Characterization of pleiotropic pet mutants defective in regulation of mitochondrial respiration and mitochondrial protein synthesis

Yeast mitochondrial respiratory activity is repressed by glucose and activated in the absence of fermentable carbon sources. I studied the pet mutants displaying pleiotropic defects in the respiratory chain to learn more about the regulatory pathways. In two of the mutants, I identified genes that provide new connections between glucose repression and mitochondrial respiration.; One of the mutants E153, harbors a mutation leading to an H55Y change in the iron binding site of the protein phosphatase Sit4p. The mitochondrial cytochrome spectrum and the expression of COX5a-lacZ indicated that the sit4 mutant escaped glucose repression. Therefore, SIT4 gene can regulate mitochondrial respiratory activity both positively and negatively. Because Sit4p is a cytoplasmic protein, and the Sit4p level remains unchanged under different growth conditions, it must regulate mitochondrial respiration through a signal transduction pathway. Microarray analysis revealed the escape of glucose repression in the sit4 mutant is the result of increased mRNA for enzymes of carbohydrate metabolism and mitochondrial respiration. The mRNA levels of many genes controlled by Mig1p, the major transcriptional repressor in the glucose repression pathway, are greatly increased in sit4 mutant grown in glucose. In the mutant, the Mig1p level is greatly reduced but MIG1 mRNA abundance is not affected. Therefore, Sit4p must affect glucose repression by controlling translation or turnover of Mig1p.; Ssn6p is another multifunctional protein participating in both glucose repression and respiratory growth. In the pet mutant E359, which also showed a respiratory growth defect, the mutation was localized to SSN6. This mutation leads to a G157D change in the fourth TPR of Ssn6p required for the Ssn6p-Rox1p interaction. Rox1p represses expression of hypoxic genes including those related to mitochondrial respiration. Because each TPR contributes to the overall protein folding, this mutation could also alter the interactions between Ssn6p and other proteins such as Mig1p. CYR1, the gene encoding adenylate cyclase, has been implicated in the regulation of glucose repression and mitochondrial respiration. The mechanisms underlying these processes are unknown. Overexpression of CYR1 or of the C terminal catalytic domain and the Srv2p binding domain of Cyr1p can suppress the growth defect of the ssn6G157D mutant. These findings indicate a link between the cAMP pathway, glucose repression, and mitochondrial respiration.