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The formation of respiratory chain complexes in mitochondria is under the proteolytic control of the m‐AAA protease

Heike Arlt, Gregor Steglich, Robert Perryman, Bernard Guiard, Walter Neupert, Thomas Langer

Author Affiliations

  1. Heike Arlt1,
  2. Gregor Steglich1,
  3. Robert Perryman2,
  4. Bernard Guiard3,
  5. Walter Neupert1 and
  6. Thomas Langer*,1
  1. 1 Institut für Physiologische Chemie der Universität München, Goethestrasse 33, 80336, München, Germany
  2. 2 Present address: Department of Neuropathology, Beaumont Hospital, Dublin 9, Ireland
  3. 3 Centre de Genetique Moleculaire CNRS, Université Pierre et Marie Curie, 91190, Gif‐sur‐Yvette, France
  1. *Corresponding author. E-mail: Langer{at}bio.med.uni-muenchen.de
  1. H.Arlt and G.Steglich contributed equally to this work

Abstract

Yta10p (Afg3p) and Yta12p (Rcal1p), members of the conserved AAA family of ATPases, are subunits of the mitochondrial m‐AAA protease, an inner membrane ATP‐dependent metallopeptidase. Deletion of YTA10 or YTA12 impairs degradation of non‐assembled inner membrane proteins and assembly of respiratory chain complexes. Mutations of the proteolytic sites in either YTA10 or YTA12 have been shown to inhibit proteolysis of membrane‐integrated polypeptides but not the respiratory competence of the cells, suggesting additional activities of Yta10p and Yta12p. Here we demonstrate essential proteolytic functions of the m‐AAA protease in the biogenesis of the respiratory chain. Cells harbouring proteolytically inactive forms of both Yta10p and Yta12p are respiratory deficient and exhibit a pleiotropic phenotype similar to Δyta10 and Δyta12 cells. They show deficiencies in expression of the intron‐containing mitochondrial genes COX1 and COB. Splicing of COX1 and COB transcripts is impaired in mitochondria lacking m‐AAA protease, whilst transcription and translation can proceed in the absence of Yta10p or Yta12p. The function of the m‐AAA protease appears to be confined to introns encoding mRNA maturases. Our results reveal an overlapping substrate specificity of the subunits of the m‐AAA protease and explain the impaired assembly of respiratory chain complexes by defects in expression of intron‐containing genes in mitochondria lacking m‐AAA protease.

Introduction

Several proteases which serve a number of different purposes have been identified in mitochondria. Specific processing peptidases cleave off targeting sequences from nuclear‐encoded mitochondrial preproteins by limited proteolysis (Pratje et al., 1994; Brunner and Neupert, 1995; Isaya and Kalousek, 1995). Misfolded polypeptides possibly harmful to mitochondrial function are removed by ATP‐dependent proteases which are present in various subcompartments of mitochondria (Langer and Neupert, 1996; Rep and Grivell, 1996; Suzuki et al., 1997). Studies in yeast also indicate crucial regulatory functions of these proteases in mitochondrial biogenesis, although target proteins in mitochondria which are under proteolytic control remain to be identified.

The examination of the stability of mitochondrial translation products provided first evidence for the presence of ATP‐dependent proteases in the inner membrane of mitochondria (Wheeldon et al., 1974; Kalnov et al., 1979; Yasuhara et al., 1994). Energy‐transducing complexes in the inner membrane are composed of nuclearly and mitochondrially encoded subunits. In the absence of a supply of cytoplasmically synthesized polypeptides, non‐assembled mitochondrial translation products are subject to rapid proteolysis. Two homologous inner membrane proteins, Yta10p (Afg3p) and Yta12p (Rca1p), were shown to be essential for the proteolytic process (Pajic et al., 1994; Arlt et al., 1996; Guélin et al., 1996). A mutation in the conserved consensus binding site for divalent metal ions in either protein abolished the degradation of non‐assembled mitochondrial translation products, suggesting that Yta10p and Yta12p exert metal‐dependent proteolytic activity (Arlt et al., 1996; Guélin et al., 1996). Both proteins form a high molecular weight complex in the inner membrane which presumably is composed of several copies of each protein (Arlt et al., 1996). Substrate polypeptides were found exclusively in association with assembled Yta10p and Yta12p. Yta10p and Yta12p harbour a conserved ATPase domain of ∼230 amino acids characteristic for the AAA family of P‐loop ATPases (for ATPases associated with diverse cellular activities) (Beyer, 1997; Patel and Latterich, 1998). Therefore, the Yta10p–Yta12p complex was termed m‐AAA protease (Arlt et al., 1996; Leonhard et al., 1996). The prefix denotes the presence of the catalytically active sites of Yta10p and Yta12p on the matrix side of the inner membrane. The m‐AAA protease belongs to a novel conserved class of membrane‐bound ATP‐dependent proteases, members of which were identified in eubacteria, mitochondria and chloroplasts (Beyer, 1997; Patel and Latterich, 1998).

The m‐AAA protease is required for the maintenance of mitochondrial functions. Cells lacking either Yta10p or Yta12p lose respiratory competence and exhibit deficiencies in the assembly of the ATP synthase and respiratory chain complexes in the inner membrane (Guélin et al., 1994; Tauer et al., 1994; Tzagoloff et al., 1994; Paul and Tzagoloff, 1995). A mutation in the consensus metal‐binding site of Yta10p or Yta12p abolished the proteolytic breakdown of non‐assembled inner membrane proteins, but neither the assembly of energy‐transducing complexes nor the respiratory competence of the cells (Arlt et al., 1996; Guélin et al., 1996). This observation was taken as evidence for additional functions of Yta10p or Yta12p which may be independent of their proteolytic activity (Arlt et al., 1996; Guélin et al., 1996). In support of this assumption, multicopy suppressors lacking proteolytic activity have been identified which maintain respiration in the absence of Yta10p or Yta12p (Rep et al., 1996a). As a stable association of non‐native polypeptides with the m‐AAA protease has been observed, this complex may exert chaperone‐like functions during the assembly of membrane protein complexes (Arlt et al., 1996).

