The goal of this study is to demonstrate turnover of the mitochondrial compartment in Saccharomyces. Our lab has identified nine complementation groups of mutations, yme (yeast mitochondrial escape) that cause a high rate of mtDNA escape compared to wild-type (Genetics 134:21-28; 1993). One of these, yme1, showed lower mtDNA escape when coupled with a disruption of the vacuolar proteinase A gene, PEP4. Strains yme2-yme9, also bearing a pep4 disruption, showed no detectable differences in mtDNA escape. A biochemical system was developed to assay for vacuole-mediated mitochondrial turnover. The vacuolar alkaline phosphatase (ALP) gene, PHO8, was localized to mitochondria in a strain bearing disruption to both genomic ALP genes. Western analysis confirmed that mALP was in the purified mitochondrial fraction. Pep4-dependent mALP activities were expected to reflect vacuole-mediated mitochondrial turnover. A yme1 strain exhibited significantly higher mALP activity than wild-type when grown in a nonfermentable carbon source but not in glucose or raffinose media. The wild-type strain displayed relative ALP activities that parallel the expected requirements for mitochondrial function in a given carbon source. This biochemical system was also used to determine if glucose catabolite inactivation induces vacuole-mediated turnover of mitochondria in wild-type yeast.