Promotion of mitochondrial membrane complex assembly by a proteolytically inactive yeast Lon.

Afg3p and Rca1p are adenosine triphosphate (ATP)-dependent metalloproteases in yeast mitochondria. Cells lacking both proteins exhibit defects in respiration-dependent growth, degradation of mitochondrially synthesized proteins, and assembly of inner-membrane complexes. Defects in growth and protein assembly, but not in degradation, were suppressed by overproduction of yeast mitochondrial Lon, an ATP-dependent serine protease. Suppression by Lon was enhanced by inactivation of the proteolytic site and was prevented by mutation of the ATP-binding site. It is suggested that the mitochondrial proteases Lon, Afg3p, and Rca1p can also serve a chaperone-like function in the assembly of mitochondrial protein complexes.

ATP-dependent proteases that also chaperone protein biogenesis.

The ATP-dependent proteases Clp and FtsH from bacteria, as well as mitochondrial homologs of FtsH and Lon from yeast, may act as chaperones; they mediate not only proteolysis, but also the insertion of proteins into membranes and the disassembly or oligomerization of protein complexes. The coordination of such processes with selective proteolysis may function in the quality control of protein biogenesis.

Intron mobility. Invasive introns.

The evidence that introns in fungal and plant organellar DNAs are mobile genetic elements has strengthened significantly; like Shaw's cockney heroine, Eliza Doolittle, they cannot help but betray their origins.

Transposition: mobile introns get into line.

Group II introns encode highly structured, frequently self-splicing RNAs; they are also mobile genetic elements. This mobility has been found to involve DNA-primed reverse transcription, with similarities to retrotransposition and telomere maintenance.

Global regulation of mitochondrial biogenesis in Saccharomyces cerevisiae: ABF1 and CPF1 play opposite roles in regulating expression of the QCR8 gene, which encodes subunit VIII of the mitochondrial ubiquinol-cytochrome c oxidoreductase.

The multifunctional DNA-binding proteins ABF1 and CPF1 bind in a mutually exclusive manner to the promoter region of the QCR8 gene, which encodes 11-kDa subunit VIII of the Saccharomyces cerevisiae mitochondrial ubiquinol-cytochrome c oxidoreductase (QCR). We investigated the roles that the two factors play in transcriptional regulation of this gene. To this end, the overlapping binding sites for ABF1 and CPF1 were mutated and placed in the chromosomal context of the QCR8 promoter. The effects on transcription of the QCR8 gene were analyzed both under steady-state conditions and during nutritional shifts. We found that ABF1 is required for repressed and derepressed transcription levels and for efficient induction of transcription upon escape from catabolite repression, independently of DNA replication. CPF1 acts as a negative regulator, modulating the overall induction response. Alleviation of repression through CPF1 requires passage through the S phase. Implications of these findings for the roles played by ABF1 and CPF1 in global regulation of mitochondrial biogenesis are discussed.

cDNA sequence of subunit VIII of ubiquinol-cytochrome-c oxidoreductase from Schizosaccharomyces pombe.

We have cloned a cDNA coding for subunit VIII of the ubiquinol-cytochrome-c oxidoreductase of Schizosaccharomyces pombe by functional complementation of the null mutant in the QCR8 gene of Saccharomyces cerevisiae. DNA sequence analysis reveals an open-reading frame of 276 bp encoding a 10.5 kDa protein with 51% amino acid sequence identity to its counterpart in S. cerevisiae.

Isolation and characterisation of the linked genes, FPS1 and QCR8, coding for farnesyl-diphosphate synthase and the 11 kDa subunit VIII of the mitochondrial bc1-complex in the yeast Kluyveromyces lactis.

The KlQCR8 gene of the yeast Kluyveromyces lactis encoding subunit VIII of the mitochondrial bc1 complex is 70.2% identical to its counterpart in Saccharomyces cerevisiae (ScQCR8). As in S. cerevisiae, chromosomal linkage between the K. lactis QCR8 and FPS1 genes is conserved, the two genes being separated by only 292 bp. Disruption of the KlQCR8 gene results in a respiratory-deficient phenotype. Compared with S. cerevisiae, expression of the KlQCR8 gene in glucose-grown cells is relatively high, yet is significantly induced when the cells are grown on non-fermentable carbon sources. The QCR8 promoters regions of the two yeasts lack overall DNA sequence similarity, but share DNA-binding sites for the transcription factors ABF1, CPF1 and HAP2/3/4. Deletion from the KlQCR8 promoter of a 93 bp region containing these sites significantly lowers mRNA levels during growth on either glucose or ethanol/glycerol, with a consequent reduction of growth rate on ethanol/glycerol.

