Saccharomyces cerevisiae as a model for the study of mitochondrial diseases and for the identification of beneficial molecules

Mitochondrial disorders are a group of clinically and genetically heterogeneous disorders, due to impairment of mitochondrial functionality induced by mutations in mitochondrial DNA or in nuclear DNA. Mitochondrial disorders may manifest at any age and in virtually any organ, although brain, skeletal muscle, liver and heart are most frequently involved because of their high-energy demand. Actually no effective therapies exist for the majority of these diseases. Here, specific models of human mitochondrial diseases were created and studied in Saccharomyces cerevisiae to evaluate the effect of missense mutations identified in patients and to discover molecules able to rescue the mitochondrial defective phenotypes induced by pathological mutations. In particular, three different pathologies associated with mutations in the human genes MPV17, YARS2, QRSL1 and GATB were studied in yeast taking advantage of the presence of the orthologous genes SYM1, MSY1, HER2 and PET112, respectively. MPV17 encodes for a protein located in the inner mitochondrial membrane, whose specific function is still elusive, taking part in a high molecular weight complex, the composition of which is unknown. Mutations in this gene result in a mitochondrial DNA depletion syndrome. Here, phenotypic analyses of seven missense mutations were performed in yeast demonstrating the deleterious effect of all the corresponding mutations in OXPHOS metabolism and mitochondrial DNA stability. The pathogenic effect of the mutations was deepened by investigating whether they prevented the correct protein localization into mitochondria or affected the stability of the proteins by determining the steady-state level on mitochondrial protein fraction. All of the Sym1 mutant proteins correctly localized into mitochondria and we demonstrated that only one mutation predominantly affects protein stability. All the other mutations compromised the Sym1 interaction with the other complex components, as demonstrated by its inability to assemble into mature complex and by the consistent fraction of the Sym1 mutant proteins found free or not in fully assembled complex, strengthening its role as protein forming part of a high molecular weight complex. To search for an MDS treatment a phenotypic screening of chemical libraries, designed to identify molecules able to rescue the OXPHOS defective phenotypes of a sym1 mutant strain, has been set up. Several potential active compounds have been found through this approach. The observation that the identified active compounds were able to rescue the oxidative growth defect also in the sym1 null mutant suggested that therapeutic effect acted through a mechanism that bypass Sym1 functions. The effect of the increased dNTPs pool was evaluated by measuring petite mutants frequency of mutant strains showing higher mtDNA stability and suggested that also in yeast the dNTPs pool is defective in sym1 mutant strains. Mutations in YARS2 gene, coding for mitochondrial tyrosyl-tRNA synthetase necessary for a correct mitochondrial protein synthesis, were identified in MLASA patients. The potential pathological role of six missense mutations recently identified was evaluated in yeast model validating their pathological role. In addition, tyrosine supplementation has been observed to be able to rescue respiratory rate in the msy1 mutant strains. QRSL1 and GATB genes code for the subunits A and B respectively of GatCAB amidotransferase complex involved in Gln-tRNA formation. In yeast Gln-tRNA was shown to result from a transamidation pathway, similar to that operating in the human mitochondria. Mutations in QRSL1 and in novel gene GATB were identified in patients affected by cardiomyopathy and lethal infantile lactic acidosis. In yeast phenotypic analyses was performed allowing the validation of the missense mutations as cause of pathology.