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• W2081640785 abstract "The F1 component of mitochondrial ATP synthase is an oligomeric assembly of five different subunits, α, β, γ, δ, and ϵ. In terms of mass, the bulk of the structure (∼90%) is provided by the α and β subunits, which form an (αβ)3 hexamer with adenine nucleotide binding sites at the α/β interfaces. We report here ultrastructural and immunocytochemical analyses of yeast mutants that are unable to form the α3β3 oligomer, either because the α or the β subunit is missing or because the cells are deficient for proteins that mediate F assembly (e.g. Atp11p, Atp12p, or Fmc1p). The F1 α1 and β subunits of such mutant strains are detected within large electron-dense particles in the mitochondrial matrix. The composition of the aggregated species is principally full-length F1 α and/or β subunit protein that has been processed to remove the amino-terminal targeting peptide. To our knowledge this is the first demonstration of mitochondrial inclusion bodies that are formed largely of one particular protein species. We also show that yeast mutants lacking the α3β3 oligomer are devoid of mitochondrial cristae and are severely deficient for respiratory complexes III and IV. These observations are in accord with other studies in the literature that have pointed to a central role for the ATP synthase in biogenesis of the mitochondrial inner membrane. The F1 component of mitochondrial ATP synthase is an oligomeric assembly of five different subunits, α, β, γ, δ, and ϵ. In terms of mass, the bulk of the structure (∼90%) is provided by the α and β subunits, which form an (αβ)3 hexamer with adenine nucleotide binding sites at the α/β interfaces. We report here ultrastructural and immunocytochemical analyses of yeast mutants that are unable to form the α3β3 oligomer, either because the α or the β subunit is missing or because the cells are deficient for proteins that mediate F assembly (e.g. Atp11p, Atp12p, or Fmc1p). The F1 α1 and β subunits of such mutant strains are detected within large electron-dense particles in the mitochondrial matrix. The composition of the aggregated species is principally full-length F1 α and/or β subunit protein that has been processed to remove the amino-terminal targeting peptide. To our knowledge this is the first demonstration of mitochondrial inclusion bodies that are formed largely of one particular protein species. We also show that yeast mutants lacking the α3β3 oligomer are devoid of mitochondrial cristae and are severely deficient for respiratory complexes III and IV. These observations are in accord with other studies in the literature that have pointed to a central role for the ATP synthase in biogenesis of the mitochondrial inner membrane. The mitochondrial inner membrane contains the ATP synthase, which utilizes a transmembrane proton gradient to catalyze ATP synthesis from inorganic phosphate and ADP. The ATP synthase has two major structural domains, an F0 component that forms a proton-permeable pore through the membrane and a peripheral, matrix-localized, F1 component where ATP is synthesized (1Boyer P.D. Annu. Rev. Biochem. 1997; 66: 717-749Crossref PubMed Scopus (1606) Google Scholar). The catalytic unit of the enzyme (F1) is composed of five different types of subunits in the stoichiometric ratio α3β3γδϵ. The three-dimensional structures of F1 from bovine heart (2Abrahams J.P. Leslie A.G. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2764) Google Scholar), rat liver (3Bianchet M.A. Hullihen J. Pedersen P.L. Amzel L.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11065-11070Crossref PubMed Scopus (224) Google Scholar), and yeast mitochondria (4Stock D Leslie A.G. Walker J.E. Science. 1999; 286: 1700-1705Crossref PubMed Scopus (1093) Google Scholar) show that the α and β subunits alternate in position within a hexamer that surrounds the amino and carboxyl termini of the γ subunit. The interfaces between α and β subunits mark the locations of three catalytic and three non-catalytic adenine nucleotide binding sites in the enzyme (1Boyer P.D. Annu. Rev. Biochem. 1997; 66: 717-749Crossref PubMed Scopus (1606) Google Scholar, 3Bianchet M.A. Hullihen J. Pedersen P.L. Amzel L.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11065-11070Crossref PubMed Scopus (224) Google Scholar, 4Stock D Leslie A.G. Walker J.E. Science. 