Proline is an amino acid that is not only required for protein synthesis but can also serve as a nitrogen source. Although proline is the least-preferred nitrogen source for many lab strains of S. cerevisiae, it is the most abundant source of nitrogen in grapes, the natural environment of wild yeast (2). When more optimal sources of nitrogen are unavailable, S. cerevisiae cells degrade proline into glutamate via the proline utilization pathway, shown here (6, 7). In the mitochondria, proline is first converted into delta-1-pyrroline-5-carboxylate (P5C) by the PUT1 gene product, proline oxidase (EC 1.5.99.8). Then, P5C is processed by the delta-1-pyrroline-5-carboxylate dehydrogenase (EC 1.5.1.12) Put2p into glutamate (6).

PUT3 encodes a pathway-specific transcriptional activator that is responsible for regulating PUT1 and PUT2 expression (7). It is a member of the Zn(II)2Cys6 family of transcription factors that has thus far been identified exclusively in fungi (8, 9). As a dimer, Put3p asymmetrically binds to a 16-bp promoter sequence, CGG-N₁₀-CCG, called UAS_PUT (10, 1). Although Put3p is constitutively bound to the PUT1 and PUT2 promoters, it is maximally active for upregulation of the PUT genes only in the presence of proline and in the absence of preferred sources of nitrogen (2, 11). Put3p regulates transcription by undergoing differential phosphorylation as a function of nitrogen source quality--hyperphosphorylation of Put3p is correlated with growth on non-preferred nitrogen sources (2). Additionally, epitope-tagging and proteolysis experiments indicate that Put3p also undergoes conformational changes in response to proline (5). Mutants lacking Put3p activity are unable to grow on proline as their sole nitrogen source (4). However, there is experimental evidence indicating that in the absence of Put3p, the Gal4p transcriptional activator is able to bind to the UAS_PUT and upregulate PUT2 expression (12).

Last updated: 2005-09-07