In an attempt to characterize the role of the m‐AAA protease in mitochondrial biogenesis, we constructed a Δyta10Δyta12 strain expressing proteolytically inactive forms of both Yta10p and Yta12p. The mutant proteins formed the same high molecular weight complex in the inner membrane as the wild‐type proteins. Respiration and the assembly of respiratory chain complexes, however, were impaired in these cells, as they were in the absence of Yta10p or Yta12p. Further analysis of cells containing proteolytically inactive variants of both Yta10p and Yta12p revealed deficiencies in the synthesis of subunit 1 of the cytochrome c oxidase (Cox1) and of cytochrome b (Cob) which both are encoded by intron‐containing genes. These results establish essential proteolytic functions of the ATP‐dependent m‐AAA protease in mitochondrial biogenesis.

Results

Cells harbouring proteolytically inactive Yta10p and Yta12p exhibit a pleiotropic phenotype similar to yta10‐ and yta12‐null mutants

Yta10p and Yta12p contain a consensus binding site for divalent metal ions, HEXXH, which is characteristic of the proteolytic centre of metal‐dependent peptidases (Jiang and Bond, 1992; Hooper, 1994; Barrett, 1997). The two histidine residues in this motif together with a variable third ligand and a water molecule mediate the binding of metal ions. The glutamate residue is dispensable for binding and serves exclusively a catalytic function during proteolysis (Vazeux et al., 1996). To examine the importance of the proteolytic activity of the m‐AAA protease for mitochondrial function, glutamate residues in the metal‐binding sites of Yta10p and Yta12p were replaced by glutamine. The mutant variants were constitutively expressed from a centromere‐based vector in Δyta10 (yta10E559Q), Δyta12 (yta12E614Q) and Δyta10Δyta12 cells (yta10E559Qyta12E614Q). Respiration of Δyta10 and Δyta12 cells was restored upon expression of Yta10E559Qp and Yta12E614Qp, respectively (Arlt et al., 1996; Guélin et al., 1996). On the other hand, yta10E559Qyta12E614Q mutant cells were respiration‐deficient (Figure 1A). Cytochrome b and cytochromes aa3 were absent in spectra of yta10E559Qyta12E614Q cells, as they were in cells lacking Yta10p, Yta12p or both (data not shown). In contrast, mitochondrial cytochromes were detectable in Δyta10 and Δyta12 cells expressing Yta10E559Qp and Yta12E614Qp, respectively (data not shown).

Figure 1.

Phenotypic analysis of yta10E559Qyta12E614Q cells. (A) Expression of Yta10E559Qp and Yta12E614Qp in Δyta10Δyta12 cells does not restore growth on non‐fermentable carbon sources. Wild‐type (‘WT’), Δyta10 (YHA101), Δyta12 (YHA201), Δyta10Δyta12 (YHA301), yta10E559Q (‘yta10EQ’; YHA103), yta12E614Q (‘yta12EQ’; YHA203) and yta10E559Qyta12E614Q cells (‘yta10EQyta12EQ’; YGS302) were grown at 30°C to logarithmic phase on selective medium supplemented with the respective auxotrophic markers and 2% glucose. Cells were isolated by centrifugation, resuspended in H2O and spotted onto YP medium containing 3% glycerol. The plates were incubated for 3 days at 30°C. (B) Inactivation of the m‐AAA protease impairs assembly of cytochrome c oxidase. The sensitivity of Cox4 to externally added trypsin was determined in mitochondria harbouring mutant m‐AAA protease as described in Materials and methods. Cox4 was present at low levels in Δyta10, Δyta12 and yta10E559Qyta12E614Q mitochondria (see Figure 3A).

To assess the assembly of the cytochrome c oxidase, we took advantage of the observation that only assembled subunits of the cytochrome c oxidase are resistant to added protease (Mason et al., 1973; Rep et al., 1996b). Mitochondria were isolated from the various mutant strains and, after solubilization with detergent, the trypsin resistance of subunit 4 of the cytochrome c oxidase (Cox4) was analysed (Figure 1B). Cox4 was protease sensitive in Δyta10 and Δyta12 cells, indicating that it was present in an unassembled state (Figure 1B). Upon expression of Yta10E559Qp in Δyta10 cells or Yta12E614Qp in Δyta12 cells, Cox4 exhibited a similar trypsin resistance to that found in wild‐type mitochondria (Figure 1B). In contrast, Cox4 was sensitive to degradation by trypsin added to detergent‐lysed mitochondria harbouring proteolytically inactive forms of both Yta10p and Yta12p (Figure 1B). Thus, similarly to cells lacking Yta10p and Yta12p, the assembly of the cytochrome c oxidase is impaired in yta10E559Qyta12E614Q mutant cells.

These experiments reveal strikingly different effects of mutations in the proteolytic sites of Yta10p and Yta12p on mitochondrial functions: the respiratory activity of yeast cells and the assembly of respiratory chain complexes remain unaffected in the presence of either proteolytically inactive Yta10p or Yta12p. In contrast, mutations in the proteolytic sites of both Yta10p and Yta12p impair respiration and the assembly of respiratory complexes, a phenotype also observed with yta10‐ and yta12‐null mutant strains. These results point to essential proteolytic functions of the m‐AAA protease in mitochondrial biogenesis, and suggest that the protease exerts residual activity after mutating the proteolytic site of one subunit.

