Joanna Rytka

Yeast Cells Lacking the Mitochondrial Gene Encoding the ATP Synthase Subunit 6 Exhibit a Selective Loss of Complex IV and Unusual Mitochondrial Morphology

Atp6p is an essential subunit of the ATP synthase proton translocating domain, which is encoded by the mitochondrial DNA (mtDNA) in yeast. We have replaced the coding sequence of Atp6p gene with the non-respiratory genetic marker ARG8m. Due to the presence of ARG8m, accumulation of ρ–/ρ0 petites issued from large deletions in mtDNA could be restricted to 20–30% by growing the atp6 mutant in media lacking arginine. This moderate mtDNA instability created favorable conditions to investigate the consequences of a specific lack in Atp6p. Interestingly, in addition to the expected loss of ATP synthase activity, the cytochrome c oxidase respiratory enzyme steady-state level was found to be extremely low (<5%) in the atp6 mutant. We show that the cytochrome c oxidase-poor accumulation was caused by a failure in the synthesis of one of its mtDNA-encoded subunits, Cox1p, indicating that, in yeast mitochondria, Cox1p synthesis is a key target for cytochrome c oxidase abundance regulation in relation to the ATP synthase activity. We provide direct evidence showing that in the absence of Atp6p the remaining subunits of the ATP synthase can still assemble. Mitochondrial cristae were detected in the atp6 mutant, showing that neither Atp6p nor the ATP synthase activity is critical for their formation. However, the atp6 mutant exhibited unusual mitochondrial structure and distribution anomalies, presumably caused by a strong delay in inner membrane fusion.

Functional analysis of RRD1 (YIL153w) and RRD2 (YPL152w), which encode two putative activators of the phosphotyrosyl phosphatase activity of PP2A in Saccharomyces cerevisiae.

Abstract In the context of the cooperative project for functional analysis of novel genes uncovered during the systematic sequencing of the Saccharomyces cerevisiae genome, we deleted two paralogous ORFs: YIL153w and YPL152w. Based on the resulting phenotypes, the corresponding genes were named RRD1 and RRD2, respectively. Rrd proteins show significant similarity to the human phosphotyrosyl phosphatase activator (PTPA). Both single mutants, rrd1Δ and rrd2Δ, were viable. Deletion of RRD1 caused pleiotropic phenotypes under a wide range of conditions, including sensitivity to Ca2+, vanadate, ketoconazole, cycloheximide and Calcofluor white, and resistance to caffeine and rapamycin. The only phenotypes found for rrd2Δ– resistance to caffeine and rapamycin – were weaker than the corresponding phenotypes of rrd1Δ. The double mutant rrd1,2Δ was inviable on rich glucose medium, but could grow in the presence of an osmotic stabilizer. The rrd1,2Δ mutant was partially rescued by inactivation of HOG1 or PBS2, suggesting an interaction between the RRD genes and the Hog1p signal transduction pathway. Introduction of slt2Δ into the rrd1,2Δ background improved the growth of rrd1,2Δ on sorbitol-containing medium, indicating that the Rrd proteins also interact with the Slt2p/Mpk1p signaling pathway. Suppression of the lethal phenotype of the rrd1,2Δ mutant by overexpression of PPH22 suggested that the products of the RRD genes function positively with catalytic subunits of PP2A. The synthetic lethality was also suppressed by the “viable” allele (SSD1-v1) of the SSD1 gene.

Isolation of the catalase A gene of Saccharomyces cerevisiae by complementation of the cta1 mutation

SummaryAs a first step in an analysis of the DNA regions involved in the control of the catalase A gene of Saccharomyces cerevisiae by glucose, heme, and oxygen this gene has been cloned. Catalase A-deficient mutants were obtained by UV mutagenesis of a ctt1 mutant strain specifically lacking catalase T. All the catalase A-deficient mutants obtained fall into one complementation group. The single recessive mutation causing specific lack of catalase A was designated cta1. Several overlapping DNA fragments complementing the cta1 mutation were obtained by transforming ctt1 cta1 double mutants with a yeast gene library in vector YEp13. Hybrid selection of RNA with the help of one of the cloned DNAs followed by in vitro translation of this RNA and identification of the protein synthesized with catalase A-specific antibodies showed that the catalase A structural gene has been cloned. A single copy of this gene is present in the yeast genome. Transcription of the catalase A gene cloned into vector YEp13 is repressed by glucose. The DNA isolated hybridizes to a 1.6 kb polyA+−RNA virtually absent from heme-deficient cells, presumably catalase A mRNA.