Isolation and characterisation of the linked genes APA2 and QCR7, coding for Ap4A phosphorylase II and the 14 kDa subunit VII of the mitochondrial bc1-complex in the yeast Kluyveromyces lactis.

We report the isolation and characterization of the KlQCR7 gene encoding subunit VII of the mitochondrial bc1 complex of the yeast Kluyveromyces lactis. The coding region is 69.3% identical to its counterpart in Saccharomyces cerevisiae (ScQCR7). Like the KlQCR8 gene (Mulder et al., accompanying paper) expression of the KlQCR7 gene during growth on glucose is high and can be further induced when cells are grown on non-fermentable carbon sources. The chromosomal linkage of the APA2 and QCR7 genes is conserved between S. cerevisiae and K. lactis. The intergenic regions containing the QCR7 promoters of the two yeasts, differ significantly in length and lack overall DNA sequence similarity, but they do share a binding site for the transcription factor complex HAP2/3/4. The KlQCR7 promoter contains, in addition, a CPF1 consensus binding site which is absent from ScQCR7. Deletion of a 35 bp region containing these two sites severely lowers the mRNA expression during growth on both glucose and ethanol/glycerol, but growth rate on both carbon sources is only mildly affected. Interestingly, in respect to the KlQCR7 gene, KlCPF1 seems to act as an important transcriptional activator, thus contrasting the proposed repressor function of ScCPF1 for the ScQCR8 gene of S. cerevisiae.

Identification of mitochondrial gene products by DNA-directed protein synthesis in vitro.

1. A cell-free system, derived from Escherichia coli is highly active in the linked transcription-translation of yeast mtDNA from both wild-type and petite strains. 2. The products of synthesis are short (Mr less than 10 000) hydrophobic polypeptides, which show a high tendency to aggregate in a specific fashion with E. coli and mitochondrial proteins. Aggregation is extremely persistent: alkali, sodium dodecyl sulphate/urea, guanidinium . HCl and carboxymethylation reduce it, but do not eliminate it completely. 3. Nevertheless, results of indirect immunoprecipitation tests suggest that antigenic determinants of cytochrome c oxidase are among the products synthesized. The immunoprecipitation appears specific by criteria including competition experiments and its absence when mtDNA from low complexity petites, retaining only the gene for 21 S rRNA and some flanking sequences, is used to programme protein synthesis. Electrophoretic analysis of material precipitated by anti-cytochrome c oxidase sera reveals four discrete polypeptides with molecular weights of 7400, 6400, 5000 and 4100, which probably represent polypeptide fragments carrying antigenic determinants of cytochrome c oxidase.

Variation, transcription and circular RNAs of the mitochondrial gene for subunit I of cytochrome c oxidase.

The gene for subunit I of cytochrome c oxidase, contained within the OX13 region of yeast mitochondrial DNA, is split and shows a remarkable variation in structure, which is strain-dependent. The most complex form so far characterized is that of the Saccharomyces cerevisiae strain KL14-4A, in which nine or possibly ten exons are separated by eight to nine introns. At least four of these are facultative, two being absent from S. cerevisiae strain D273-10B (sequenced by Bonitz et al., 1980) and a further two lacking from the gene in Saccharomyces carlsbergensis. The complexity of the gene in KL14-4A is also reflected in its transcript pattern. RNA blot hybridization with isolated and cloned DNA fragments of the OX13 region permits visualization of more than 60 RNAs, which show overlapping and discontinuous hybridization behaviour. In the less complex strains D273-10B and S. carlsbergensis, this number is 20 and 11, respectively. These RNAs are most likely intermediates in processing events leading to the appearance of the mature messenger RNA for cytochrome c oxidase subunit I, which we identify as a 2100-nucleotide transcript (18SE). Most of the processing events are dependent on mitochondrial protein synthesis and do not constitute a single obligatory processing pathway. Like other yeast mitochondrial mRNAs, the 18 S RNA contains a long, untranslated 5' flanking sequence (approximately 400 nucleotides). One unusual aspect of splicing events involving OX13 transcripts is the accumulation of three of the excised introns as single-stranded RNA circles. These abundant and stable transcripts appear to be covalently closed. The simplest assumption is that they arise as (by)-products of splicing, but secondary ligation events have not been excluded. Their function is as yet unknown.

Isolation, characterization and regulation of expression of the nuclear genes for the core II and Rieske iron-sulphur proteins of the yeast ubiquinol-cytochrome c reductase.