Mutations in the proteolytic sites of Yta10p and Yta12p do not affect m‐AAA protease assembly or substrate binding

Yta10p and Yta12p appear to perform their proteolytic function only after assembly into a high molecular weight complex in the inner membrane (Arlt et al., 1996). To exclude indirect effects of mutations in the proteolytic sites, complex formation of Yta10E559Qp and Yta12E614Qp was examined. Mitochondria were isolated from wild‐type and yta10E559Qyta12E614Q cells and gel filtration experiments were performed after solubilization of mitochondrial membranes. Proteolytically inactive Yta10p or Yta12p, like the wild‐type proteins, co‐eluted from the sizing column in two peaks corresponding to apparent molecular masses of ∼1000 and 250 kDa (Figure 2A). The large complex contains assembled Yta10p and Yta12p, while the 250 kDa form may correspond to a homooligomeric assembly intermediate of both proteins (Arlt et al., 1996).

Figure 2.

Assembly and substrate binding of proteolytically inactive m‐AAA protease. (A) Yta10E559Qp and Yta12E614Qp form a high molecular weight complex in the inner membrane. Wild‐type (‘WT’) and yta10E559Qyta12E614Q (YGS302) cells were solubilized and mitochondrial extracts were fractionated by Superose 6 gel chromatography as described (Arlt et al., 1996). Yta10p and Yta12p were detected in the eluate fractions by immunoblotting using a chemiluminescence detection system. Protein amounts, determined by laser densitometry, are given as a percentage of total Yta10p or Yta12p in the eluate. Hsp60 (840 kDa), thyroglobulin (670 kDa), apoferritin (443 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa) and carboanhydrase (29 kDa) were used as standards for calibration. (B) Co‐immunoprecipitation of Yta10E559Qp and Yta12E614Qp. Mitochondria (200 μg) isolated from yta10E559Qyta12E614Q (YGS302) cells were solubilized and, after a clarifying spin, co‐immunoprecipitations were performed with pre‐immune and Yta10p‐ and Yta12p‐specific antisera as described (Arlt et al., 1996). The precipitates were analysed by SDS–PAGE and immunoblotting with Yta10p‐ and Yta12p‐specific antisera. (C) Cross‐linking of substrate polypeptides to proteolytically inactive m‐AAA protease. Mitochondrially encoded polypeptides were labelled with [35S]methionine for 20 min at 25°C in wild‐type (‘WT’), Δyta10 (YHA101), Δyta12 (YHA201) and yta10E559Qyta12E614Q (YGS302) mitochondria and subjected to chemical cross‐linking using 1,5‐difluoro‐2,4‐dinitrobenzene (100 μM) as previously described (Arlt et al., 1996). Association with Yta10p or Yta12p was determined by immunoprecipitation with Yta10p‐ or Yta12p‐specific antisera, respectively. After SDS–PAGE, radioactivity in the precipitate was quantified using a phosphorimaging system to determine the translation efficiency. The amount of protein that was used in the immunoprecipitation was adjusted according to the incorporated radioactivity. 35S‐Labelled polypeptides bound to Yta10p or Yta12p in wild‐type mitochondria were set to 100% of control.

Assembly of Yta10E559Qp and Yta12E614Qp was demonstrated directly by co‐immunoprecipitation in further experiments. After solubilization of yta10E559Qyta12E614Q mitochondria with Triton X‐100, extracts were incubated with pre‐immune, Yta10p‐ and Yta12p‐specific antisera. The precipitates were analysed by Western blotting. Immunoprecipitation of Yta12E614Qp with Yta10p‐specific antiserum and of Yta10E559Qp with Yta12p‐specific antiserum was observed (Figure 2B). In contrast, Yta10E559Qp and Yta12E614Qp were not precipitated with pre‐immune serum (Figure 2B). These results demonstrate that the assembly of the m‐AAA protease is not affected by the mutations in the proteolytic sites of its subunits.

The binding of substrate polypeptides to the proteolytically inactive m‐AAA protease was examined by chemical cross‐linking (Figure 2C). Mitochondrially encoded polypeptides were synthesized in the presence of [35S]methionine in wild‐type and yta10E559Qyta12E614Q mitochondria which were then subjected to chemical cross‐linking using 1,5‐difluoro‐2,4‐dinitrobenzene. After solubilization of the mitochondria, Yta10E559Qp and Yta12E614Qp were immunoprecipitated with Yta10p‐ and Yta12p‐specific antisera, respectively. Binding of substrate polypeptides occurred with similar efficiency to the proteolytically active and inactive forms of Yta10p and Yta12p (Figure 2C). This finding also substantiates the association of Yta10E559Qp with Yta12E614Qp, as substrate binding was strongly reduced in Δyta10 and Δyta12 mitochondria and thus occurs only to the assembled m‐AAA protease (Figure 2C).

We conclude from these results that the replacement of the glutamic acid residues in the consensus metal‐binding sites of Yta10p or Yta12p by glutamine does not interfere with the assembly of the m‐AAA protease nor with the binding of substrate polypeptides to the complex. Thus, the impaired respiratory competence of yta10E559Qyta12E614Q cells suggests crucial proteolytic functions of the m‐AAA protease for the assembly of the respiratory chain.

Mitochondrially encoded subunits of the cytochrome c oxidase do not accumulate in mitochondria lacking m‐AAA protease

In order to identify processes in mitochondria which are under the proteolytic control of the m‐AAA protease, we further determined its role in the biogenesis of the cytochrome c oxidase. Cox4 accumulated at strongly reduced levels in Δyta10, Δyta12 and yta10E559Qyta12E614Q mitochondria (Figure 3A). Similarly, the mitochondrially encoded subunits Cox1, Cox2 and Cox3 were not detectable in mitochondria lacking active m‐AAA protease (Figure 3A). In agreement with the growth phenotype and the cytochrome spectra, however, subunits of the cytochrome c oxidase were present at wild‐type levels in yta10E559Q and yta12E614Q mitochondria (Figure 3A).

Figure 3.