1999; 286: 1700-1705Crossref PubMed Scopus (1093) Google Scholar). In Saccharomyces cerevisiae, all five F1 subunits are encoded by nuclear genes (5Takeda M. Vassarotti A. Douglas M.G. J. Biol. Chem. 1985; 260: 15458-15465Abstract Full Text PDF PubMed Google Scholar, 6Takeda M. Chen W.J. Salzgaber J. Douglas M.G. J. Biol. Chem. 1986; 261: 15126-15133Abstract Full Text PDF PubMed Google Scholar, 7Guélin E. Rep M. Grivell L.A. FEBS Lett. 1996; 381: 42-46Crossref PubMed Scopus (81) Google Scholar, 8Giraud M-F. Velours J. Eur. J. Biochem. 1994; 222: 851-859Crossref PubMed Scopus (38) Google Scholar, 9Paul M.F. Ackerman S. Yue J. Arselin G. Velours J. Tzagoloff A. J. Biol. Chem. 1994; 269: 26158-26164Abstract Full Text PDF PubMed Google Scholar) and are synthesized in the cytoplasm and then imported into mitochondria (10Tokatlidis K. Schatz G. J. Biol. Chem. 1999; 274: 35285-35288Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Like other nuclear-encoded mitochondrial proteins, the F1 subunits are imported to the matrix compartment as unfolded polypeptide chains. After the mitochondrial targeting signal (if present) has been removed, their folding is facilitated by the Hsp60 and Hsp10 proteins (11Hendrick J.P. Hartl F.U. Annu. Rev. Biochem. 1993; 62: 349-384Crossref PubMed Scopus (1477) Google Scholar). The final steps in the formation of functional F1 require two proteins called Atp11p and Atp12p (reviewed in Ref. 12Ackerman S.H. Biochim. Biophys. Acta. 2002; 1555: 101-105Crossref PubMed Scopus (48) Google Scholar). Both F1 α and β subunits were found as aggregated proteins in sedimentation profiles of mitochondrial extracts of yeast mutants that are deficient for either Atp11p or Atp12p (13Ackerman S.H. Tzagoloff A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4986-4990Crossref PubMed Scopus (131) Google Scholar). Yeast two-hybrid screens and affinity tag precipitation experiments have shown that Atp11p binds selectively to the β subunit of F1 (14Wang Z.G. Ackerman S.H. J. Biol. Chem. 2000; 275: 5767-5772Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) and that Atp12p binds selectively to the α subunit of F1 (15Wang Z.G. Sheluho D. Gatti D.L. Ackerman S.H. EMBO J. 2000; 19: 1486-1493Crossref PubMed Google Scholar). The binding determinants for Atp11p in the β subunit, and those for Atp12p in the α subunit, are in the adenine nucleotide binding domains of the F1 proteins (14Wang Z.G. Ackerman S.H. J. Biol. Chem. 2000; 275: 5767-5772Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 15Wang Z.G. Sheluho D. Gatti D.L. Ackerman S.H. EMBO J. 2000; 19: 1486-1493Crossref PubMed Google Scholar) and are predicted to be located specifically at the hydrophobic regions of these domains that will ultimately be sequestered at the α/β subunit interfaces in the assembled F1 oligomer. Under this point of view, Atp11p and Atp12p are proposed to serve as molecular chaperones and to protect the two F1 proteins from non-productive interactions by masking their hydrophobic surfaces until incorporation of α and β monomers into α3β3 oligomers. Atp11p and Atp12p are present in the mitochondria at a concentration that is at least 100-fold lower than the steady state level of F1 proteins (16White M. Ackerman S.H. Arch. Biochem. Biophys. 1995; 319: 299-304Crossref PubMed Scopus (14) Google Scholar, 15Wang Z.G. Sheluho D. Gatti D.L. Ackerman S.H. EMBO J. 2000; 19: 1486-1493Crossref PubMed Google Scholar), which is reasonable in view of the fact that the F1 subunits are assembled into the oligomer as soon as they are imported from the cytoplasm. In other words, there are no pools of unassembled α and β subunits in the mitochondrial matrix (17Burns D.J. Lewin A.S. J. Biol. Chem. 1986; 261: 12066-12073Abstract Full Text PDF PubMed Google Scholar). This may explain why when either Atp11p or Atp12 is absent, both F1 subunits aggregate. Indeed, when one subunit aggregates as a direct consequence of the lack of its chaperone protein, the steady state level of the “partner” subunit builds up and eventually far exceeds the level of the remaining chaperone. Consistent with this interpretation, the α subunit aggregates in strains lacking the β subunit gene, and the β subunit aggregates when the α subunit is not expressed (13Ackerman S.H. Tzagoloff A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4986-4990Crossref PubMed Scopus (131) Google Scholar). In contrast, the solubility of the α and β subunits is not modified in strains lacking one of the other F1 subunits, δ (8Giraud M-F. Velours J. Eur. J. Biochem. 