A high copy number of yeast γ-glutamylcysteine synthetase suppresses a nuclear mutation affecting mitochondrial translation

Abstract

A new temperature-sensitive nuclear mutant affecting the biogenesis of functional mitochondria has been identified. This pet mutant was formerly characterized by a complete block of mitochondrial translation at the restrictive temperature. The analysis of mitochondrial transcripts demonstrates the accumulation of precursors for the small ribosomal RNA. Transformation of the mutant with plasmids from gene banks identified a chromosomal DNA fragment which can restore growth at the restrictive temperature. A reading frame of 2034 base pairs was found to be responsible for complementation of the mutant phenotype. Sequence analysis identified this gene as the γ-glutamylcysteine synthetase of yeast. This enzyme catalyses the first reaction in the γ-glutamyl cycle for the synthesis of glutathione. Disruption of yeast γ-glutamylcysteine synthetase causes a drastic reduction of growth on glucose medium. The insertion mutants were not able to grow on plates with glycerol as the sole carbon source indicating the special dependence of mitochondria on this substance. Crosses between the pet-ts mutant and the disruption mutant produced diploid cells with a complementation of all their genetic defects indicating that the pet-ts mutation and the insertion mutation are located in different genes. This finding demonstrates that the cloned yeast gene acts as an extragenic suppressor when present on a high-copy-number plasmid inside the pet mutant.