Mitochondrially encoded subunits of the cytochrome c oxidase do not accumulate in the absence of m‐AAA protease activity. (A) Steady‐state levels of cytochrome c oxidase subunits in yta10 and yta12 mutant mitochondria. Mitochondrial proteins (30 μg) were analysed by SDS–PAGE and immunoblotting. (B) Labelling of mitochondrially encoded subunits in cells harbouring mutant m‐AAA protease. Mitochondrial translation products were labelled in vivo in wild‐type (‘WT’), Δyta10 (YHA101), yta10E559Q (YHA103), Δyta12 (YHA201), yta12E614Q (YHA203) and yta10E559Qyta12E614Q (YGS302) cells for 20 min at 30°C as described (Douglas et al., 1979; McKee and Poyton, 1984; Langer et al., 1995). Incorporation of [35S]methionine was determined using a phosphoimaging system. The translation efficiency in Δyta10, Δyta12 and yta10E559Qyta12E614Q cells was on average reduced to ∼30, 25 and 20%, respectively, when compared with wild‐type cells. Synthesis of Cox1, Cox2 and Cox3 was quantified and corrected for the translation efficiency in the various strains. Labelling of Cox1, Cox2 and Cox3 in wild‐type cells was set to 100% of control. The average of several experiments is given (mean ± SEM). n indicates the number of experiments.

If the m‐AAA protease act only post‐translationally, non‐assembled subunits might be subject to rapid proteolysis. Alternatively, defects in the synthesis of mitochondrially encoded subunits in the absence of the m‐AAA protease have to be considered. To examine the latter possibility, labelling of mitochondrial translation products with [35S]methionine was performed in cells lacking Yta10p and Yta12p (Figure 3B). Cox1, Cox2 and Cox3 were synthesized at greatly reduced levels in Δyta10 and Δyta12 cells when compared with wild‐type cells (Figure 3B). Although a decreased translation efficiency of all mitochondrially encoded proteins was observed in these cells, this did not account for the reduced labelling of Cox1, Cox2 and Cox3 in the absence of either Yta10p or Yta12p (see quantification in Figure 3B). The expression of the mitochondrially encoded subunits of the cytochrome c oxidase was also impaired in yta10E559Qyta12E614Q cells and, therefore, depends on the proteolytic activity of the m‐AAA protease. The enzymatic activity of one subunit of the m‐AAA protease, however, is sufficient to ensure the accumulation of Cox1, Cox2 and Cox3 as they were synthesized at wild‐type levels in Δyta10 or Δyta12 mitochondria harbouring Yta10E559Qp or Yta12E614Qp, respectively (Figure 3A).

The m‐AAA protease is required for the expression of COX1 in mitochondria

As degradation of Cox2 and Cox3 has been observed in the absence of Cox1 (McEwen et al., 1986; Van Dyck et al., 1998), it is conceivable that the m‐AAA protease is required primarily for efficient synthesis of Cox1. Therefore, the expression of the COX1 gene was examined in more detail. The m‐AAA protease may affect transcription or translation. As COX1 represents a mosaic gene harbouring several introns (Costanzo and Fox, 1990; Grivell, 1995), defects in pre‐mRNA processing in the absence of the protease also have to be considered.

Mitochondrial RNA (mtRNA) was isolated from cells containing mutations in YTA10, YTA12 or both and analysed by Northern hybridization with probes specific for COX1 and COX2. Inactivation of the m‐AAA protease did not affect the accumulation of COX2 mRNA which was present at wild‐type levels in mitochondria lacking m‐AAA protease (Figure 4A). In contrast, transcripts of ∼2.1 kb corresponding to mature COX1 mRNA were hardly detectable in Δyta10 and Δyta12 mitochondria (Figure 4A). Although wild‐type levels accumulated in mitochondria harbouring either Yta10E559Qp or Yta12E614Qp, mature COX1 transcripts were almost completely absent in yta10E559Qyta12E614Q cells (Figure 4A). Thus, the deficiency in the formation of mature COX1 mRNA results from the loss of the proteolytic activity of the m‐AAA protease.

Figure 4.

Inactivation of the m‐AAA protease impairs the maturation of COX1 pre‐mRNA. (A) Northern blot analysis of COX1 and COX2 transcripts in yta10 and yta12 mutant mitochondria. mtRNA was extracted from wild‐type (‘WT’), Δyta10 (YGS101), Δyta12 (YGS201), yta10E559Q (YGS103), yta12E614Q (YGS203) and yta10E559Qyta12E614Q (YGS302) mitochondria and analysed by Northern hybridization with a COX1‐specific exon probe and with a COX2 probe as a gel loading control. (B) Northern blot analysis of COX1 and COX2 mRNAs in Δyta10 and Δyta12 mitochondria harbouring an intronless mitochondrial genome. mtRNA was isolated from wild‐type (‘WT’) and from Δyta10 and Δyta12 strains containing (‘Δyta10’, YGS101; ‘Δyta12’, YGS201) or lacking (‘Δyta10i‐less’, YGS108; ‘Δyta12i‐less’, YGS208) introns and analysed as in (A). (C) Synthesis of Cox1 in Δyta10 and Δyta12 mitochondria harbouring an intronless mitochondrial genome. Labelling of mitochondrial translation products was performed in wild‐type and Δyta10 and Δyta12 cells containing (‘Δyta10’, YGS101; ‘Δyta12’, YGS201) or lacking (‘Δyta10i‐less’, YGS108; ‘Δyta12i‐less’, YGS208) introns as in Figure 3B. Cox1 synthesis was quantified and corrected for differences in the translation efficiency as in Figure 3B. Cox1 synthesis in wild‐type cells was set to 100% of control. The average of several experiments is given (mean ± SEM). n indicates the number of experiments.

