Abstract
Gln3p is a GATA-type transcription factor responsive to different nitrogen nutrients and starvation in yeast Saccharomyces cerevisiae. Recent evidence has linked TOR signaling to Gln3p. Rapamycin causes dephosphorylation and nuclear translocation of Gln3p, thereby activating nitrogen catabolite repressible-sensitive genes. However, a detailed mechanistic understanding of this process is lacking. In this study, we show that Tor1p physically interacts with Gln3p. An intact TOR kinase domain is essential for the phosphorylation of Gln3p, inhibition of Gln3p nuclear entry and repression of Gln3p-dependent transcription. In contrast, at least two distinct protein phosphatases, Pph3p and the Tap42p-dependent phosphatases, are involved in the activation of Gln3p. The yeast pro-prion protein Ure2p binds to both hyper- and hypo-phosphorylated Gln3p. In contrast to the free Gln3p, the Ure2p-bound Gln3p is signifcantly resistant to dephosphorylation. Taken together, these results reveal a tripartite regulatory mechanism by which the phosphorylation of Gln3p is regulated.
Highlights
Yeast cells have sophisticated transcriptional programs to regulate the expression of genes encoding proteins responsible for the transport and degradation of small nitrogenous compounds in response to different nitrogen nutrients
Cells respond to the preferred nitrogen sources by decreasing the expression of genes associated with transport and degradation of poor nitrogen sources
We show that TOR binds to Gln3p via its HEAT domain
Summary
Yeast cells have sophisticated transcriptional programs to regulate the expression of genes encoding proteins responsible for the transport and degradation of small nitrogenous compounds in response to different nitrogen nutrients. Glutamine prevents the expression of GLN1 and GAP1, structural genes for glutamine synthetase and the general amino acid permease Their expression is derepressed with glutamate as the sole nitrogen source. Gln3p activity is inhibited by Ure2p, a yeast pro-prion protein in the presence of preferred nitrogen sources (1, 2). TOR signaling to NCR genes has been linked to the GATA-type transcription factor Gln3p and its inhibitor Ure2p (17–19). Rapamycin causes rapid dephosphorylation and nuclear entry of Gln3p (18) It is not clear what the role of TOR is with regard to the regulation of Gln3p. The Tap42p1⁄7Sit4p protein phosphatase complex has been suggested to mediate TOR signaling to Gln3p (18), there is evidence indicating that it is not involved (19). A tripartite regulatory mechanism is involved in governing the repression and activation of Gln3p
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