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 (3). When more optimal sources of nitrogen are unavailable, S. cerevisiae cells degrade proline into glutamate via the proline utilization pathway, shown here (4, 5). 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 (4).

Proline uptake is carried out mainly by two nitrogen-regulated permeases, the general amino acid permease Gap1p (6) and the high-affinity proline permease Put4p (7). The PUT4 gene product facilitates the import not only of proline, but of alanine and glycine as well (8). Put4p also mediates the transport of gamma-amino butyric acid (9, 10) and the toxic proline analog azetidine-2-carboxylate (2). Null mutations in PUT4 do not prevent growth on high concentrations of proline due to proline uptake by low affinity transport systems such as Gap1p (11). In contrast, mutations in the cytoplasmic N-terminal region of Put4p impede its degradation, leading to increased alanine, glycine, and proline uptake during beer brewing (12). Because proline is not normally taken up during fermentation (1), this results in an increased fermentation rate without compromised beer quality (12).

PUT4 is regulated at the level of transcription by nitrogen catabolite repression (NCR) (13, 14), which prevents the utilization of proline as a nitrogen source if better nitrogen compounds such as ammonia, asparagine or glutamine are present. PUT4 downregulation by NCR is mediated by the transcription factors Ure2p and Dal80p (1, 15, 16). In the presence of proline and the absence of a preferred nitrogen source, NCR is released by the transcriptional activator Gln3p and the co-activator Hfi1p (16, 17). Put4p is also regulated posttranslationally. The gene products of SEC13, LST4, LST7, are LST8 are necessary for proper localization of Put4p from the Golgi to the plasma membrane (18). Additionally, the kinases Npr1p and Pho85p are required for proline permease activation, although direct phosphorylation of Put4p by these enzymes has yet to be shown (19, 1, 20). Put4p is also regulated through ubiquitination and subsequent degradation mediated by the Rsp5p ubiquitin protein ligase and the Doa4p ubiquitin isopeptidase (21, 22, 23).

Last updated: 2005-09-07