To examine whether the function of the m‐AAA protease is related to the presence of introns in the COX1 gene, we generated Δyta10 and Δyta12 cells harbouring an intronless mitochondrial genome. mtRNA was isolated and analysed by Northern blotting with a COX1 exon probe (Figure 4B). Mature COX1 transcripts accumulated in these cells at levels similar to wild‐type cells. Thus, transcription could proceed normally in the absence of the m‐AAA protease and only the maturation of intron‐containing COX1 transcripts was impaired upon inactivation of the protease. Moreover, labelling of mitochondrial translation products revealed that the synthesis of Cox1 was at least partially restored in Δyta10 and Δyta12 cells carrying an intronless mitochondrial genome (Figure 4C). Apparently, translation of mature COX1 mRNA can also occur in the absence of the m‐AAA protease.

Taken together, these results establish the requirement for the m‐AAA protease for the expression of the mitochondrially encoded COX1 gene. The apparent absence of precursor and mature forms of COX1 mRNA in mitochondria lacking the m‐AAA protease may reflect a crucial role for the protease in the stabilization of intron‐containing COX1 transcripts. However, it is also conceivable that the m‐AAA protease directly regulates processing of COX1 pre‐mRNAs. Splicing defects in the absence of the protease may result in the degradation of COX1 pre‐mRNAs, thereby preventing the accumulation of non‐processed transcripts in these mitochondria.

Inactivation of the m‐AAA protease impairs the synthesis of Cob in mitochondria

In addition to the cytochrome c oxidase, a deficiency in the assembly of the bc1 complex has been observed in the absence of either Yta10p or Yta12p (Tauer et al., 1994; Tzagoloff et al., 1994). Mitochondrially encoded cytochrome b (Cob), a subunit of the bc1 complex, is encoded by an intron‐containing gene in mitochondria, as is Cox1. Therefore, a role for the m‐AAA protease in the expression of COB in mitochondria had to be considered. Indeed, Cob was not detectable by immunoblotting in Δyta10 or Δyta12 mitochondria, while two other subunits of the bc1 complex, cytochrome c1 and Qcr6p, were present at wild‐type levels (Figure 5A). The dependence on the proteolytic activity of the m‐AAA protease was examined in cells harbouring Yta10E559Qp, Yta12E614Qp or both (Figure 5A). Wild‐type levels of Cob accumulated in yta10E559Q and yta12E614Q mitochondria, whereas Cob was not detectable in yta10E559Qyta12E614Q mitochondria (Figure 5A). Thus, the accumulation of Cob depends on the proteolytic activity of the m‐AAA protease, suggesting an impaired synthesis of Cob in the absence of the protease.

Figure 5.

Defective expression of COB in mitochondria lacking m‐AAA protease activity. (A) Steady‐state level of Cob in yta10 and yta12 mutant mitochondria. Mitochondrial proteins (60 μg) were analysed by SDS–PAGE and immunoblotting. (B) Northern blot analysis of COB and COX2 transcripts in mitochondria harbouring mutant m‐AAA protease. mtRNA was extracted from wild‐type (‘WT’), Δyta10 (YGS101), Δyta12 (YGS201), yta10E559Q (YGS103), yta12E614Q (YGS203) and yta10E559Qyta12E614Q (YGS302) mitochondria and analysed by Northern hybridization with a COB‐specific exon probe and with a COX2 probe as a gel loading control. (C) Northern blot analysis of COB transcripts in Δyta10 and Δyta12 mitochondria containing intronless mtDNA. mtRNA was isolated from wild‐type (‘WT’) and from Δyta10 and Δyta12 strains containing (‘Δyta10’, YGS101; ‘Δyta12’, YGS201) or lacking (‘Δyta10i‐less’, YGS108; ‘Δyta12i‐less’, YGS208) introns and analysed with a COB‐specific probe as in (A). Mature COB mRNA was quantified using a phosphorimaging system and is given as a percentage of wild‐type of control. (D) Steady‐state level of Cob in Δyta10 and Δyta12 cells harbouring an intronless mtDNA. Mitochondria (60 μg), isolated from cells described in (B), were subjected to SDS–PAGE and analysed by immunoblotting.

mtRNA was isolated from cells carrying mutations in YTA10, YTA12 or both and analysed with a probe specific for COB. With wild‐type cells and cells harbouring Yta10E559Qp or Yta12E614Qp, the probe hybridized with transcripts of 2.2 kb corresponding to mature COB mRNA (Figure 5B). In contrast, mature COB transcripts accumulated at greatly reduced levels in mitochondria of Δyta10, Δyta12 or yta10E559Qyta12E614Q cells (Figure 5B). These results demonstrate that the efficient formation of mature COB transcripts depends on proteolysis by the m‐AAA protease. Similarly to COX1 transcripts, the requirement for the m‐AAA protease for the stabilization of COB transcripts is restricted to intron‐containing COB pre‐mRNAs. mtRNA was isolated from Δyta10 and Δyta12 cells harbouring an intronless mitochondrial genome and analysed by Northern hybridization with a COB‐specific probe. Similar levels of mature COB transcripts were detected in wild‐type, Δyta10 and Δyta12 cells (Figure 5C).

To examine whether expression of COB is restored in Δyta10 and Δyta12 mitochondria containing an intronless mitochondrial genome, steady‐state levels of Cob were determined (Figure 5D). Cob accumulated in mitochondria which lack Yta10p or Yta12p but harbour intronless mtDNA (Figure 5D). We conclude from these results that removal of introns from the mitochondrial genome alleviates the dependence of COB expression on the m‐AAA protease. Thus, the function of the protease is linked directly to the presence of introns in this gene.

