Academic journal article Genetics

Sit4 and PP2A Dephosphorylate Nitrogen Catabolite Repression-Sensitive Gln3 When TorC1 Is Up- as Well as Downregulated

Academic journal article Genetics

Sit4 and PP2A Dephosphorylate Nitrogen Catabolite Repression-Sensitive Gln3 When TorC1 Is Up- as Well as Downregulated

Article excerpt

Saccharomyces cerevisiae lives in an ever-changing and, at times, hostile, boom and bust nutritional environment. Sophisticated regulatory systems have evolved to rapidly cope with these changes while preserving intracellular homeostasis. Target of Rapamycin Complex 1 (TorC1), is a serine/threonine kinase complex that is activated/upregulated by excess nitrogen, and inhibited/downregulated by limiting nitrogen growth conditions (Figure 1) (Beck and Hall 1999; Hughes Hallett et al. 2014; González and Hall 2017). Two of the many downstream targets regulated by TorC1 are Gln3 and Gat1-GATA-family transcription activators responsible for expression of many Nitrogen Catabolite Repression- (NCR-) sensitive genes (Cooper 1982; Beck and Hall 1999; Cardenas et al. 1999; Hardwick et al. 1999; Bertram et al. 2000). Among these genes are most of those that function to transport, degrade, and interconvert poor nitrogen sources scavenged from adverse nitrogen environments (Cooper 1982, 2002).

In a nitrogen-replete environment, Gln3 is cytoplasmic (bound in a Gln3-Ure2 complex) and NCR-sensitive transcription is repressed (Figure 1). In nitrogen-limiting conditions, Gln3 relocates to the nucleus and activates/ derepresses transcription (Hofman-Bang 1999; Magasanik and Kaiser 2002; Cooper 2004; Broach 2012; Ljungdahl and Daignan-Fornier 2012; Conrad et al. 2014; Swinnen et al. 2014; González and Hall 2017; Zhang et al. 2018). In the presence of excess nitrogen, TorC1 is active, binds to, and phosphorylates, Tor Associated Protein 42 (Tap42). Phosphorylated PTap42 in turn forms complexes with the Sit4 and PP2A phosphatases (Figure 1) (DiComo and Arndt 1996; Jiang and Broach 1999). PTap42-Sit4 and PTap42PP2A complexes bound to TorC1 are enzymatically inactive (Yan et al. 2006). TorC1 also phosphorylates Gln3 in vitro and an intact TorC1 kinase domain is required to sequester Gln3 within the cytoplasm (Figure 1) (Bertram et al. 2000). PP2A is a heterotrimer, Pph21/22-Tpd3-Ccd55/Rts1, in which Pph21/22 are redundant catalytic subunits (Zabrocki et al. 2002; Zaman et al. 2009).

When TorC1 is inhibited with rapamycin, or nitrogenlimitation/starvation, the PTap42-Sit4 and PTap42-PP2A complexes dissociate from TorC1 (Figure 1) (Wang et al. 2003; Yan et al. 2006). Thus freed, they dephosphorylate Gln3, which dissociates from the cytoplasmic Gln3-Ure2 complex, enters the nucleus, and activates NCR-sensitive transcription (Figure 1) (Blinder et al. 1996; Beck and Hall 1999; Cardenas et al. 1999; Hardwick et al. 1999; Bertram et al. 2000; Kulkarni et al. 2001). Treating cells with the glutamine synthetase inhibitor methionine sulfoximine (Msx) also elicits nuclear Gln3 localization (Crespo et al. 2002; Kulkarni et al. 2006; Georis et al., 2011a; Tate and Cooper 2013).

The requirements of Sit4 and PP2A for nuclear Gln3 localization, however, are dependent on how nitrogen limiting conditions are established (Tate and Cooper 2013). For rapamycin-treated, glutamine-grown cells, both Sit4 and PP2A are required (Beck and Hall 1999; Bertram et al. 2000; Tate et al. 2006a, 2009, 2010; Tate and Cooper 2013). Parenthetically, even though Sit4 can dephosphorylate Gln3 in rapamycin-treated, glutamine-grown cells lacking PP2A, Gln3 cannot enter the nucleus (Tate et al. 2009). In limiting nitrogen or short-term nitrogen starvation (1-4 hr depending on the strain assayed), only Sit4 is required (Tate and Cooper 2013). Finally, neither Sit4 nor PP2A is required for nuclear Gln3 localization after long-term nitrogen starvation that correlates with G-1 arrest of the cell cycle (~8-10 hr of starvation), or in cells treated with Msx (Tate and Cooper 2013).

The course of Gln3 once it is in the nucleus correlates with the glutamine concentration (Rai et al. 2015). When glutamine levels are highest, i.e., when glutamine is the sole nitrogen source, or a glutamine analog (GAGM, L-glutamic acid-g-monohydroxamate) is provided, Gln3 can exit from the nucleus in the absence of binding to its GATA-target sequences situated in the promoters of NCR-sensitive genes (Rai et al. …

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