Genes for the enzymes that metabolize galactose in Saccharomyces cerevisiae are strongly induced by galactose and tightly repressed by glucose. Because glucose also represses mitochondrial activity, we examined if derepression of the GAL] galactokinase gene requires physiologically active mitochondria. The effect of mitochondria on the expression of GAL1 was analyzed by a novel approach in which the activity of the organelles was altered by functional expression of URF13, a mitochondrial protein unique to the Texas-type cytoplasmic male sterility phenotype in maize. Mitochondrial targeting and functional expression of the URF13 protein in yeast result in a decrease of the mitochondrial membrane potential similar to those observed in cells treated with mitochondrial inhibitors such as antimycin A or sodium azide. Activation of URF13 in galactose-induced cells results in the inhibition of GAL1 expression in the absence of repressing concentrations of glucose. Our data reveal the existence of a regulatory pathway that connects the derepression of the GAL1 gene with mitochondrial activity. (c) 2005 Elsevier Inc. All rights reserved.