The m‐AAA protease affects the splicing of a maturase‐encoding COB intron

To characterize further the role of the m‐AAA protease in the maturation of COB transcripts, we analysed mtRNA from wild‐type and Δyta10 cells with a probe specific for intron bI1 of COB, a group II intron which forms a stable lariat structure upon splicing (Costanzo and Fox, 1990; Perlman, 1990; Grivell, 1995). The probe hybridized with a transcript of ∼0.8 kb corresponding to the excised lariat regardless of the presence of Yta10p in mitochondria (Figure 6A). Thus, the m‐AAA protease is not required for the splicing of this intron. Notably, the excised intron accumulated at greatly increased levels in Δyta10 mitochondria when compared with wild‐type cells (Figure 6A). The impaired synthesis of Cob protein in the absence of the m‐AAA protease most likely causes an upregulation of transcription of COB resulting in the accumulation of the excised intron in these cells.

Figure 6.

Splicing of the maturase‐encoding COB intron bI2 depends on the m‐AAA protease. (A) Northern blot analysis of COB transcripts in Δyta10 strains harbouring different numbers of introns in the COB gene. mtRNA was extracted from mitochondria of wild‐type (‘WT’), Δyta10 (YGS101), Δyta10i‐less (YGS108) and Δyta10 mitochondria harbouring a COB gene with introns bI1 and bI2 only (YGS110). The introns present in the COB gene in the various strains are indicated. mtRNA was analysed with a COB‐specific exon probe (‘COB’), a probe specific for intron bI1 (‘bI1’) of the COB gene and with a COX2‐specific probe as a gel loading control. The excised intron bI1 was detectable in wild‐type mitochondria only upon prolonged exposure of the membrane. (B) Steady‐state levels of Cob in Δyta10 mitochondria harbouring different numbers of introns in the COB gene. Mitochondria isolated from wild‐type and Δyta10 strains described in (A) were subjected to SDS–PAGE and analysed by immunoblotting. Cob protein was detectable at very low levels in Δyta10 harbouring a COB gene with introns bI1 and bI2 upon prolonged exposure of the membrane.

The splicing of many mitochondrial introns depends on mRNA maturases which are encoded within the same intron (Grivell and Schweyen, 1989; Costanzo and Fox, 1990; Pel and Grivell, 1993). To examine the role of the m‐AAA protease in the splicing of a maturase‐encoding intron, YTA10 was disrupted in a yeast strain with mtDNA containing only introns bI1 and bI2 in the COB gene. In contrast to intron bI1, an mRNA maturase is encoded by intron bI2 (Lazowska et al., 1980). mtRNA was isolated from these cells and analysed with an exon‐specific COB probe and a probe specific for the intron bI1 (Figure 6A). Similarly to Δyta10 cells harbouring the complete COB gene, the excised intron bI1 accumulated in these cells, demonstrating efficient splicing of this intron (Figure 6A). In contrast, mature COB mRNA accumulated at strongly reduced levels in mitochondria lacking Yta10p, while precursor transcripts of ∼4.0 and 3.2 kb were detected in these cells (Figure 6A). Furthermore, Cob protein was hardly detectable by immunoblotting of mitochondria isolated from these cells (Figure 6B). Thus, maturation of COB transcripts harbouring only introns bI1 and bI2 still depends on the m‐AAA protease. As intron bI1 is excised normally in the absence of the protease, these results demonstrate the requirement for the m‐AAA protease for the efficient splicing of the maturase‐encoding COB intron bI2.

Proteolytically inactive m‐AAA protease does not maintain the respiratory competence of cells harbouring an intronless mitochondrial genome

The synthesis of both Cox1 and Cob was restored in Δyta10 and Δyta12 cells upon removal of introns from the corresponding genes. Still, these cells were respiratory deficient and cytochromes aa3 and b were absent, indicating impaired complex assembly (data not shown). This finding is reminiscent of other proteins involved in mitochondrial mRNA splicing whose inactivation was found to impair the respiratory competence of cells containing intronless mtDNA (Seraphin et al., 1989; Wiesenberger et al., 1992). Expression of proteolytically inactive Yta10p and Yta12p in Δyta10Δyta12 cells did not maintain respiration nor were cytochromes detectable in spectra of these cells (data not shown). Thus, in addition to the synthesis of Cox1 and Cob, the assembly of respiratory chain complexes appears to be under the proteolytic control of the m‐AAA protease.

Discussion

Cells lacking Yta10p or Yta12p are respiratory deficient and exhibit defects in the proteolysis of non‐assembled inner membrane proteins and the assembly of respiratory chain complexes (Guélin et al., 1994; Pajic et al., 1994; Tauer et al., 1994; Tzagoloff et al., 1994). Our results explain this pleiotropic phenotype by the loss of essential proteolytic functions of the m‐AAA protease. Mutations in the proteolytic centre of both Yta10p and Yta12p, similarly to deletions of either YTA10 or YTA12, lead to loss of the respiratory competence of the cells. Inactivation of the m‐AAA protease impairs the synthesis of the mitochondrially encoded respiratory chain subunits Cox1 and Cob and, thereby, the assembly of the cytochrome c oxidase and the bc1 complex in the inner membrane. Thus, the biogenesis of the respiratory chain is under the proteolytic control of the ATP‐dependent m‐AAA protease.

The m‐AAA protease represents a large proteolytic complex composed of Yta10p and Yta12p. Cells carrying proteolytically inactive variants of both Yta10p and Yta12p are respiratory deficient, while mutating either protein alone did not interfere with the respiratory competence of the cells. The genetic interaction of mutations in the proteolytic sites of Yta10p and Yta12p allows several conclusions on the function of both proteins in mitochondria. First, the complex of Yta10p and Yta12p represents the proteolytically active structure which ensures cellular respiration. In agreement with this finding, substrate polypeptides have been found exclusively in association with assembled Yta10p and Yta12p (Arlt et al., 1996). Secondly, the m‐AAA protease exerts residual proteolytic activity after inactivation of either Yta10p and Yta12p, which is sufficient to maintain respiratory competence but not to mediate the degradation of non‐assembled inner membrane proteins (Arlt et al., 1996; Guélin et al., 1996). It is conceivable that the proteolysis of polypeptides by the m‐AAA protease requires multiple cleavage events which may depend on the coordinated activity of both subunits. The activity of one subunit of the m‐AAA protease, on the other hand, may allow the processing or clipping of polypeptides, apparently sufficient to maintain respiration. Indeed, Yta10p was shown to mediate the cleavage of a model substrate if assembled with proteolytically inactive Yta12p (Leonhard et al., 1996). Thirdly, the subunits of the m‐AAA protease, Yta10p and Yta12p, exert overlapping substrate specificity. The respiratory competence of the cells is maintained regardless of which subunit of the protease exerts proteolytic activity. The specificities of the two subunits of the m‐AAA protease, however, are not identical. Differential effects of mutations in Yta10p and Yta12p on the degradation of a model substrate protein have been observed (Leonhard et al., 1996).