1994; 222: 851-859Crossref PubMed Scopus (38) Google Scholar, 18Duvezin-Caubet S. Caron M. Giraud M-F Velours J. di Rago J.-P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13235-13240Crossref PubMed Scopus (45) Google Scholar), γ (9Paul M.F. Ackerman S. Yue J. Arselin G. Velours J. Tzagoloff A. J. Biol. Chem. 1994; 269: 26158-26164Abstract Full Text PDF PubMed Google Scholar), or ϵ (19Guélin E. Chevallier J. Rigoulet M. Guérin B. Velours J. J. Biol. Chem. 1993; 268: 161-167Abstract Full Text PDF PubMed Google Scholar). Interestingly, in the absence of the γ subunit, there is evidence to suggest αβ oligomers may exist (9Paul M.F. Ackerman S. Yue J. Arselin G. Velours J. Tzagoloff A. J. Biol. Chem. 1994; 269: 26158-26164Abstract Full Text PDF PubMed Google Scholar). Thus, it appears that aggregation of F1 α and β subunits occurs under conditions in which formation of α β heterodimers is not possible, either because the α or the β subunit is missing or because a protein (i.e. Atp11p or Atp12p) that specifically protects the α and β subunits from non-productive interactions is not present. Fmc1p is a protein of yeast mitochondria that is required for the formation of the F1 unit in cells cultured at 37 °C (20Lefebvre-Legendre L. Vaillier J. Ben Habdelhak H. Velours J. Slonimski P.P. di Rago J.-P J. Biol. Chem. 2001; 276: 6789-6796Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). In mitochondria of Δfmc1 cells grown at the restrictive temperature, the F1 α and β subunits sediment in sucrose gradient as large protein aggregates. Fmc1p seems to be necessary for Atp12p stability and/or activity at elevated temperature as suggested by the fact that Δfmc1 cells grown at 37 °C show a marked reduction in Atp12p content and recover the capacity to form the F1 unit by overexpression of Atp12p (20Lefebvre-Legendre L. Vaillier J. Ben Habdelhak H. Velours J. Slonimski P.P. di Rago J.-P J. Biol. Chem. 2001; 276: 6789-6796Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). In this study we have performed an ultrastructural and immunocytochemical analysis of yeast mutants that are unable to assemble the F1 α3β3 oligomer. Electron micrographs of Δatp1, Δatp2, Δatp11, Δatp12, and Δfmc1 yeast cells all show the presence of large, electron-dense particles in the mitochondrial matrix. The inclusion bodies are immunodecorated in situ with antibodies against the F1 α and/or β subunits (as appropriate) and, following purification via sucrose gradients, are shown by silver-stained gels to be composed almost exclusively of F1 protein. Furthermore, there is evidence that mitochondrial biogenesis is grossly defective in these mutant strains as indicated microscopically by the absence of cristae and by spectral analysis, which shows a marked reduction in cytochromes c1, b, and aa3 relative to wild type yeast. Strains and Media—Rich galactose medium (2% galactose, 1% yeast extract, 2% bactopeptone, 40 mg/liter of adenine) was used for growing yeast. The genotypes of the Δfmc1 mutant (MC6) and its parental wild type strain (MC1) are given in Ref. 20Lefebvre-Legendre L. Vaillier J. Ben Habdelhak H. Velours J. Slonimski P.P. di Rago J.-P J. Biol. Chem. 2001; 276: 6789-6796Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar; those of the Δatp11 and Δatp12 mutants are in Ref. 13Ackerman S.H. Tzagoloff A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4986-4990Crossref PubMed Scopus (131) Google Scholar. The Δatp1 and Δatp2 strains were from the Euroscarf collection. Biochemical Methods—Mitochondria were prepared as described in Ref. 21Guérin B. Labbe P. Somlo M. Methods Enzymol. 