Yeast as a system for modeling mitochondrial disease mechanisms and discovering therapies.

ABSTRACT Mitochondrial diseases are severe and largely untreatable. Owing to the many essential processes carried out by mitochondria and the complex cellular systems that support these processes, these diseases are diverse, pleiotropic, and challenging to study. Much of our current understanding of mitochondrial function and dysfunction comes from studies in the bakers yeast Saccharomyces cerevisiae. Because of its good fermenting capacity, S. cerevisiae can survive mutations that inactivate oxidative phosphorylation, has the ability to tolerate the complete loss of mitochondrial DNA (a property referred to as ‘petite-positivity’), and is amenable to mitochondrial and nuclear genome manipulation. These attributes make it an excellent model system for studying and resolving the molecular basis of numerous mitochondrial diseases. Here, we review the invaluable insights this model organism has yielded about diseases caused by mitochondrial dysfunction, which ranges from primary defects in oxidative phosphorylation to metabolic disorders, as well as dysfunctions in maintaining the genome or in the dynamics of mitochondria. Owing to the high level of functional conservation between yeast and human mitochondrial genes, several yeast species have been instrumental in revealing the molecular mechanisms of pathogenic human mitochondrial gene mutations. Importantly, such insights have pointed to potential therapeutic targets, as have genetic and chemical screens using yeast. Summary: In this Review, we discuss the use of budding yeast to understand mitochondrial diseases and help in the search for their treatments.

A Yeast Model of the Neurogenic Ataxia Retinitis Pigmentosa (NARP) T8993G Mutation in the Mitochondrial ATP Synthase-6 Gene

NARP (neuropathy, ataxia, and retinitis pigmentosa) and MILS (maternally inherited Leigh syndrome) are mitochondrial disorders associated with point mutations of the mitochondrial DNA (mtDNA) in the gene encoding the Atp6p subunit of the ATP synthase. The most common and studied of these mutations is T8993G converting the highly conserved leucine 156 into arginine. We have introduced this mutation at the corresponding position (183) of yeast Saccharomyces cerevisiae mitochondrially encoded Atp6p. The “yeast NARP mutant” grew very slowly on respiratory substrates, possibly because mitochondrial ATP synthesis was only 10% of the wild type level. The mutated ATP synthase was found to be correctly assembled and present at nearly normal levels (80% of the wild type). Contrary to what has been reported for human NARP cells, the reverse functioning of the ATP synthase, i.e. ATP hydrolysis in the F1 coupled to F0-mediated proton translocation out of the mitochondrial matrix, was significantly compromised in the yeast NARP mutant. Interestingly, the oxygen consumption rate in the yeast NARP mutant was decreased by about 80% compared with the wild type, due to a selective lowering in cytochrome c oxidase (complex IV) content. This finding suggests a possible regulatory mechanism between ATP synthase activity and complex IV expression in yeast mitochondria. The availability of a yeast NARP model could ease the search for rescuing mechanisms against this mitochondrial disease.

Probing the Active-site Residues in Saccharomyces cerevisiae Ferrochelatase by Directed Mutagenesis IN VIVO AND IN VITRO ANALYSES

