The roles of PUT3, URE2, and GLN3 regulatory proteins in the proline utilization pathway ofSaccharomyces cerevisiae

Abstract

The yeast Saccharomyces cerevisiae can use alternative nitrogen sources such as allantoin, urea, γ-aminobutyrate, or proline when preferred nitrogen sources such as asparagine, glutamine, or ammonium ions are unavailable in the environment. To use proline as the sole nitrogen source, cells must activate the expression of the proline transporters and the genes that encode the catabolic enzymes proline oxidase (PUT1) and Δ1-pyrroline-5-carboxylate dehydrogenase (PUT2). Transcriptional activation of the PUT genes requires the PUT3 regulatory protein, proline, and relief from nitrogen repression. PUT3 is a 979 amino acid protein that binds a short DNA sequence in the promoters of PUT1 and PUT2, independent of the presence of proline. The functional domains of PUT3 have been studied by biochemical and molecular tests and analysis of activator-constitutive and activator-defective mutant proteins. Mutations in the URE2 gene relieve nitrogen repression, permitting inducer-independent transcription of the PUT genes in the presence of repressing nitrogen sources. The GLN3 protein that activates the expression of many genes in alternative nitrogen source pathways is not required for the expression of the PUT genes under inducing, derepressing conditions (proline) or noninducing, repressing conditions (ammonia). Although it has been speculated that the URE2 protein antagonizes the action of GLN3 in the regulation of many nitrogen assimilatory pathways, URE2 appears to act independently of GLN3 in the proline-utilization pathway. Key words: Saccharomyces cerevisiae, proline utilization, nitrogen repression.

Effect of 21 Different Nitrogen Sources on Global Gene Expression in the YeastSaccharomyces cerevisiae

“…The PUT1 and PUT2 genes (encoding, respectively, proline oxidase and ⌬-1-pyrroline-5-carboxylate dehydrogenase) are involved in proline catabolism and are targets of the Put3 transcription factor activated by intracellular proline (18,35,36,60). As expected, both genes show higher expression on proline than on the other tested media.…”

“…For instance, the UGA1 and UGA4 genes are specifically inducible by GABA (121) and the CAR1 gene by arginine (40). Likewise, DAL4, DAL5, DAL7, DUR1,2, and DUR3 are inducible by allophanate (a product of urea degradation) (25) and PUT1 and PUT2 by proline (18). Other genes in this first category are subject to transcriptional regulations acting on wider sets of genes.…”

“…Thus, several nitrogenous compounds, such as arginine (39), proline (18), serine, threonine (59), urea, allantoin (25), ␥-aminobutyric acid (GABA) (121), and the aromatic amino acids (62), specifically induce the transcription of the genes involved in their utilization. On the other hand, nitrogen catabolite repression (NCR) is typically exerted on the many genes involved in the utilization of nonpreferential nitrogen sources when a good nitrogen source (e.g., ammonium, glutamine, and asparagine) is available in the medium (26,84,136).…”

Crystal structure of a PPR1-DNA complex: DNA recognition by proteins containing a Zn2Cys6 binuclear cluster.

“…This lysine is con served in all but one of the homologous proteins con taining a Zn^CySg region (for review, see Johnston 1987;Andre 1990). The one exception, PUT3, (Brandiss and Magasanik 1979;Brandriss 1987) has a histidine in the corresponding position. Modeling suggests that the his tidine could make compensatory interactions.…”

“…In addition to GAL4 and PPRl, these include PUTS (Brandriss and Magasanik 1979;Brandriss 1987), LEU3 (Zhou et al 1987;Friden et al 1988), and the Kluyveromyces lactis homolog of GAL4, LAC9 (Wray et al 1987;Halvorsen et al 1991). LAC9, which is extremely similar to GAL4 within its linker and dimerization, has naturally occurring sites with spacing identical to those for GAL4 (Wray et al 1987;Halvorsen et al 1991).…”