NPR1 Kinase and RSP5-BUL1/2 Ubiquitin Ligase Control GLN3-dependent Transcription in Saccharomyces cerevisiae

Abstract

The GATA transcription factors GLN3 and GAT1 activate nitrogen-regulated genes in Saccharomyces cerevisiae. NPR1 is a protein kinase that controls post-Golgi sorting of amino acid permeases. In the presence of a good nitrogen source, TOR (target of rapamycin) maintains GLN3 and NPR1 phosphorylated and inactive by inhibiting the type 2A-related phosphatase SIT4. We identified NPR1 as a regulator of GLN3. Specifically, loss of NPR1 causes nuclear translocation and activation of GLN3, but not GAT1, in nitrogen-rich conditions. NPR1-mediated inhibition of GLN3 is independent of the phosphatase SIT4. We also demonstrate that the E3/E4 ubiquitin-protein ligase proteins RSP5 and BUL1/2 are required for GLN3 activation under poor nitrogen conditions. Thus, NPR1 and BUL1/2 antagonistically control GLN3-dependent transcription, suggesting a role for regulated ubiquitination in the control of nutrient-responsive transcription.In yeast, the quality of the available nitrogen source controls the expression of genes encoding proteins required for the uptake and assimilation of nitrogenous compounds. This regulatory process is mediated by four GATA transcription factors, the activators GLN3 and GAT1/NIL1 and the repressors DAL80 and DEH1/NIL2/GZF3. GLN3 is the major activator of nitrogen-regulated genes (for recent reviews, see Refs. 1 and 2). URE2 is a GLN3-binding protein that retains GLN3 in the cytoplasm to prevent its nuclear translocation and activation during growth under good nitrogen conditions.The two homologous kinases TOR1 1 and TOR2 are central controllers of cell growth, and their inactivation with rapamycin causes physiological changes characteristic of nutrientstarved cells (3, 4). A TOR signaling pathway regulates the expression of nitrogen-regulated genes by inhibiting GLN3 and GAT1 (5). In the presence of a preferred nitrogen source such as NH 4 ϩ or glutamine, TOR promotes complex formation between GLN3 and URE2 by maintaining GLN3 in the phosphorylated state necessary for URE2 binding (5). Upon nitrogen limitation or rapamycin treatment, the TOR-controlled phosphatase SIT4 causes GLN3 dephosphorylation, and GLN3 dissociates from URE2 and translocates into the nucleus to activate target genes (5).NPR1 encodes a Ser/Thr kinase that controls the post-Golgi sorting and degradation of amino acid permeases. NPR1 inversely regulates GAP1, the general amino acid permease, and TAT2, a tryptophan permease (2). In cells grown in poor nitrogen sources, NPR1 is dephosphorylated by the TOR-modulated SIT4 phosphatase to become active (6). In turn, active NPR1 allows GAP1 to reach the plasma membrane and causes the rapid degradation of TAT2. In cells shifted to a good nitrogen source medium, plasma membrane GAP1 is internalized and pre-existing permease is no longer delivered to the plasma membrane, whereas TAT2 is stabilized at the plasma membrane (7-9). GAP1 down-regulation occurs via its ubiquitination by the E3 ubiquitin protein ligase RSP5 in conjunction with the RSP5-binding proteins BUL1 and BUL2 (8,10,11).Here, we ...

Regulation of Amino Acid, Nucleotide, and Phosphate Metabolism in Saccharomyces cerevisiae

“…Consistent with this notion, there is accumulating evidence that rapamycin exerts its effects in a manner that does not faithfully mimic nitrogen starvation (Cox et al 2004;Crespo et al 2004;Kulkarni et al 2006;Georis et al 2008Georis et al , 2009aTate et al 2009Tate et al , 2010. For example, in direct opposition to rapamycin treatment, a functional myc-tagged Gln3 construct becomes hyperphosphorylated during nitrogen and carbon starvation (Cox et al 2004;Kulkarni et al 2006), and the phosphorylation status of Gln3 does not affect its ability to bind Ure2 (Bertram et al 2000).…”

“…There are several examples where this has been problematic. For example, in ammonium-grown cells, the mutational inactivation of NPR1 results in Gln3-dependent derepression of NCR-sensitive genes (Crespo et al 2004). The kinase activity of Npr1 is required for proper post-transcriptional control of several ammonium-sensitive permeases (Boeckstaens et al 2007).…”

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“…Instead, damaged membrane proteins are targeted for endocytosis and pass through the multivesicular body (MVB) and to the lysosome (in yeast, the vacuole), where they are eventually degraded (Piper & Luzio, 2007; Zhao et al ., 2013). Bul1 plays an important role in this plasma membrane quality control system by facilitating the Rsp5 ‐ dependent polyubiquitination of multiple protein targets which directs their sorting to the vacuole for degradation (Yashiroda et al ., 1996; De Craene et al ., 2001; Helliwell et al ., 2001; Crespo et al ., 2004; Merhi & André, 2012; O'Donnell, 2012; Zhao et al ., 2013). Disruption of this membrane protein quality control pathway has been previously linked to accelerated aging (Fabrizio et al ., 2010), while the Rsp5/Bul1 node, in particular, has been shown to ameliorate the effects of protein aggregation in a model of neurodegenerative diseases (Tardiff et al ., 2013).…”

“…Gln3 is a transcription factor that regulates nitrogen metabolism, is directly regulated by both TOR and Bul1, and is located on chromosome 5. During growth on a nonpreferred nitrogen source, Bul1 and its functionally similar binding partner Bul2 are required for nuclear translocation of Gln3 (Crespo et al ., 2004). Emphasizing the complexity of nutrient signaling, however, the inhibition of TOR by rapamycin treatment activates Gln3 via a mechanism that is not dependent on Bul1/Bul2 (Crespo et al ., 2004).…”

“…During growth on a nonpreferred nitrogen source, Bul1 and its functionally similar binding partner Bul2 are required for nuclear translocation of Gln3 (Crespo et al ., 2004). Emphasizing the complexity of nutrient signaling, however, the inhibition of TOR by rapamycin treatment activates Gln3 via a mechanism that is not dependent on Bul1/Bul2 (Crespo et al ., 2004). Bul1/Bul2 have additional roles in nutrient signaling upstream of Gln3 nuclear translocation.…”