In yeast it is known that membrane-bound inositol lipids play important roles in adaptation to environmental stresses. More recently it has been shown that soluble inositol polyphosphates, especially inositol pyrophosphates play important roles in diverse cellular processes, such as cell wall maintenance, vacuolar morphogenesis, resistance to salt stress and also mediate homologous DNA recombination in yeast (Luo et al., 2001; Dubois et al., 2002). Arg82p and Kcs1p are two inositol polyphosphate kinases (Saiardi et al., 1999; 2000; York et al., 1999). Arg82p converts Ins(1,4,5)P3 to Ins(1,3,4,5)P4 and Ins(1,4,5,6)P4, which are converted to Ins(1,3,4,5,6)P5. Kcs1p converts Ins(1,3,4,5,6)P5 and InsP6 to different inositol pyrophosphates, PPInsP4, PPInsP5 and (PP)2InsP3 (Saiardi et al., 1999; 2000) (Fig. 1). Cells deleted of ARG82 or KCS1 genes display severe growth defect at high temperature, vacuolar fragmentation, increased leakiness of cellular phosphatase and hypersensitivity to salt stress (Dubois et al., 2002). A strain impaired in Arg82p kinase activity not only presents a strong reduction in Ins(1,3,4,5)P4, Ins(1,4,5,6)P4, Ins(1,3,4,5,6)P5 and InsP6 pools, but also a significant reduction of inositol pyrophosphates, whereas impairing Kcs1p kinase activity only decreases the synthesis of inositol pyrophosphates (Dubois et al., 2002). Consequently, the defects observed in both deleted strains would result from very low amounts of inositol pyrophosphates.
Arg82p plays a second important role in the cell by stabilizing Mcm1p a protein essential for cell viability, and controlling G1/S and G2/M cell cycle transitions (Althoefer et al., 1995; Oehlen et al., 1996; McInerny et al., 1997), mating (Jarvis et al., 1989), osmotolerance (Kuo et al., 1997), recombination (Elble and Tye, 1992), minichromosome maintenance (Passmore et al., 1988) and arginine metabolism (Messenguy and Dubois, 1993). Arg82p stabilizes another MADS-box protein Arg80p, which is also required for the formation of a regulatory complex with the arginine sensor Arg81p, at the ‘arginine boxes’ present in the co-regulated arginine genes (Amar et al., 2000; El Bakkoury et al., 2000). Thus, for the arginine metabolism Arg82p acts as a protein chaperone, and Dubois et al. (2000; 2002) have shown that Arg82p kinase activity was not required for this function. However, it was not established whether the kinase activity of Arg82p was required for Mcm1p-dependent gene expression.
The cell response to diverse stresses requires efficient MAP kinase signalling pathways (Banuett, 1998) which could be perturbed in strains lacking inositol pyrophosphates. To identify the regulatory network of Arg82p and Kcs1p, we conducted genome wide analysis. Among a set of genes whose expression was increased or decreased in strains deleted of ARG82 or KCS1 genes compared to wild-type strain, two families of genes emerged strikingly. In arg82Δ or kcs1Δ cells, genes controlled in response to the quality of the nitrogen source (NCR) were downregulated, whereas genes controlled by phosphate availability (PHO) were upregulated in high phosphate medium. In this study we also show that the control of NCR and PHO genes require the kinase domains of Arg82p and Kcs1p, but not the polyaspartate stretch in Arg82p involved in Mcm1p stabilization.