the yeast bc 1 complex from the laboratories of Berden [30] and Tzagoloff [31]. It has been demonstrated previously that gene expression, import of proteins into mitochondria and sorting to the inner membrane are not influenced by the absence of subunits of the bc 1 complex [19,20,30]. Thus, this experimental strategy allows the determination of which subunits are present in the inner mitochondrial membrane independent of previous steps in bc 1 complex assembly. Defects in the mitochondrial membrane composition of bc 1 subunits in the deletion strains can be ascribed to an altered process of assembly of the multisubunit complex in the inner mito- chondrial membrane. The bc 1 subunits that are imported but not assembled into the multisubunit complex or subcomplexes thereof are probably more susceptible to proteolysis, as previously proposed [19,20,30,32]. This is reflected in decreased amounts or absence of the non- assembled subunits in the mitochondrial membranes. With all of the single and double deletion mutants there appeared to be a strict correlation in the amounts of Fig. 4. Subunit composition of mitochondrial membranes from a yeast mutant lacking mitochondrial DNA and a yeast mutant with a truncated cytochrome b gene. The wild-type (WT), q°, and CKL57 yeast strains were grown on YPD medium and mitochondrial membranes were analyzed by SDS/PAGE and Western blotting with antibodies to the subunits of the yeast bc 1 complex indicated on the left side of the blots. The Western blots are shown in panels A and B and the relative amounts of each of the subunits determined by densitometry scanning of the stained Western blots of the q° and CKL57 membranes are shown in panels C and D, respectively. Ó FEBS 2004 Assembly of the yeast cytochrome bc 1 complex (Eur. J. Biochem. 271) 1215 cytochrome b, subunit 7 and subunit 8. Deletion of the gene for any one of these proteins caused a strong decrease or the disappearance of the other two components. Accordingly, the double deletion mutant VZ6, in which both QCR7 and QCR8 had been deleted, showed no cytochrome b.This agrees with the crystal structures that show that these two supernumerary subunits both interact with cytochrome b. As shown in Fig. 1B, subunit 7 envelopes the N-terminus of cytochrome b within the membrane near the inner mem- brane surface. Subunit 8 exhibits a single transmembrane helix that spans the membrane parallel to cytochrome b, interacting extensively with transmembrane helices G and H1 and also interacting with helix a of cytochrome b parallel to the inner membrane surface. This structural relationship and the coincidental behavior of these three subunits in the deletion strains lend support to previous suggestions [13,30,31] that cytochrome b, subunit 7 and subunit 8 may form a nucleating subcomplex in the lipid bilayer of the inner mitochondrial membrane, around which the other subunits are assembled (Fig. 5). Subunit 8 interacts with several other subunits of the complex in addition to cytochrome b [6]. Our results with the single deletion mutant lacking subunit 8 extend the previous findings of Maarse coworkers [19]. In addition to the strong decrease or disappearance of subunit 7, cytochrome b and ISP as reported previously [19], we observed the disappearance of subunit 6 and a strong decrease of subunit 9 and both core proteins. Accordingly, the QCR8 gene deletion resulted in the most severe phenotype among the single deletion strains tested. In the deletion mutant lacking subunit 7 we found an almost complete lack of cytochrome b, subunit 8 and ISP, in agreement with previous studies [20]. However, unlike previous results [20], we also found a significant decrease of both core proteins, and low levels of subunits 6 and 9. In fact, concomitant and significant decreases of almost all remaining subunits, except cytochrome c 1 ,wereobserved. These results were confirmed by those obtained with the double deletion strain VZ6 (D7/D8). The results with the deletion mutant lacking subunit 7 further corroborate the interdependence among subunits 7, 8 and cytochrome b and the role of this core subcomplex in organizing the cytochrome bc 1 complex. It was proposed previously that the N-terminus of subunit 7 plays an important role during the assembly of the cytochrome bc 1 complex [33,34]. In support of this proposal, it is the N-terminal 30 amino acids of subunit 7 that envelopes the N-terminus of cytochrome b near the matrix side of the inner membrane (Fig. 1B). Cytochrome c 1 appears to be the cytochrome bc 1 com- ponent least influenced by the absence of other subunits of the complex. In fact, only marginal variations in cyto- chrome c 1 were observed in the single deletion mutants tested, except for the increase of the intermediate form of c 1 in the strain lacking subunit 6. Subunit 6 is an acidic protein that interacts with cytochrome c 1 on the cytosolic surface of the membrane. The retardation in c 1 maturation in the absence of subunit 6 suggests that the apo- cytochrome must associate with this subunit before the c-type heme can be inserted. The formation of a subcom- plex between cytochrome c 1 and subunit 6 has previously been proposed on the basis of biochemical [35] and genetic evidence [18]. Interestingly, the D6/D7 double deletion strain is the only one showing a complete lack of mature cytochrome c 1 and also showed only a small amount of the cytochrome c 1 intermediate form. This is probably due to the combination of two phenomena, the maturation delay caused by the absence of subunit 6, and the pleiotropic effects due to the deletion of QCR7, including almost complete disappearance of cytochrome b and subunit 8. Similar effects, including the presence of the intermediate form of cytochrome c 1 along with a complete lack of the mature form, were previously seen in the QCR6 deletion strain grown at nonpermissive temperatures [36]. In that study, also a complete block of cytochrome c 1 maturation was found together with a simultaneous lack of both subunits 6 and 8 and low levels of cytochrome b. Together, these results suggest that the absence of subunit 6 delays cytochrome c 1 maturation while the absence of the cytochrome b subcomplex (formed by Fig. 5. Schematic model summarizing the putative cytochrome bc 1 subcomplexes involved in bc 1 complex assembly. The double arrows indicate that the sequence of events by which the three subcomplexes associate to form a subcomplex containing both cytochromes b and c 1 prior to insertion of ISP and subunit 10 (Qcr10p) in the inner mito- chondrial membrane is currently not known. 