1979; 55: 149-159Crossref PubMed Scopus (193) Google Scholar. Ultrasonic irradiation of mitochondria was performed at 4 °C for 10 s using a 75TS Annemasse sonicator at 120 V with a 5-mm diameter tip. Sucrose gradient sedimentation of sonicated mitochondria was done using previously described conditions (13Ackerman S.H. Tzagoloff A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4986-4990Crossref PubMed Scopus (131) Google Scholar). SDS/PAGE was according to Ref. 22Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar. Western blot analyses were performed as described (23Arselin G. Vaillier J. Graves P.V. Velours J. J. Biol. Chem. 1996; 271: 20284-20290Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) using the ECL detection kit from Amersham Biosciences. Acrylamide gels were silver stained as in Ref. 24Ansorge W. Stathakos D. Electrophoresis. Walter de Gruyter & Co., Berlin1983: 235-242Google Scholar. Antibodies against F1 α and F1 β subunits were used at a 1/100,000 dilution. Freezing and Freeze Substitution for Ultrastructural Studies—The yeast pellets were placed on the surface of a copper electron microscopy grid (400 mesh) coated with formvar. Each loop was very quickly submersed in precooled liquid propane and held at -180 °C by liquid nitrogen. The loops were then transferred to a precooled solution of 4% osmium tetroxide in dry acetone in a 1.8-ml polypropylene vial at -82 °C for 48 h (substitution fixation), warmed gradually to room temperature, and washed three times in dry acetone. Specimens were stained for 1 h with 1% uranyl acetate in acetone at 4 °C in a black room. Following another rinse in dry acetone, the loops were infiltrated progressively with araldite (epoxy resin; Fluka). Ultrathin sections were contrasted with lead citrate. Immunogold Electron Microscopy—Yeast were cryofixed as described for ultrastructural studies and freeze substituted with acetone plus 0.1% glutaraldehyde or methanol plus 0.5% uranyl acetate for 3 days at -82 °C. Samples were rinsed with acetone or methanol at -20 °C, embedded progressively at -20 °C in LR Gold resin (EMS). Resin polymerization was carried out at -20 °C for 3 days under UV illumination. Ultrathin LR Gold sections were collected on nickel grids coated with formvar. Sections were first incubated for 5 min with 1 mg/ml glycine and then 5 min with fetal calf serum (1:20). The grids were incubated for 45 min at room temperature with antibodies against α-F1 subunit (1:2000), β-F1 subunit (1:3500), or porin (1:2000) and then incubated for 45 min at room temperature with anti-rabbit or anti-mouse IgG, respectively, conjugated to 10 nm gold particles (BioCell). The sections were rinsed with distilled water and contrasted 5 min with 2% uranyl acetate in water, followed by 1% lead citrate for 1 min. Specimens were observed with a Philips CM10 (80 kV) or Tecnai Biotwin (120 kV) electron microscope (SERCOMI, University Victor Segalen Bordeaux 2). Cytochrome Spectral Analysis—Spectra were recorded for whole cells frozen at liquid nitrogen temperature with a Cary 128 spectrophotometer after reduction of the cytochromes by dithionite as previously described (25Claisse M.L. Pajot P.F. Eur. J. Biochem. 1974; 49: 49-59Crossref PubMed Scopus (39) Google Scholar). Ultrastructural and Immunocytochemical Analyses of the Δfmc1 Mutant—Yeast cells were embedded in araldite resin and examined for ultrastructure by transmission electron microscopy. Morphometric analysis is optimized through the use of araldite resin, which although not amenable to immunolabeling proteins enables the use of osmium tetroxide during the fixation process. This provides for excellent membrane contrast. Electron micrographs of Δfmc1 cells grown at 37 °C revealed the presence, in 70% of the cell sections examined (Table I), of abnormal electron-dense particles that were located exclusively in the mitochondrial matrix (Fig. 1, A-C). The particles are noted to be largely globular in shape, with rather circular or oval contour, and range in size from 50 to 500 nm. There were no such electron-dense particles observed in electron micrographs of Δfmc1 grown at 28 °C nor were there any seen in cells of the wild type parental strain cultured at 28 or 37 °C (Table I).Table IFrequency of inclusion bodies in yeast cellsStrainsGrowth temperaturePercent cell sections with IBaIB, inclusion bodies.Percent mitochondrial profiles with IBaIB, inclusion bodies.