Elke Pratje

Oxa1p mediates the export of the N‐ and C‐termini of pCoxII from the mitochondrial matrix to the intermembrane space

Oxa1p is a mitochondrial protein reported to be involved in the assembly of the cytochrome oxidase complex. In the absence of a functional Oxa1p, subunit II of the cytochrome oxidase accumulates as its precursor form (pCoxII). Using mitochondria isolated from a yeast strain bearing a temperature sensitive mutation in the Oxa1p, pet ts1402, we have analyzed the function of the Oxa1p protein. We demonstrate that the accumulation of pCoxII in the pet ts1402 mitochondria does not reflect a compromised Imp1p activity in this mutant. Furthermore, measurement of the membrane potential has shown it to be sufficient to support the export of CoxII from the matrix. Rather, we found that newly synthesized pCoxII accumulates in the matrix of the pet ts1402 mitochondria, because export across the inner membrane is inhibited in the pet ts1402 mitochondria. In conclusion, Oxa1p mediates the export of the N‐ and C‐termini of the mitochondrially encoded subunit II of cytochrome oxidase from the matrix to the intermembrane space.

A novel two-step mechanism for removal of a mitochondrial signal sequence involves the mAAA complex and the putative rhomboid protease Pcp1.

The yeast protein cytochrome c peroxidase (Ccp1) is nuclearly encoded and imported into the mitochondrial intermembrane space, where it is involved in degradation of reactive oxygen species. It is known, that Ccp1 is synthesised as a precursor with a N-terminal pre-sequence, that is proteolytically removed during transport of the protein. Here we present evidence for a new processing pathway, involving novel signal peptidase activities. The mAAA protease subunits Yta10 (Afg3) and Yta12 (Rca1) were identified both to be essential for the first processing step. In addition, the Pcp1 (Ygr101w) gene product was found to be required for the second processing step, yielding the mature Ccp1 protein. The newly identified Pcp1 protein belongs to the rhomboid-GlpG superfamily of putative intramembrane peptidases. Inactivation of the protease motifs in mAAA and Pcp1 blocks the respective steps of proteolysis. A model of coupled Ccp1 transport and N-terminal processing by the mAAA complex and Pcp1 is discussed. Similar processing mechanisms may exist, because the mAAA subunits and the newly identified Pcp1 protein belong to ubiquitous protein families.

Inner membrane protease I, an enzyme mediating intramitochondrial protein sorting in yeast.

Several precursors transported from the cytoplasm to the intermembrane space of yeast mitochondria are first cleaved by the MAS‐encoded protease in the matrix space and then by additional proteases that have not been characterized. We have now developed a specific assay for one of these other proteases. The enzyme is an integral protein of the inner membrane; it requires divalent cations and acidic phospholipid for activity, and is defective in yeast mutant pet ts2858 which accumulates an incompletely processed cytochrome b2 precursor. The protease contains a 21.4 kd subunit whose C‐terminal part is exposed on the outer face of the inner membrane. An antibody against this polypeptide inhibits the activity of the protease. As overproduction of the polypeptide does not increase the activity of the protease in mitochondria, the enzyme may be a hetero‐oligomer. This ‘inner membrane protease I’ shares several key features with the leader peptidase of Escherichia coli and the signal peptidase of the endoplasmic reticulum.

PET1402, A NUCLEAR GENE REQUIRED FOR PROTEOLYTIC PROCESSING OF CYTOCHROME OXIDASE SUBUNIT 2 IN YEAST

The nuclear mutation pet ts1402 prevents proteolytic processing of the precursor of cytochrome oxidase subunit 2 (cox2) in Saccharomyces cerevisiae. The structural gene PET1402 was isolated by genetic complementation of the temperature-sensitive mutation. DNA sequence analysis identified a 1206-bp open reading frame, which is located 215 by upstream of the PET122 gene. The DNA sequence of PET1402 predicts a hydrophobic, integral membrane protein with four transmembrane segments and a typical mitochondrial targeting sequence. Weak sequence similarity was found to two bacterial proteins of unknown function. Haploid cells containing a null allelle of PET1402 are respiratory deficient.

Mitochondrial DNA of Chlamydomonas reinhardtii: the structure of the ends of the linear 15.8-kb genome suggests mechanisms for DNA replication

The mitochondrial genome of Chlamydomonas reinhardtii is a linear double-stranded DNA of 15.8 kb. With the exception of the termini its DNA sequence has been published. Here we describe the unique structure of the two termini determined from cloned fragments or, for the very terminal sequences, by the Maxam and Gilbert method after 5′ labeling of uncloned terminal fragments. The 15.8-kb DNA is characterized by terminal inverted repeats of 531 or 532 bp in length including long 3′ extensions. The 3′ single-stranded extensions of the left and right ends are non-complementary, identical in sequence, and comprise 39 to 41 nucleotides. Remarkably, the linear genome possesses in addition an internal 86-bp repeat of the two outermost sequences. The unusual structure of the 15.8-kb DNA termini is compared with those of other linear mitochondrial DNAs. Possible mechanisms of 15.8-kb DNA replication are discussed.

Mitochondrial DNA of Chlamydomonas reinhardtii: the gene for apocytochrome b and the complete functional map of the 15.8 kb DNA.