1216 V. Zara et al. (Eur. J. Biochem. 271) Ó FEBS 2004 cytochrome b, subunit 7 and subunit 8) hinders the insertion of mature cytochrome c 1 into the complex. However, when the cytochrome b subcomplex is missing, but the gene encoding subunit 6 is not deleted, as in several of the single and double deletion strains, mature cytochrome c 1 is present in the mitochondrial membranes in considerable amount. As reported previously [18,37], the strain lacking the gene for subunit 6 showed only moderate defects in the levels of most of the other subunits of the bc 1 complex when grown at permissive temperatures. However, we found that subunit 9 was present in this deletion strain at about only one-third of the normal level, which suggests that subunit 6 stabilizes subunit 9, although the crystal structure shows that these two subunits do not interact directly [6]. Deletion of the gene encoding subunit 9 resulted in a respiratory deficient yeast strain with very low bc 1 complex activity, particularly at high temperatures [15,29]. In this strain we found a significant decrease of both cytochrome c 1 and subunit 6. Interestingly, previous studies suggested an interaction between subunit 9 and cytochrome c 1 [24,38,39]. Taken together, these results suggest that a subcomplex between cytochrome c 1 and the two supernumerary subunits 6 and 9 is possible (Fig. 5). This would be consistent with the crystal structure, which shows that these two supernumerary subunits interact with cytochrome c 1 [6]. The level of ISP was significantly influenced in almost all of the deletion strains. This catalytic subunit was present in very low amounts in the D7, D8andD9 single deletion mutants, and in all of the double deletion mutants prepared in this study. The extensive loss of ISP in the yeast strain lacking the gene for subunit 9 is in agreement with previous results indicating that this catalytic subunit is protease- sensitive in the absence of subunit 9 [29]. In addition, recent findings show a synergistic interaction between cyto- chrome b and subunit 9 in yeast mitochondria [40]. These authors proposed a stabilizing role of subunit 9 on the interactions among the catalytic subunits of the cyto- chrome bc 1 complex, especially at high temperatures. In this regard, it is noteworthy that the level of cytochrome b increased in the strain lacking the gene for subunit 9 when the cells were grown at 25 °C instead of 30 °C. In addition, less dramatic changes in subunit composition were found in cytochrome b mutant strains grown at 25 °C instead of 30 °C (Results). A critical effect of the temperature on the level of various subunits of cytochrome bc 1 complex is therefore evident in the yeast strains in which the genes for subunit 6, subunit 9 and cytochrome b had been deleted. Core protein 1 and core protein 2 interact with each other and with the membrane-embedded subunits of the bc 1 complex and protrude, almost completely, into the mito- chondrial matrix [6]. In contrast to previous results with several yeast bc 1 complex mutants [31], the amounts of core 1 and core 2 proteins were significantly influenced by the absence of other subunits of the bc 1 complex. Deletion of the genes for subunit 7, subunit 8 or subunit 9 caused a strong reduction of the two core proteins in the mito- chondrial membranes (Fig. 2 and Table 3). These results were confirmed by those obtained with the double deletion strains (Fig. 3 and Table 4). Furthermore, deletion of the gene for cytochrome b caused a decrease of both core proteins (Fig. 4). The low levels of both core proteins found in this study may be due to the fact that we examined mitochondrial membranes instead of mitochondria. Using mitochondria there is still the possibility to detect proteins in transit and not yet inserted into the inner mitochondrial membrane. The fact that both core proteins decreased by the same extent in the various deletion strains suggests that they probably form a subcomplex as hypothesized previ- ously [30,31] (Fig. 5). Our results allow some insight into the sequence of events in assembly of the bc 1 complex. Two of the supernumerary subunits, 7 and 8, along with cytochrome b,appeartoplay an important role in the structural organization of the bc 1 complex. This suggests that these subunits associate at an early step in the assembly pathway. In contrast, the supernumerary subunit 10 seems to play only a minor role in the overall structure of the bc 1 complex. Deletion of the QCR10 gene has no effect on the composition of bc 1 subunits in the mitochondrial membrane. This subunit is readily lost during purification and is not present in the crystal structure of the bc 1 complex [6]. This suggests that subunit 10 is in a peripheral location on the bc 1 complex and that it is added late in the assembly pathway. In general, our results agree with and extend the model for the assembly of the yeast bc 1 complex proposed by Berden and coworkers [30]. In fact, these authors proposed the existence of three distinct subcomplexes, essentially con- firmed by the present data (Fig. 5). In addition, our results revealed a strict interdependence between the cytochrome b subcomplex and the supernumerary subunit Qcr6p. It is also evident that Qcr9p plays an important role in the temperature-sensitive stabilization of the yeast bc 1 complex. 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