The proteolytic activity of the m‐AAA protease is crucial for the expression of the intron‐containing COX1 and COB genes in mitochondria. While transcription proceeds normally in the absence of the protease, mature‐sized transcripts of COX1 and COB are present at strongly decreased levels in cells lacking Yta10p or Yta12p. The dependence on the m‐AAA protease is alleviated in cells harbouring an intronless mitochondrial genome, indicating that the protease regulates the processing and/or stability of COX1 and COB precursor transcripts. Unspliced species of COX1 and COB transcripts did not accumulate in the absence of m‐AAA protease activity, suggesting that the stability of transcripts harbouring several introns is affected in these mitochondria. Intron‐containing COB precursor RNAs over‐accumulate, however, in the absence of the m‐AAA protease in mitochondria containing only intron bI1 and bI2 in the COB gene. The same phenomenon has been observed previously upon inactivation of other nuclear genes involved in the splicing of COB or COX1 transcripts (Seraphin et al., 1988; Pel et al., 1990; Wiesenberger et al., 1992). These results thus demonstrate that the m‐AAA protease is required for the splicing of intron bI2 encoding an mRNA maturase. The stability or activity of a protein involved in the splicing of a maturase‐encoding intron is apparently under the proteolytic control of the m‐AAA protease in mitochondria. mRNA maturases are synthesized as fusion proteins with the peptide products of the preceding exons and, presumably after proteolytic activation, mediate the splicing of the intron by which they are encoded (Costanzo and Fox, 1990; Perlman, 1990; Grivell, 1995). The m‐AAA protease may catalyse the processing of at least some of these fusion proteins, thereby releasing active maturase.

The experiments described here establish the requirement for the m‐AAA protease for the splicing of the intron bI2 of the COB gene and thereby also for the removal of subsequent COB introns. The impaired splicing of COB transcripts is sufficient to explain the deficiency in the maturation of COXI pre‐mRNA as the maturase encoded by the intron bI4 of the COB gene is required for the splicing of both intron bI4 itself and intron aI4 of the COXI gene (Dhawale et al., 1981; Banroques et al., 1987). A more general role for the protease during the splicing of maturase‐encoding introns remains, therefore, to be demonstrated. Inactivation of the m‐AAA protease did not result in the over‐accumulation of specific COB and COX1 precursor transcripts in cells with a COB gene containing several introns. The requirement for the protease is apparently not confined to one particular intron. Rather, a cumulative effect on the splicing of several introns has to be envisaged.

Low levels of mature COX1 and COB transcripts were detected in mitochondria in the absence of the m‐AAA protease. Other proteins may partially substitute for the m‐AAA protease in this case. PIM1 protease, an ATP‐dependent Lon‐like protease in the mitochondrial matrix space (Suzuki et al., 1994; Van Dyck et al., 1994), is a likely candidate; its overexpression was found to restore the respiratory competence of cells lacking m‐AAA protease (Rep et al., 1996a). The analysis of the proteolytic breakdown of misfolded polypeptides in mitochondria provided biochemical evidence for overlapping substrate specificities of both proteases (Savel'ev et al., 1998). pim1 mutant cells, similarly to Δyta10 and Δyta12 cells, exhibit deficiencies in the synthesis of Cox1 and Cob (Van Dyck et al., 1998). PIM1 protease is required for the stability and splicing of intron‐containing pre‐mRNAs of either gene and, in addition, for the translation of mature COX1 transcripts (Van Dyck et al., 1998). Thus, the expression of the intron‐containing COX1 and COB genes is under the proteolytic control of two ATP‐dependent proteases in mitochondria.

Similarly to PIM1 protease, overexpression of Oxa1p has been reported to suppress the respiratory deficiency of cells lacking m‐AAA protease (Rep et al., 1996a). Oxa1p facilitates the membrane insertion of integral inner membrane proteins from the matrix side, but evidently does not exert proteolytic activity (He et al., 1997; Hell et al., 1997). Our results explain impaired respiration in the absence of the m‐AAA protease by the loss of essential proteolytic functions and, therefore, suggest an indirect suppressive effect of Oxa1p. Further studies are required to elucidate the molecular consequences of an Oxa1p overproduction.

The deficiency in the assembly of the cytochrome c oxidase and the bc1 complex in the absence of Yta10p or Yta12p can be explained satisfactorily by defects in the synthesis of Cox1 and Cob. The respiratory competence of cells harbouring an intronless mitochondrial genome, however, was not maintained upon inactivation of the m‐AAA protease, pointing to additional post‐translational functions of the m‐AAA protease. While expression of COB and COX1 was restored in Δyta10 or Δyta12 mitochondria harbouring intronless mtDNA, the cytochrome spectra of these cells indicate impaired complex assembly. Moreover, the F1F0‐ATPase did not assemble in cells lacking m‐AAA protease despite the synthesis of mitochondrially encoded subunits of this complex (Tzagoloff et al., 1994; Paul and Tzagoloff, 1995; Arlt et al., 1996). Although its precise role remains to be characterized, our results point to essential proteolytic functions of the m‐AAA protease also in these processes.