Cloning and mapping of the yeast nuclear genes for the core II (Mr 40 000) and Rieske iron-sulphur proteins of the mitochondrial ubiquinol-cytochrome c reductase, and comparison with the genomic regions in nuclear DNA from which they are derived, show that the genes are likely to be present in single copies and that they are not closely linked. They have been reintroduced into yeast cells on multi-copy plasmids and, similar to results obtained for the Mr 11 000 subunit [Van Loon et al., EMBO J. 2 (1983) 1765-1770], increase in the dosage of either gene prompts discoordinate synthesis of the encoded protein. Quantitative analysis of transformants carrying extra copies of the gene for the iron-sulphur protein shows that messenger RNA level, rate of synthesis and steady-state concentration of the protein correlate well with each other. This indicates that its level, in contrast to that of the Mr 11 000 subunit, is only determined by the concentration of its messenger RNA. Over-production of these proteins does not interfere with mitochondrial function as judged from growth rates of transformed cells on non-fermentable media. The excess Mr 40 000 protein is imported into the mitochondrion, showing that import of this subunit is not obligatorily coupled to complex assembly.

Isolation and characterization of nuclear genes coding for subunits of the yeast ubiquinol-cytochrome c reductase complex.

Nuclear genes coding for the Mr 17 000, 14 000 and 11 000 subunits of the ubiquinol-cytochrome c reductase complex (complex III) in yeast have been isolated from a clone bank of yeast nuclear DNA by use of a mRNA hybridization-competition assay. This is based on our observations that levels of mRNAs for these subunits are much reduced during glucose repression and in cytoplasmic petite mutants and the procedure should be of general application for the isolation of other inducible or repressible genes coding for mRNAs present at low levels in the cell. A first characterization of the clones is presented. The genes are not closely linked in the genome and those coding for Mr 14 000 and 11 000 subunits are present in unique genomic environments, which suggests that there are only single copies of each in the nuclear genome.

MBA1 encodes a mitochondrial membrane-associated protein required for biogenesis of the respiratory chain.

The yeast MBA 1 gene (Multi-copy Bypass of AFG3) is one of three genes whose overexpression suppresses afg3-null and rca1-null mutations. Bypass of AFG3 and RCA1, whose products are essential for assembly of mitochondrial inner membrane enzyme complexes, suggests a related role for MBA1. The predicted translation product is a 30 kDa hydrophilic protein with a putative mitochondrial targeting sequence and no homology to any sequence in protein or EST databases. Gene disruption leads to a partial respiratory growth defect, which is more pronounced at temperatures above 30 degrees C. Concomitantly, amounts of cytochromes b and aa3 are reduced. A C-terminal c-myc-tagged MBA1 gene product is functional and is found associated with the mitochondrial inner membrane, from which it can he extracted by carbonate, but not by high salt. These observations give further support to a role of MBA1 in assembly of the respiratory chain.

Topological organization of subunits VII and VIII in the ubiquinol-cytochrome c oxidoreductase of Saccharomyces cerevisiae.

To determine the topology of subunit VIII of the yeast ubiquinol-cytochrome c oxidoreductase in the mitochondrial inner membrane, an epitope has been introduced in the N-terminal half of this protein. Previous topology studies had shown that at least the C-terminus faces the intermembrane space [Hemrika and Berden (1990) Eur. J. Biochem. 192, 761-765]. Based on sensitivity of the protein to proteinase K digestion we now suggest that the N-terminus of subunit VIII is similarly oriented, implying that this subunit does not span the membrane. Despite this, however, subunit VIII cannot be extracted from the membrane even after treatment with 0.1 M Na2CO3 at pH 11.5, showing that the protein is integrally embedded in the membrane. A similar behaviour was displayed by another low molecular weight protein of the complex, subunit VII, which faces the matrix side. A model for the topology of these subunits in the membrane is discussed with respect to the structure of the complex and their involvement in quinone binding.

Identification of additional homologues of subunits VII and VIII of the ubiquinol-cytochrome c oxidoreductase enables definition of consensus sequences.

The Candida utilis QCR7 gene encoding subunit VII of the ubiquinol-cytochrome c oxidoreductase was isolated by functional complementation of the Saccharomyces cerevisiae subunit VII-null mutant. Several other subunit VII homologues as well as homologues for subunit VIII were identified by screening the GenBank database. Some of these homologues for subunit VII could only be identified as such using a consensus sequence that was derived from the multiple sequence alignment. Definition of the consensus should facilitate further analysis of structure/function relationships in this protein.

Afg3p, a mitochondrial ATP-dependent metalloprotease, is involved in degradation of mitochondrially-encoded Cox1, Cox3, Cob, Su6, Su8 and Su9 subunits of the inner membrane complexes III, IV and V.

