Isolation of the nuclear yeast genes for citrate synthase and fifteen other mitochondrial proteins by a new screening method.

To isolate nuclear genes specifying imported mitochondrial proteins, a yeast genomic clone bank was screened by an RNA hybridization-competition assay. This assay exploited the fact that mRNAs for imported mitochondrial proteins are enriched in polysomes which are bound to the mitochondrial surface in cycloheximide-inhibited yeast cells. Clones selectively hybridizing to these enriched mRNAs were further screened by hybrid-selected translation and immunoprecipitation with monospecific antisera against individual mitochondrial proteins. Thirty-six clones were isolated which contained complete or partial copies of 16 different genes for imported mitochondrial proteins. Several of these clones caused expression of the corresponding precursor polypeptide in Escherichia coli or over-expression of the corresponding mature protein in yeast. The gene for the matrix enzyme citrate synthase was sequenced; the derived amino acid sequence of the precursor polypeptide revealed an amino-terminal extension containing basic but no acidic residues.     

Biosynthesis of the ubiquinol-cytochrome c reductase complex in yeast. Characterization of precursor forms of the 44-kDa, 40-kDa and 17-kDa subunits and identification of individual messenger RNAs for these and other imported subunits of the complex

The mitochondrial ubiquinol--cytochrome c reductase complex (complex III or cytochrome bc1 complex) is thought to consist of eight subunits, seven of which are specified by nuclear genes and synthesized in the cytoplasm. We have studied the synthesis of five of the nuclear-encoded subunits both in vivo and in vitro and show that of these the 44-kDa, 40-kDa and 17-kDa subunits are synthesized with cleavable extensions, while the 14-kDa and 11-kDa proteins are synthesized without detectable extra sequences. The sizes of the pre-sequences, as determined by the relative mobility of the precursor proteins in sodium dodecyl sulphate/polyacrylamide gels, range from 0.5-kDa for the 44-kDa and 40-kDa subunits to 9-kDa for the 17-kDa subunit. The existence in vivo of precursor forms to the 44-kDa, 40-kDa and 17-kDa subunits implies that import is at least partially a post-translational process. The precursor of the 44-kDa subunit can be processed post-translationally in vitro by isolated mitochondria. The messenger RNAs for subunits of the complex have been studied. Those coding for the 44-kDa, 40-kDa, 14-kDa and 11-kDa proteins and cytochrome c1 are of different sizes, indicating that each of these subunits is synthesized as a separate protein, rather than as part of a polyprotein precursor.     

MRS3 and MRS4, two suppressors of mtRNA splicing defects in yeast, are new members of the mitochondrial carrier family.

When present in high copy number plasmids, the nuclear genes MRS3 and MRS4 from Saccharomyces cerevisiae can suppress the mitochondrial RNA splicing defects of several mit- intron mutations. Both genes code for closely related proteins of about Mr 32,000; they are 73% identical. Sequence comparisons indicate that MRS3 and MRS4 may be related to the family of mitochondrial carrier proteins. Support for this notion comes from a structural analysis of these proteins. Like the ADP/ATP carrier protein (AAC), the mitochondrial phosphate carrier protein (PiC) and the uncoupling protein (UCP), the two MRS proteins have a tripartite structure; each of the three repeats consists of two hydrophobic domains that are flanked by specific amino acid residues. The spacing of these specific residues is identical in all domains of all proteins of the family, whereas spacing between the hydrophobic domains is variable. Like the AAC protein, the MRS3 and MRS4 proteins are imported into mitochondria in vitro and without proteolytic cleavage of a presequence and they are located in the inner mitochondrial membrane. In vivo studies support this mitochondrial localization of the MRS proteins. Overexpression of the MRS3 and MRS4 proteins causes a temperature-dependent petite phenotype; this is consistent with a mitochondrial function of these proteins. Disruption of these genes affected neither mitochondrial functions nor cellular viability. Their products thus have no essential function for mitochondrial biogenesis or for whole yeast cells that could not be taken over by other gene products. The findings are discussed in relation to possible functions of the MRS proteins in mitochondrial solute translocation and RNA splicing.     

Location and nucleotide sequence of frdB, the gene coding for the iron-sulphur protein subunit of the fumarate reductase of Escherichia coli

The frdB gene, encoding the iron-sulphur protein subunit of fumarate reductase, has been located and its complete nucleotide sequence determined. The identity of the gene was confirmed by protein chemical studies and determination of the NH2-terminal sequence of the FrdB protein. The frdB gene is situated distal to and partially overlapped by frdA which codes for the flavoprotein subunit of the reductase. Its reading frame contains 244 codons and predicts a protein of Mr 27092. In composition, the FrdB protein is strikingly similar to the corresponding subunit of the related flavoenzyme, succinate dehydrogenase. Analysis of the protein's primary structure revealed several features characteristic of iron-sulphur proteins.     

