Moving proteins from the cytosol into mitochondria.

Mitochondria of the yeast Saccharomyces cerevisiae contain at least 750 different proteins, which perform diverse roles. Most of these proteins (approx. 99%) are translated on cytosolic ribosomes, and their import into mitochondria is essential for mitochondrial function. Proteinaceous machineries of great complexity, the so-called translocases, in the mitochondrial membranes mediate the import of these proteins.

Ssb1 chaperone is a [PSI+] prion-curing factor.

Yeast SUP7' or SUP11 nonsense suppressors have no phenotypic expression in strains deficient in the isopentenylation of A37 in tRNA. Here we show that such strains spontaneously produce cells with a nonsense suppressor phenotype which is related to the cytoplasmically inherited determinant and manifests all the key features of the [PSI+] prion. A screen of a multicopy yeast genomic library for genes that inactivate the [PSI+]-related suppressor phenotype resulted in the isolation of the SSB1 gene. Moreover, we demonstrate that multicopy plasmid encoding the Ssb1 chaperone cures cells of the [PSI+] prion.

Prion-dependent switching between respiratory competence and deficiency in the yeast nam9-1 mutant.

Nam9p is a protein of the mitochondrial ribosome. The respiration-deficient Saccharomyces cerevisiae strain MB43-nam9-1 expresses Nam9-1p containing the point mutation S82L. Respiratory deficiency correlates with a decrease in the steady level of some mitochondrially encoded proteins and the complete lack of mitochondrially encoded cytochrome oxidase subunit 2 (Cox2). De novo synthesis of Cox2 in MB43-nam9-1 is unaffected, indicating that newly synthesized Cox2 is rapidly degraded. Respiratory deficiency of MB43-nam9-1 is overcome by transient overexpression of HSP104, by deletion of HSP104, by transient exposure to guanidine hydrochloride, and by expression of the C-terminal portion of Sup35, indicating an involvement of the yeast prion [PSI(+)]. Respiratory deficiency of MB43-nam9-1 can be reinduced by transfer of cytosol from S. cerevisiae that harbors [PSI(+)]. We conclude that nam9-1 causes respiratory deficiency only in combination with the cytosolic prion [PSI(+)], presenting the first example of a synthetic effect between cytosolic [PSI(+)] and a mutant mitochondrial protein.

Coupling of mitochondrial translation with the formation of respiratory complexes in yeast mitochondria.

In contrast to most other eukaryotic organisms, yeast can survive without respiration. This ability has been exploited to investigate nuclear genes required for expression of mitochondrial DNA. Availability of complete Saccharomyces cerevisiae genomic sequence has provided additional help in detailed molecular analysis. Seven of the eight major products encoded by mitochondrial DNA are hydrophobic subunits of respiratory complexes in the inner membrane. Localization of the translation process in the same cellular compartment ensures synthesis of mitochondrially encoded proteins near sites of their assembly into multimeric respiratory complexes. Association of mitochondrial ribosomes with the membrane is mediated by mRNA-specific translational activators, that are involved in the recognition of initiation codon. The newly synthesized mitochondrial proteins are transferred to membrane by a specific export system. This review discusses the role of membrane-localized factors responsible for quality control and turnover of mitochondrially synthesized subunits as well as for assembly of respiratory complexes.

Expression of the yeast NAM9 gene coding for mitochondrial ribosomal protein.

We studied expression of the NAM9 gene of Saccharomyces cerevisiae that was previously reported to code for a mitochondrial ribosomal protein. Increase in NAM9 gene dosage is accompanied by the increase in both mRNA and protein. The levels of the NAM9 transcript and protein are both reduced in cells growing on glucose as compared to cells growing on galactose as a carbon source. Nam9p accumulates to the same level in rho(o) and rho(+) cells. These results confirm previous data indicating diverse regulation of different mitochondrial ribosomal protein genes and suggest that expression of Nam9p is not co-ordinated with the expression of other mitochondrial ribosomal components.

Isolation and functional characterization of mutant ferrochelatases in Saccharomyces cerevisiae.

Ferrochelatase is a mitochondrial inner membrane-bound enzyme that catalyzes the incorporation of ferrous iron into protoporphyrin, the last step in protoheme biosynthesis. It is encoded by the HEM15 gene in the yeast Saccharomyces cerevisiae. Five hem15 mutants causing defective heme synthesis and protoporphyrin accumulation were investigated. The mutations were identified by sequencing the mutant hem15 alleles amplified in vitro from mutant genomic DNA. A single nucleotide change, causing an amino acid substitution, was found in each mutant. The substitution L62F caused a five-fold increase in Vmax and 32-fold and four-fold increases in the KM's for protoporphyrin and metal. Replacements of the conserved G47 by S and S102 by F increased the KM for protoporphyrin 10-fold without affecting the affinity for metal or enzyme activity. Two amino acid changes, L205P and P221L, produced a thermosensitive phenotype. In vivo heme synthesis, the amount of immunodetected protein, and ferrochelatase activity measured in vitro were more affected in cells grown at 37 degrees C than at 30 degrees C. The effects of these mutations on the enzyme function are discussed with respects to ferrochelatase structure and mechanism of action.