The yeast AFG3 gene encodes an ATP-dependent metalloprotease belonging to a subgroup of the AAA-family. This protease has been suggested to be essential for a metal- and ATP-dependent breakdown of incompletely mitochondrially synthesized polypeptides in the inner membrane, a process proposed to be important for mitochondrial function (Pajic et al. (1994) FEBS Lett. 353, 201-206). Here, we confirm the proteolytic activity by site-directed mutagenesis and demonstrate that the proteins Cox1, Cox3, Cob, Su6, Su8 and Su9 are substrates of Afg3p. Surprisingly, this proteolytic activity is not required for respiratory function and thus presumably also not essential for mitochondrial biogenesis.

The aromatic nature of residue 66 of the 11-kDa subunit of ubiquinol-cytochrome c oxidoreductase of the yeast Saccharomyces cerevisiae is important for the assembly of a functional enzyme.

Transformation of multi- and single-copy plasmids carrying a mutated version (LTN2, region 66-YWYWW-70 replaced by SASAA) of QCR8, the gene encoding the 11-kDa subunit ubiquinol-cytochrome c oxidoreductase of Saccharomyces cerevisiae, to a QCR8(0) strain indicated the importance of this aromatic region for the assembly of a functional enzyme. Sequencing of plasmids giving spontaneous restoration of growth to some colonies among the single-copy LTN2 transformants showed that changing the sequence SASAA into the sequence FASAA could, to a large extent, overcome the observed assembly defect, indicating the importance of the aromatic nature of residue 66.

The mitochondrial inner membrane AAA metalloprotease family in metazoans.

Three metalloproteases belonging to the AAA superfamily (Yme1p, Afg3p and Rca1p) are involved in protein turnover and respiratory chain complex assembly in the yeast inner mitochondrial membrane. Analysis of the completed genome sequences of Caenorhabditis elegans and Drosophila melanogaster indicates that this gene family typically comprises 3-4 members in metazoans. Phylogenetic analysis reveals three main branches represented, respectively, by Saccharomyces cerevisiae YME1, human SPG7 (paraplegin) and S. cerevisiae AFG3 and RCA1. mt-AAA metalloproteases are weak candidates for several previously studied Drosophila mutants. A full elucidation of the cellular and physiological roles of mt-AAA metalloproteases in metazoans will require the creation of targeted mutations.

Increased synthesis and decreased stability of mitochondrial translation products in yeast as a result of loss of mitochondrial (NAD(+))-dependent isocitrate dehydrogenase.

We have previously demonstrated that the yeast Krebs cycle enzyme NAD(+)-dependent isocitrate dehydrogenase (Idh) binds specifically and with high affinity to the 5'-untranslated leader sequences of mitochondrial mRNAs in vitro and have proposed a role for the enzyme in the regulation of mitochondrial translation [Elzinga, S.D.J. et al. (2000) Curr. Genet., in press]. Although our studies initially failed to reveal any consistent correlation between idh disruption and mitochondrial translational activity, it is now apparent that compensatory extragenic suppressor mutations readily accumulate in idh disruption strains thereby masking mutant behaviour. Now, pulse-chase protein labelling of isolated mitochondria from an Idh disruption mutant lacking suppressor mutations reveals a strong (2-3-fold) increase in the synthesis of mitochondrial translation products. Strikingly, the newly synthesised proteins are more short-lived than in mitochondria from wild-type cells, their degradation occurring with a 2-3-fold reduced half-life. Enhanced degradation of translation products is also a feature of yeast mutants in which tethering/docking of mitochondrial mRNAs is disturbed. We therefore suggest that binding of Idh to mitochondrial mRNAs may suppress inappropriate translation of mitochondrial mRNAs.

The aromatic domain 66(YWYWW)70 of subunit VIII of the yeast ubiquinol-cytochrome c oxidoreductase is important for both assembly and activity of the enzyme.

The aromatic character of the region 66(YWYWW)70 of the 11-kDa subunit VIII of ubiquinol-cytochrome c oxidoreductase (bc1 complex) of the yeast Saccharomyces cerevisiae has previously been demonstrated to be important for assembly of a functional complex [Hemrika et al. (1994) FEBS Lett. 344, 15-19]. Especially the aromatic nature of residue 66 appeared to be relevant, as the very low level (5%) of bc1 complex in the mutant 66(SASAA)70 was restored to nearly 70% of the wild-type level in a phenotypic revertant with the sequence 66(FASAA)70. In the present study, three other site-directed mutants (66(SAYAA)70, 66(SASAW)70 and 66(SWYWW)70) were constructed and analysed. The data indicate that the presence of one aromatic residue is enough for a substantial level of assembly and that its position modulates the level of both assembly and electron transfer activity. The results also confirm the relevance of this region of subunit VIII for the formation of the Q(out) reaction domain, as reported by Hemrika et al. [(1993) Eur. J. Biochem. 215, 601-609]. It is further shown that the lowered specific activity of the mutant described by these authors is not due to the introduction of a cysteine in the sequence of subunit VIII.