The regulator of the yeast proline utilization pathway is differentially phosphorylated in response to the quality of the nitrogen source.

TLDR: There are two signals, proline induction and quality of nitrogen source, impinging on Put3p that act synergistically for maximum expression of the proline utilization pathway, and this report demonstrates by phosphatase treatment of immunoprecipitates of extracts metabolically labeled with32P or 35S that Put3P is a phosphoprotein.

Abstract: Saccharomyces cerevisiae cells can sense the quality of the nitrogen source in their environment, enabling them to utilize preferred nitrogen-containing compounds over nonpreferred ones or to express pathways for the utilization of alternative nitrogen sources when the preferred ones have been consumed. Although very little is known about the sensing mechanism itself, work over the last decade has led to the discovery of a set of regulatory proteins, the GATA factors, whose role is to regulate, in both positive and negative directions, the expression of pathways of nitrogen assimilation in yeast. These proteins, Gln3p (26), Nil1p/Gat1p (10, 44), Dal80p/Uga43p (12, 13), and Nil2p/Gzf3p/Deh1p (11, 34, 42), are involved in a complex set of regulatory loops, competition for GATA binding sites, and possibly even some autoregulation. Recently, the coactivator Ada1p, isolated as Gan1p, was identified as a link between the GATA binding proteins and the basal transcriptional machinery (41). Global nitrogen repressor Ure2p is believed to interact with Gln3p to obtain appropriate expression of a variety of nitrogen assimilatory pathways (3; reviewed by Magasanik [23]). In their natural habitat, S. cerevisiae cells are found on grapes and in grape must, a nitrogen-poor environment where the most abundant nitrogen source is proline (2). Although proline is the least-preferred nitrogen source for many laboratory yeast strains and although its utilization results in the slowest growth rates, yeast cells have evolved a regulatory circuit that enables them to use the proline in the environment when preferred nitrogen sources are no longer available. The flux of proline into yeast cells is controlled by the activities of the general amino acid permease Gap1p and the proline-specific permease, Put4p (21). These permeases are regulated by nitrogen repression and do not respond to proline induction (17, 21, 43). The enzymes of the proline utilization pathway are induced by the presence of proline (6), and their levels reflect internal proline levels. The PUT1 and PUT2 genes encoding the enzymes of the pathway are regulated by Put3p, a member of the Zn(II)2Cys6 binuclear cluster protein family (4, 6, 7, 15, 24, 25, 40, 45, 49) and a close relative of Gal4p, the activator of the galactose utilization pathway. In vivo, Put3p binds the promoters of PUT1 and PUT2 in the presence or absence of proline and without regard to the quality of the nitrogen sources present in the growth medium (1) but activates transcription to a maximum level when proline is the sole source of nitrogen. PUT1 and PUT2 are repressed by Ure2p and in some, but not all, strain backgrounds are regulated by some of the GATA factors (9, 14, 50). This report presents the results of studies on wild-type and regulation-defective mutant Put3 proteins in cells grown in media containing different nitrogen sources. We show that Put3p is differentially phosphorylated as a function of the quality of the nitrogen source and that the slowest-migrating species of Put3p are correlated with elevated transcriptional activity. Analysis of the Put3p phosphoforms of activator-defective and activator-constitutive mutants leads to the suggestion that altered phosphorylation status may be one of two signals (proline induction being the other) that is required for maximum transcriptional activity by Put3p.