Roles ofURE2andGLN3in the Proline Utilization Pathway inSaccharomyces cerevisiae

The yeast Saccharomyces cerevisiae can use alternative nitrogen sources such as arginine, urea, allantoin, g-aminobutyrate, or proline when preferred nitrogen sources like glutamine, asparagine, or ammonium ions are unavailable in the environment. Utilization of alternative nitrogen sources requires the relief of nitrogen repression and induction of specific permeases and enzymes. The products of theGLN3andURE2genes are required for the appropriate transcription of many genes in alternative nitrogen assimilatory pathways. GLN3 appears to activate their transcription when good nitrogen sources are unavailable, and URE2 appears to repress their transcription when alternative nitrogen sources are not needed. The participation of nitrogen repression and the regulators GLN3 and URE2 in the proline utilization pathway was evaluated in this study. Comparison of PUT gene expression in cells grown in repressing or derepressing nitrogen sources, in the absence of the inducer proline, indicated that both PUT1 and PUT2 are regulated by nitrogen repression, although the effect onPUT2is comparatively small. Recessive mutations inURE2elevated expression of the PUT1andPUT2genes 5- to 10-fold when cells were grown on a nitrogen-repressing medium. Although PUT3, the proline utilization pathway transcriptional activator, is absolutely required for growth on proline as the solenitrogensource,aput3ure2strainhadsomewhatelevatedPUTgeneexpression,suggestinganeffectofthe ure2mutation in the absence of thePUT3product.PUT1andPUT2gene expression did not require the GLN3 activator protein for expression under either repressing or derepressing conditions. Therefore, regulation of thePUTgenesbyURE2doesnotrequireafunctionalGLN3protein.Theeffectoftheure2mutationonthePUT genes is not due to increased internal proline levels. URE2 repression appears to be limited to nitrogen assimilatory systems and does not affect genes involved in carbon, inositol, or phosphate metabolism or in mating-type control and sporulation. The proline utilization pathway in Saccharomyces cerevisiae enables cells to use proline as the sole source of nitrogen when preferred nitrogen sources are not available in the environment. The proline utilization enzymes proline oxidase and D 1 -pyrroline-5-carboxylate dehydrogenase, encoded by the nuclear genes PUT1 and PUT2, respectively, convert proline to glutamate in mitochondria (9‐11). The expression of thePUT genes is regulated by the PUT3 activator protein, which responds to the presence of proline in the medium and increases the transcription of thePUT1andPUT2genes (7, 8, 67). The PUT3 protein constitutively binds to the upstream activation sequences in the promoters of bothPUT1andPUT2genes in vitroandinvivobutactivatestranscriptiononlyinthepresence of proline (2, 44, 45, 58). Early studies on the proline utilization pathway concluded that the structural genes PUT1 and PUT2 were not regulated by nitrogen repression (10). We speculated that the proline transporters were inactive in the presence of good nitrogen sources and proline could not enter the cell, a conclusion subsequently proved by Jauniaux et al. (35). From our measurementsoflowprolineoxidaseactivityincellsgrownonurea