Steady state analysis of mitochondrial RNA after growth of yeast Saccharomyces cerevisiae under catabolite repression and derepression

The steady state levels of mitochondrial rRNAs, 5 tRNAs, the 9 S RNA, and the RNA products from the genes coding for subunits 6 and 9 of the ATP synthase, cytochrome b, and subunit 1 of cytochrome oxidase have been determined after growth of yeast under conditions of respiratory repression or derepression. The analysis indicates that the mitochondrial rRNAs are present in 2000 or 9000 copies/cell in repressed or derepressed yeast, respectively. The levels of the other RNAs also differed to a similar extent, with the exception of the level of the tRNAfMet which differs by only 1.7-fold. The levels of the individual protein coding RNAs varied from 480 copies/cell for the Oli-1 RNA to 100 copies/cell for the Oli-2 RNA under derepressive conditions and from 130 copies/cell to 33 copies/cell for the same RNAs in glucose repressive conditions. The levels of the tRNAs varied even more markedly, ranging from 4200 copies/cell for the tRNAPhe to 240 copies/cell for the tRNACys after growth in derepressive conditions and from 800 copies/cell for the tRNAfMet to 30 copies/cell for the tRNACys of glucose repressed yeast. These results indicate that glucose repression uniformly decreases the levels of the individual mitochondrial RNAs studied. This decrease is related to a lower synthesis of mitochondrial RNA in the glucose repressed cells as compared to derepressed cells.     

Nuclear genes coding the yeast mitochondrial adenosine triphosphatase complex. Isolation of ATP2 coding the F1-ATPase beta subunit

A yeast nuclear pet mutant of Saccharomyces cerevisiae lacking any detectable mitochondrial F1-ATPase activity was genetically complemented upon transformation with a pool of wild type genomic DNA fragments carried in the yeast Escherchia coli shuttle vector YEp 13. Plasmid-dependent complementation restored both growth of the pet mutant on a nonfermentable carbon source as well as functional mitochondrial ATPase activity. Characterization of the complementing plasmid by plasmid deletion analysis indicated that the complementing gene was contained on adjoining BamH1 fragments with a combined length of 3.05 kilobases. Gel analysis of the product of this DNA by in vitro translation in a rabbit reticulocyte lysate programmed with yeast mRNA hybrid selected by the plasmid revealed a product which could be immunoprecipitated by antisera against the beta subunit of the yeast mitochondrial ATPase complex. A comparison of the protein sequence derived from partial DNA sequence analysis indicated that the beta subunit of the yeast mitochondrial ATPase complex exhibits greater than 70% conservation of protein sequence when compared to the same subunit from the ATPase of E. coli, beef heart, and chloroplast. The gene coding the beta subunit (subunit 2) of yeast mitochondrial adenosine triphosphatase is designated ATP2. The utilization of cloned nuclear structural genes of mitochondrial proteins for the analysis of the post-translational targeting and import events in organelle assembly is discussed.     

A novel mitochondrial DEAD box protein (Mrh4) required for maintenance of mtDNA in Saccharomyces cerevisiae

In a screen of nuclear genes that assist splicing of mitochondrial localized group II introns in yeast we isolated low-copy number suppressors of splicing and respiratory-deficient point mutants of intron aI5gamma, the last intron of the gene encoding cytochrome c oxidase subunit I. One of the genes found contains the open reading frame (ORF) YGL064c that has previously been proposed to encode a putative RNA helicase of the DEAD box family. Deletion of the ORF gives rise to 100% cytoplasmic petites, indicating that the protein plays an essential role in the mitochondrial RNA metabolism. Overexpression of YGL064c-GFP fusions clearly revealed a mitochondrial localization of the protein. The gene encodes the fourth putative RNA helicase of Saccharomyces cerevisiae implicated in a mitochondrial function and was therefore termed MRH4 (for mitochondrial RNA helicase).     

Biosynthesis of the ubiquinol-cytochrome c reductase complex in yeast. Discoordinate synthesis of the 11-kd subunit in response to increased gene copy number

In wild-type yeast cells, steady-state concentrations of subunits of the ubiquinol-cytochrome c reductase complex (complex III) and the levels of their translatable mRNAs change coordinately in response to the need for mitochondrial function. Despite this, re-introduction of the cloned gene for one of the subunits (11 kd) into cells by transformation with a free-replicating plasmid results in the discoordinate synthesis of this subunit only, without effects on either the synthesis or degradation of the other subunits. The overproduced subunit is associated with the mitochondrial fraction, yet does not interfere with mitochondrial function, as judged by the growth of transformed cells on nonfermentable media. Quantitative analysis of both mRNA and protein levels suggests that both translational controls and elevated turnover of excess protein contribute to a partial compensation for the effects of increased gene dosage in transformed cells. These contain approximately 30 copies of the cloned gene and 15-30 times the normal level of its mRNA. Nevertheless, synthesis of the 11-kd protein is only 6- to 8-fold higher than normal, and steady-state levels are increased only 5- to 10-fold. These findings imply that synthesis of the various subunits of complex III is not tightly coupled and that for the 11-kd subunit at least, the level of mRNA is likely to be the most important means of regulating protein level. Fine-tuning may be additionally achieved by control of translation and degradation of excess protein which is not assembled in the complex.