Ferrochelatase is a mitochondrial inner membrane-bound enzyme that catalyzes the insertion of ferrous iron into protoporphyrin, the terminal step in protoheme biosynthesis. The functional/structural roles of 10 invariant amino acid residues were investigated by site-directed mutagenesis in the yeast Saccharomyces cerevisiae ferrochelatase. The mutant enzymes were expressed in a yeast strain lacking the ferrochelatase gene HEM15 and in Escherichia coli. The kinetic parameters of the mutant enzymes were determined for the enzymes associated with the yeast membranes and the enzymes in the bacterial soluble fraction. They were compared with the in vivo functioning of the mutant enzymes. The main conclusions are the following. Glu-314 is critical for catalysis, and we suggest that it is the base responsible for abstracting the N-pyrrole proton(s). His-235 is essential for metal binding. Asp-246 and Tyr-248 are also involved in metal binding in a synergistic manner. The K for protoporphyrin was also increased in the H235L, D246A, and Y248L mutants, suggesting that the binding sites of the two substrates are not independent of each other. The R87A, Y95L, Q111E, Q273E, W282L, and F308A mutants had 1.2-2-fold increased V and 4-10-fold increased K values for protoporphyrin, but the amount of heme made in vivo was 10-100% of the normal value. These mutations probably affected the geometry of the active center, resulting in improper positioning of protoporphyrin.

Diamino Acid Derivatives of Porphyrins Penetrate into Yeast Cells, Induce Photodamage, but Have No Mutagenic Effect

The yeast Saccharomyces cerevisiae was used as a model eukaryotic organism to study the uptake of diamino acid derivatives of porphyrins and their phototoxicity with particular emphasis on possible mutagenic effects. The water‐soluble hematoporphyrin derivatives diarginate (HpD[Arg]2) and 1‐arginin di(N‐amino acid)‐protoporphyrinate used in this study are effective photosensitizers in tumor photodynamic therapy. Depending on the amino acid substituent, the porphyrin derivatives differ in their affinity for yeast cells. It is shown that HpD(Arg)2 and PP(Met)2 (Arg)2 penetrate into the yeast cell and are metabolized. Both compounds sensitize yeast cells to photodamage but have no mutagenic effect on nuclear or mitochondrial genomes.

Disruption of six novel yeast genes located on chromosome II reveals one gene essential for vegetative growth and two required for sporulation and conferring hypersensitivity to various chemicals.

A PCR‐based method for targeted gene deletion by kanMX4 module was used to construct complete deletion mutants of six individual open reading frames from chromosome II: YBR128c, YBR131w, YBR133c, YBR137w, YBR138c and YBR142w. The ORFs were deleted in two diploid strains, FY1679 and W303. Sporulation and tetrad analysis revealed that only one ORF, YBR142w, encoding a putative DEAD‐box RNA helicase, is an essential gene. A systematic phenotypic analysis of the deleted mutants was carried out. Homozygous diploids ybr128cΔ/ybr128cΔ and ybr131wΔ/ybr131wΔ did not sporulate. The ybr131cΔ mutant whether haploid or homozygous diploid, in addition displayed an increased sensitivity to Caffeine, Calcium and Zinc, and to emphasize this phenotype we named the gene CCZ1. ORF YBR133c was independently reported by others as Histone Synthetic Lethal (HSL7) (Ma et al., 1996). We found that the aberrant morphology characteristic for ybr133cΔ (hsl7Δ) cells was observed in W303 but not in FY1679 genetic background. Furthermore, we observed that deletion of YBR133c had a pleiotropic effect under a wide range of conditions, including increased sensitivity to calcium, caffeine, calcofluor white, vanadate and verapamil. The effects of the deletion were reinforced in W303 background. We found no phenotypic effects of the two remaining deletions, ybr137wΔ and ybr138cΔ. Copyright

Haemoprotein formation in yeast

SummaryA procedure was described for the isolation of mutants affected in the regulation of catalase activity. Two such mutants, cgr 1 and cgr 2 were obtained. Both of them show catalase activity that is resistant to repression by glucose, but is sensitive to anoxia to the same extent as the wild type.

The CHiPS Domain – Ancient Traces for the Hermansky–Pudlak Syndrome

Hermansky–Pudlak syndrome (HPS) is a rare disorder caused by malfunctions of lysosomes and specialized lysosome‐related organelles, resulting primarily in oculocutaneous albinism and bleeding diathesis. The majority of the HPS genes have been described as novel, but herein we report the identification of a conserved protein family which includes human HPS4, as well as distant homologs for other HPS genes. Our results suggest that the cellular machinery involved in the HPS syndrome is ancient.