°CWild type280.0 (n = 50)0.0 (n = 243)Wild type370.0 (n = 50)0.0 (n = 234)Δfmc1280.0 (n = 20)0.0 (n = 101)Δfmc13770.0 (n = 40)33.0 (n = 109)Δatp112844.0 (n = 25)23.5 (n = 81)Δatp122840.0 (n = 35)18.4 (n = 103)Δatp12816.0 (n = 25)9.3 (n = 43)Δatp22820.0 (n = 25)9.4 (n = 64)a IB, inclusion bodies. Open table in a new tab Immunogold electron microscopy of Δfmc1 cells grown at 37 °C provided insight into the nature of the inclusion bodies that accumulate in this strain at elevated temperature. First, the membranes surrounding the inclusion bodies were shown to be labeled with antibodies against porin, a marker protein for the mitochondrial outer membrane (Fig. 1D). This result provides evidence that the inclusion bodies are intramitochondrial. The use of antibodies against the F1 α subunit or the F1 β subunit (Fig. 1, E and F, respectively) resulted in a heavy and uniform labeling of all the inclusion bodies (Table II), showing the presence and co-existence within them of both F1 subunits. Outside of the inclusion bodies, the Δfmc1 mitochondrial sections gave a rather weak immunogold response (gold particles/μm2) in comparison to wild type cells (62 ± 25 versus 378 ± 72 with antibodies against subunit α; 29 ± 17 versus 225 ± 40 with antibodies against subunit β), and no significant extra-mitochondrial labeling (<10) was detected for either strain (Table II). There was virtually no immunogold labeling observed under conditions in which primary antibody was omitted from the experiment (Fig. 1G).Table IIDistribution of immunogold particles in yeast cellsSubcellular compartmentStrainsIBMitochondria minus IBExtramitochondrialAnti-F1αAnti-F1βAnti-F1αAnti-F1βAnti-F1αAnti-F1βΔfmc1721 ± 771044 ± 11462 ± 2529 ± 176 ± 14 ± 1Δatp111028 ± 2051016 ± 17242 ± 199 ± 310 ± 24 ± 3Δatp130 ± 271339 ± 36034 ± 1530 ± 105 ± 13 ± 1Δatp21099 ± 10125 ± 1587 ± 337 ± 57 ± 24 ± 1Wild type378 ± 72225 ± 406 ± 22 ± 1 Open table in a new tab Ultrastructural and Immunocytochemical Analyses of Δatp11, Δatp12, Δatp1, and Δatp2 Mutants—Electron micrographs of araldite thin cell sections showed the presence of abnormal electron-dense particles in the mitochondrial matrix of Δatp11, Δatp12, Δatp1, and Δatp2 mutants grown at 28 °C (data shown for Δatp11 and Δatp12, Fig. 2, A-C), in ∼20-40% of the cell sections examined (Table I). Those in Δatp11 and Δatp12 mutants were all heavily and uniformly stained with antibodies against the F1 α or F1 β subunit (data shown for Δatp11 immunogold labeling, Fig. 2, D and E). These results indicate that the two F1 subunits co-localize within the same inclusion bodies of Δatp11 and Δatp12 in a manner similar to what is observed in Δfmc1 cells cultured at 37 °C. Inclusion bodies of the Δatp1 mutant reacted strongly to antibodies against the β subunit (Fig. 2G) but not to those against the α subunit (panel F), which is expected because the α subunit is not made in this mutant. Conversely, the inclusion bodies in the Δatp2 mutant, which are devoid of the β subunit, were stained only with antibodies against the α subunit (panel H) and not with those against the β subunit (panel I). There was essentially no immune response to F1 protein outside of the inclusion bodies in Δatp11, Δatp12, Δatp1, and Δatp2 mutants (Table II) cell sections, which suggests that these structures contain most if not all of the α and/or β subunits present in the cells. Purification and Protein Composition Analysis of the Inclusion Bodies—To determine whether the inclusion bodies contain other proteins in addition to the F1 α and β subunits, sonically disrupted samples of Δfmc1 mitochondria were fractionated on a discontinuous (20-80%) sucrose gradient using centrifugation conditions shown previously (13Ackerman S.H. Tzagoloff A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4986-4990Crossref PubMed Scopus (131) Google Scholar) to separate high molecular weight protein aggregates from bulk membrane and soluble proteins. The profile for Δfmc1 proteins in the fractions collected from a discontinuous sucrose gradient was analyzed in a silver-stained SDS-polyacrylamide gel (Fig. 3A, left side). Following the premise that some soluble proteins may be inadvertently trapped with the aggregated material, fractions of high sucrose percentage (fractions 8 and 9) were diluted out, the insoluble material was collected by centrifugation, and proteins were visualized on a silver-stained SDS gel (Fig. 3A, middle). The superscript “c” in the lane headings indicates that the samples applied to this gel were 9-fold more concentrated relative to samples 8 and 9 of the adjacent gel described above. Western analysis of fraction 8 (designated by superscript “w”) shows