The PUT) gene was isolated by functional complementation of a put) (proline oxidase-deficient) mutation in Saccharomyces cerevisiae. Three independent clones with overlapping inserts of 6.8, 10.5, and 11 kilobases (kb) were isolated from S. cerevisiae genomic libraries in YEp24 (2,um) and YCp5O (CEN) plasmids. The identity of the PUT) gene was determined by a gene disruption technique, and Southern hybridization and genetic analyses confirmed that the bona fide gene had been cloned. Plasmids containing the PUT) gene restored regulated levels of proline oxidase activity to put) recipient strains. The PUT) DNA was present in a single copy in the yeast genome and encoded a transcript of ca. 1.5 kb. Si nuclease protection experiments were used to determine the direction of transcription of the PUT) message and to localize its 5' and 3' termini within a subcloned 3-kb DNA fragment. Approximately 50-fold more PUT)-specific mRNA was detected in induced (proline-grown) cells than in uninduced (ammonia-grown) cells. A yeast strain carrying the previously identified put3 regulatory mutation that caused constitutive levels of proline oxidase activity was found to have sevenfold elevated PUT) mRNA levels under noninducing conditions. The absence of a functional electron transport system in vegetative petite (rho-) strains interfered with their ability to use proline as a nitrogen source. Although these strains were Put-and made no detectable proline oxidase activity, PUT) message was detected under inducing conditions. The PUT) gene was mapped distal to the GAL2 gene on chromosome XII by tetrad analysis.Metabolic conversion of proline to glutamate in Saccharomyces cerevisiae cells permits them to grow on a medium containing proline as the sole source of nitrogen. This two-step pathway occurs inside mitochondria and is catalyzed by the nuclearly encoded, cytoplasmically synthesized, and mitochondrially imported enzymes, proline oxidase and A'-pyrroline-5-carboxylate (P5C) dehydrogenase (7,8,10). The activity levels of both enzymes are induced by proline, while the two transport systems that bring proline into the cell, the proline-specific (PUT4) permease and the general amino acid permease, are regulated not by proline induction but by nitrogen catabolite repression (18).Several mutations in nuclear genes that affect proline utilization have been identified and characterized. Mutations in the PUT] gene result in a deficiency in proline oxidase activity, and those in the PUT2 gene cause a reduction in P5C dehydrogenase activity (7). The put3 mutation results in inducer-independent expression of both of these enzymes (8).The PUT2 gene has been cloned and characterized (6), sequenced (17), and recently identified as the structural gene for P5C dehydrogenase (J. Kaput and M. C. Brandriss, unpublished results). The gene is regulated at the level of RNA by proline, and the increase in enzyme activity seen in the put3 mutant is correlated with an increase in PUT2-specific mRNA in that strain (6).This report describes the isolati...