SummaryWe have sequenced the termini of the mitochondrial genome of Chlamydomonas reinhardtii and now present the DNA sequence of the gene for apocytochrome b. This gene is the thirteenth gene of the linear 15.8 kb DNA and appears to be the last one of the mt genome. The deduced protein sequence of 381 amino acid residues shows 56%, 48.6% and 48% identity with the apocytochrome b proteins of maize, Drosophila yakuba and mouse, respectively. RNA analysis reveals a transcript of about 1250 nucleotides. It is now possible to present the complete protein-coding capacity, the pattern of codon utilization for all eight protein genes, and the complete functional map of the mitochondrial 15.8 kb DNA of C. reinhardtii. One surprising feature is the absence of mitochondrial genes for ATPase and subunits II and III of cytochrome oxidase. No more than three tRNA genes appear to be present on the 15.8 kb mitochondrial DNA.

Mitochondrial inner membrane protease 1 of Saccharomyces cerevisiae shows sequence similarity to the Escherichia coli leader peptidase

SummaryThe nuclear yeast mutant pet ts2858 is defective in the removal of pre-sequences from the mitochondrially encoded cytochrome oxidase subunit II (COXII) and the processing intermediate of cytochrome b2 (Cytb2), a nuclear gene product. In order to identify the genetic lesion in this mutant we have cloned and characterized a DNA region which complements the pet ts2858 mutation. The DNA sequence revealed three open reading frames, one of which is responsible for the complementation. A 570 by reading frame represents the structural gene PET2858, as demonstrated by in vitro mutagenesis, gene expression from a foreign promoter, and allelism tests. PET2858 encodes a 21.4 kDa protein, which is essential for growth on non-fermentable carbon sources and for the proteolytic processing of COXII and the Cytb2 intermediate. When the N-terminus of the PET2858 protein is fused to a reporter protein, the resulting hybrid molecule is imported into mitochondria. Interestingly, the N-terminal half of the deduced PET2858 protein exhibits 30.7% amino acid identity to the leader peptidase of Escherichia coli. These results suggest that PET2858 codes for a mitochondrial inner membrane protease (IMP1) or at least a subunit of it. This protease is involved in protein processing and export from the mitochondrial matrix.

A nuclear mutation prevents processing of a mitochondrially encoded membrane protein in Saccharomyces cerevisiae.

Subunit II of cytochrome oxidase is encoded by the mitochondrial OXI1 gene in Saccharomyces cerevisiae. The temperature‐sensitive nuclear pet mutant ts2858 has an apparent higher mol. wt. subunit II when analyzed on lithium dodecylsulfate (LiDS) polyacrylamide gels. However, on LiDS‐6M urea gels the apparent mol. wt. of the wild‐type protein exceeds that of the mutant. Partial revertants of mutant ts2858 that produce both the wild‐type and mutant form of subunit II were isolated. The two forms of subunit II differ at the N‐terminal part of the molecule as shown by constructing and analyzing nuclear ts2858 and mitochondrial chain termination double mutants. The presence of the primary translation product in the mutant and of the processed form in the wild‐type lacking 15 amino‐terminal residues was demonstrated by radiolabel protein sequencing. Comparison of the known DNA sequence with the partial protein sequence obtained reveals that six of the 15 residues are hydrophilic and, unlike most signal sequences, this transient sequence does not contain extended hydrophobic parts. The nuclear mutation ts2858 preventing post‐translational processing of cytochrome oxidase subunit II lies either in the gene for a protease or an enzyme regulating a protease.

Som1, a third component of the yeast mitochondrial inner membrane peptidase complex that contains Imp1 and Imp2.

Abstract The mitochondrial inner membrane peptidase Imp is required for proteolytic processing of the mitochondrially encoded protein Cox2, the nucleus-encoded Cyt b2, Mcr1, and Cyt c1, and possibly other proteins, during their transport across the mitochondrial membranes. The peptidase contains two catalytic subunits, Imp1 and Imp2. The small protein Som1 was previously shown to affect the function of Imp1, but the precise role of Som1 remained unknown. Using mutants deleted for IMP1, IMP2 and SOM1, we show here that the Som1 protein is absent in the imp1Δ mutant, whereas the level of the Imp1 subunit of the peptidase is only slightly reduced in the som1 null mutant. The Som1 protein is not essential for proteolytic processing of Cyt b2, while the two other known Imp1 substrates, Cox2 and Mcr1, are not processed in the absence of Som1. Proteolytic processing of Cyt c1 by the Imp2 subunit, and of Ccp by an as yet unidentified peptidase, is not impaired in the som1 deletion mutant. By crosslinking and co-immunoprecipitation assays we demonstrate that the Imp1 and Som1 proteins physically interact. We conclude from our results that stabilisation of Som1 and correct Imp1 function is mediated by a direct interaction between the Imp1 and Som1 proteins, suggesting that Som1 represents a third subunit of the Imp peptidase complex.

Allelism studies of mitochondrial mutants resistant to antimycin A or funiculosin in Saccharomyces cerevisiae

SummaryA rapid procedure has been employed to isolate a large number of mitochondrial mutants resistant to antimycin A or funiculosin. A total of 15 antimycin A resistance mutations has been mapped by allelism tests. The mutations belong to two new mitochondrial loci, designated AI and AII. All funiculosin resistance mutations studied up to now map at locus AII. Thus mitochondrial funiculosin resistance might allow the specific selection of mutations in AII. Recombination between the two antimycin A resistance loci AI and AII occurs at frequencies from 8 to 21%. Apparently the two loci are not linked to PAR1, RIB1, RIB3, OLI1, and OLI2. Mutants of the two loci AI and AII have been characterized by measurements of oxygen consumption. Analysis of cytochrome spectra indicates that the mutations affect the cytochrome bc1 complex of the mitochondrial respiratory chain.