the α and β subunits of F1 among the proteins observed at the margin of 60:80% sucrose. By comparison with a sample of purified F1FO, it is noteworthy that the α and β subunit proteins in the aggregate are fully processed to the mature size and show no evidence of proteolytic breakdown. Similar results were obtained with Δatp11 mitochondria (not shown). The nature of the material trapped at the 60:80% sucrose interface was analyzed further (data shown for Δatp11, Fig. 3B). The left and middle lanes show silver-stained gels for fraction 8 collected from a discontinuous sucrose gradient pre-loaded with Δatp11 disrupted mitochondria. This fraction corresponds to the 60:80% sucrose interface (see panel A). Comparable amounts of sample were loaded in the lanes marked “8” and “8Tx”, the difference being that the latter sample shows the profile for detergent-insoluble particulate material that is recovered from fraction 8 following extraction of the sample with Triton X-100 (details for this experiment are given in the legend to Fig. 3). Notably, with the exception of two bands, a comparison of lane 8Tx with lane 8 shows that most of the proteins observed in the original sample are removed by the detergent, which suggests that the 60:80% sucrose region of the gradient is contaminated with membrane material. The identification of the prominent aggregated proteins as the α and β subunits of F1 is confirmed by the Western blot (lane 8w) shown at the right of panel B. Although the F1 α and β subunits are clearly the predominant species in the aggregated fraction, there is evidence of a minor amount of other proteins as well. None of these proteins reacted, in Western blot analyses, with antibodies against subunits γ and 4 of the F1F0 ATP synthase (data not shown). We did not test any other ATP synthase subunit as they fail to accumulate in the Δatp11 mutant. In addition, we did not detect any immunological signal for Fmc1p or Atp12p (data not shown). Efforts were also made to determine whether Atp11p accumulates with F1 β or if Atp12p accumulates with F1 α, in the aggregated mitochondrial protein fraction from Δatp1 or Δatp2, respectively. To detect the chaperone proteins by Western blot analysis in these experiments we used transformed Δatp1 and Δatp2 strains that express ATP11 or ATP12 from a multicopy plasmid. In a control experiment, we found that even in an otherwise wild type background, a subpopulation of the over-produced Atp11p and Atp12p protein is insoluble and migrates to a high density position in sucrose gradients (data not shown). As such, even though there is also a minor peak for Atp11p or for Atp12p at the 60:80% sucrose region in gradients run with mitochondria from Δα or Δβ yeast (data not shown), these results may be due exclusively to an artifact of overproducing the chaperone proteins and are not considered to be informative on aspects related to Atp11p and Atp12p action in F1 assembly. The Loss of the F1 Unit Coincides with the Absence of the Cristal Membrane—The inner membrane system of mitochondria is known to consist of two contiguous but distinct membranes, one that opposes the outer membrane (the inner boundary membrane) and one that forms tubules or lamellae in the interior (the cristal membrane) (26Frey T.G. Renken C.W. Perkins G.A. Biochim. Biophys. Acta. 2002; 1555: 196-203Crossref PubMed Scopus (130) Google Scholar). Cristae organization allows greater amounts of membrane-bound energy-transducing enzymes to be packed in the interior of the organelle. An illustration of this has been the demonstration that more than 90% of respiratory complexes III and V of bovine heart mitochondria reside in the cristal membrane rather than at the organelle periphery (27Gilkerson R.W. Selker J.M. Capaldi R.A. FEBS Lett. 2003; 546: 355-358Crossref PubMed Scopus (225) Google Scholar). Fig. 4 shows mitochondrial cristae morphology in a respiratory-competent yeast wild type strain (panel A) and the distribution along this membrane of F1 subunits α (panel B) and β (panel C). An interesting observation in this study was the absence of any apparent cristal membrane in mutants where F1 synthesis is virtually abolished (Δfmc1, Δatp1, Δatp2, Δatp11, and Δatp12; shown for Δatp1 in Fig. 4D). This finding is consistent with previous work that has provided evidence that