Materials and methods

Yeast strains and growth conditions

Yeast strains used in this study are derivatives of W303 and are summarized in Table I. Cells were grown at 30°C on YP medium (1% yeast extract, 2% peptone) containing 2% galactose and 0.5% lactate or on minimal medium (0.7% yeast nitrogen base containing ammonium sulfate) supplemented with the auxotrophic requirements.

View this table:
Table 1. Yeast strains

YTA10 (YGS101) and YTA12 (YGS201) were disrupted by PCR‐targeted homologous recombination using the heterologous markers HIS3MX6 or KanMX4 (Wach et al., 1994). The complete open reading frame of YTA12 or an internal fragment of YTA10 (bp 150–2170) were replaced by the disruption cassettes. Homologous recombination was verified by PCR. To generate a W303 strain containing an intronless mitochondrial genome (YGS6), a ρ0 derivative of W303‐1B was generated by ethidium bromide treatment (Fox et al., 1991) and transformed with the plasmid YCplac22 (CEN, TRP1). This strain was crossed with kar167‐1 cells harbouring intronless mtDNA (Seraphin et al., 1987), and haploid cytoductants were isolated on selective medium lacking tryptophan (YGS6) (Conde and Fink, 1976; Berlin et al., 1991). The strains YGS108 and YGS208 were generated by PCR‐targeted deletion of YTA10 or YTA12 in these cells using the HIS3MX6 disruption cassette. The Δyta10 strain harbouring only intron bI1 and bI2 (YGS110) is a derivative of the strain CKST9 (Golik et al., 1995).

To allow co‐expression of Yta10E559Qp and Yta12E614Qp, a 4 kb DNA fragment containing the ADH1 promotor and YTA10E559Q was isolated from YCplac111/ADH1. YTA10E559Q (Arlt et al., 1996) by restriction digestion with SpeI and HindIII and cloned into the centromere‐based plasmid YCplac22 (Gietz and Sugino, 1988). To generate yta10E559Qyta12E614Q cells (YGS302) or yta10E559Qyta12E614Q cells containing intronless mtDNA (YGS305), plasmids encoding Yta10E559Qp or Yta12E614Qp were transformed into the Δyta12 strains YGS203 or YGS208, respectively. YTA10 was then disrupted using the HIS3MX6 or the KanMX4 cassette by PCR‐targeted homologous recombination. The maintenance of mtDNA was examined by mating with mit tester strains and by labelling of mitochondrial translation products in vivo.

Assessing assembly of cytochrome c oxidase

Mitochondria (300 μg) were resuspended at a concentration of 5 mg/ml in SEM buffer (250 mM sucrose, 1 mM EDTA, 10 mM MOPS–KOH pH 7.2) containing 0.8% (v/v) laurylmaltoside and solubilized by vigorous mixing for 30 min at 4°C. After a clarifying spin for 30 min at 110 000 g, the sample was divided. Mitochondria (50 μg) were diluted 3‐fold with SEM buffer, and trypsin was added at the concentrations indicated. Trypsin digestion was performed for 20 min at 4°C and then inhibited by incubating with soybean trypsin inhibitor (600 μg/ml) for 5 min at 4°C. The trichloroacetic acid (TCA)‐precipitated samples were analysed by SDS–PAGE and immunoblotting with a polyclonal antiserum directed against Cox4. Cox4 present in solubilized mitochondria without adding trypsin was set to 100%.

Gel electrophoresis and Western blot analysis

Standard procedures were employed for SDS–PAGE and immunoblotting. For denaturation prior to electrophoresis, mitochondrial proteins were incubated for 30 min at 4°C under vigorous mixing in SDS sample buffer. Goat anti‐rabbit antibodies conjugated to horseradish peroxidase (Sigma) and a chemiluminescence kit (Amersham) were used for immunodetection. Polyclonal antisera directed against Cob and Cox3 were raised against purified proteins kindly provided by U.Brandt (University of Frankfurt). The Cob antiserum cross‐reacted with cytochrome c1 in mitochondria. Cox1‐specific antiserum was obtained from Molecular Probes.

Miscellaneous

The following procedures were performed as previously described: determination of cytochrome spectra (Claisse et al., 1970); isolation of mitochondria (Herrmann et al., 1994; Zinser and Daum, 1995); blue native gel electrophoresis (Schägger and von Jagow, 1991; Schägger, 1995); gel filtration analysis of mitochondrial extracts (Arlt et al., 1996); co‐immunoprecipitation of Yta10p and Yta12p (Arlt et al., 1996); labelling of mitochondrial translation products (Douglas et al., 1979; McKee and Poyton, 1984; Langer et al., 1995); chemical cross‐linking of substrate polypeptides to the m‐AAA protease (Arlt et al., 1996); and Northern hybridization analysis (Van Dyck et al., 1998).

The following DNA fragments were used as probes for the Northern blot analysis: COB exon probe, pA12/Mb2 (Nobrega and Tzagoloff, 1980); COXI exon probe, pCOX1/A4‐I corresponding to a DNA fragment from COXI containing exon A4 and part of intron aI4 (kindly provided by A.Tzagoloff); COXII probe, PCR‐amplified 689 bp internal DNA fragment of COXII; and COB intron probes, bI1 and pYJL12 (Lazowska et al., 1989).

Acknowledgements

We thank Drs J.Lazowska and A.Tzagoloff for plasmids and yeast strains, and Dr U.Brandt for purified Cox3 and Cob protein. The excellent technical assistance of Petra Robisch and Alexandra Weinzierl is gratefully acknowledged. R.P. was a recipient of a Senior Research Fellowship of the European Union. The work was supported by grants from the Deutsche Forschungsgemeinschaft (La918/1‐2; SFB 184/B21) to T.L.

References