In Saccharomyces cerevisiae the nicotinic acid moiety of NAD + can be synthesized from tryptophan using the kynurenine pathway or incorporated directly using nicotinate phosphoribosyl transferase (NPT1). We have identified the genes that encode the enzymes of the kynurenine pathway and for BNA5 (YLR231c) and BNA6 (YFR047c) confirmed that they encode kynureninase and quinolinate phosphoribosyl transferase respectively. We show that deletion of genes encoding kynurenine pathway enzymes are co-lethal with the v vnpt1, demonstrating that no other pathway for the synthesis of nicotinic acid exists in S. cerevisiae. Also, we show that under anaerobic conditions S. cerevisiae is a nicotinic acid auxotroph. ß

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 ARG8 m . Due to the presence of ARG8 m , accumulation of ؊ / 0 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 steadystate 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.In the mitochondrial inner membrane, the F 1 F 0 -type ATP synthase produces ATP from ADP and inorganic phosphate by using the energy of the transmembrane electrochemical proton gradient generated by the respiratory chain in the course of electron transfer to oxygen. The ATP synthase harbors two major structural domains, a transmembrane component (F 0 ) containing a proton-permeable pore and a peripheral, matrixlocalized, catalytic component (F 1 ) where the ATP is synthesized (1-4). In the F 0 , the core of the proton channel consists of a ring of c subunits (ten in yeast (4)) and one a subunit (Atp6p). Proton movement through this channel coincides with rotation of the subunit c ring (5-9), which results in conformational changes favoring ATP synthesis in the F 1 (1).Due to its good fermenting capacity the yeast Saccharomyces cerevisiae has been extensively used as a genetic system for the study of the mitochondrial ATP synthase (for reviews see Refs. 10 and 11). As in most eukaryotes, the yeast ATP synthase has a dual genetic origin, nuclear and mitochondrial. The yeast mitochondrial ATP synthase genes (ATP6, ATP9, and ATP8) encode the proton channel subunits a and c (usually referred to in yeast as Atp6p and Atp9p), respectively, and a third F 0 subunit (Atp8p) of unknown function. Dozens of mutations in the nuclear ATP synthase genes have provided much information on their protein products (10, 11). In contrast, only a very few mutants of the mitochondrial ATP synthase genes have been reported. Random generation of respiratory growth-deficient yeast strains issued fro...

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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 baker's 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.

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, rrd1delta and rrd2delta, 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 rrd2delta - resistance to caffeine and rapamycin - were weaker than the corresponding phenotypes of rrd1delta. The double mutant rrd1,2delta was inviable on rich glucose medium, but could grow in the presence of an osmotic stabilizer. The rrd1,2delta 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 slt2delta into the rrd1,2delta background improved the growth of rrd1,2delta on sorbitol-containing medium, indicating that the Rrd proteins also interact with the Slt2p/Mpk1p signaling pathway. Suppression of the lethal phenotype of the rrd1,2delta 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.