complex V plays a key role in the formation of this membrane (see “Discussion”). As a consequence, the mutants with F1 assembly defects were expected to show a decreased content in respiratory complexes III (bc1 cytochromes) and IV (aa3 cytochromes), which are residents of the inner membrane. This was investigated with the Δfmc1 mutant by spectral analysis of whole cells. A strong deficit in cytochromes b, c1, and aa3 was indeed seen in this mutant when the cells were grown at 37 °C, whereas no difference was observed between mutant and wild type cultivated at 28 °C (Fig. 5). As reported previously (13Ackerman S.H. Tzagoloff A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4986-4990Crossref PubMed Scopus (131) Google Scholar), Δatp11 and Δatp12 mutants are also grossly deficient for cytochromes of the bc1 and cytochrome c oxidase respiratory proteins. It must be stressed here that Δatp11, Δatp12, and Δfmc137 °C cultures do not produce high levels of cytoplasmic petites, which are cells that bear large deletions in mitochondrial DNA (ρ-) or lack completely the mitochondrial genome (ρ0). The petite population is only 1-2% in Δatp11 and Δatp12 cell cultures and 15-20% in Δfmc1 cells cultured at 37 °C. Therefore, the observed spectral deficiency in these mutants is not caused by a lack of mitochondrial DNA, which codes for apocytochromes b, a, and a3. Rather, the spectra likely reflect a deficit in respiratory proteins that results from an underdevelopment of the inner membrane. Notably, cytochrome c, a soluble cytochrome of the intermembrane space, was not decreased in the Δfmc1 cells but instead was found to be present in significantly higher amounts relative to the wild type control cells (Fig. 5). This is similar to what is observed for wild type ρ-/ρ° cells, which also lack cristal membranes (28Yotsuyagani Y. J. Ultrastruc. Res. 1962; 7: 141-158Crossref PubMed Scopus (38) Google Scholar, 29Stevens B. Strathern J.N. Jones E.W. Broach J.R. The Molecular Biology of the Yeast Saccharomyces cerevisiae: Life Cycle and Inheritance. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York1981: 471-504Google Scholar) and accumulate high levels of cytochrome c (30Slonimski P.P. Ephrussi B. Ann. Inst. Pasteur. 1949; 77: 47-63Google Scholar). We show that the α and β subunits of mitochondrial ATP synthase are sequestered as inclusion bodies in the mitochondrial matrix when they cannot be incorporated into α3β3 oligomers. This phenotype is associated with two types of yeast mutants, those that lack either the α or the β subunit (Δatp1 and Δatp2 strains) and those that are deleted for a molecular chaperone function associated with assembly of α β oligomers (Δatp11, Δatp12, and Δfmc1 strains). Unassembled F1 α and β subunits were shown to be present together within the same inclusion bodies in Δatp11 and Δfmc1 mutants (Figs. 1 and 2) and to be the principal protein species of these structures (Fig. 3). This observation does not necessarily imply a co-aggregation phenomenon, because Δatp1 and Δatp2 strains show inclusion bodies of pure β or α subunits, respectively (Fig. 2). To our knowledge this is the first demonstration of mitochondrial inclusion bodies that are formed largely of a particular protein species. The accumulation of unassembled α and β subunits in mitochondria of F1 assembly-defective mutants is a curious phenomenon. The turnover of mitochondrial energy-transducing complex subunits is considered necessary to prevent the accumulation of single subunits and subcomplexes in the inner membrane, which may disturb assembly processes or change the properties of this membrane (for reviews see Refs. 31Rep M. Grivell L.A. Curr. Genet. 1996; 30: 367-380Crossref PubMed Scopus (101) Google Scholar and 32Van Dyck" @default.
• W2081640785 created "2016-06-24" @default.
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• W2081640785 creator A5013985152 @default.
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• W2081640785 date "2005-05-01" @default.
• W2081640785 modified "2023-10-17" @default.
• W2081640785 title "Failure to Assemble the α3 β3 Subcomplex of the ATP Synthase Leads to Accumulation of the α and β Subunits within Inclusion Bodies and the Loss of Mitochondrial Cristae in Saccharomyces cerevisiae" @default.
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