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.

The target of rapamycin protein (TOR) is a highly conserved ataxia telangiectasia-related protein kinase essential for cell growth. Emerging evidence indicates that TOR signaling is highly complex and is involved in a variety of cellular processes. To understand its general functions, we took a chemical genomics approach to explore the genetic interaction between TOR and other yeast genes on a genomic scale. In this study, the rapamycin sensitivity of individual deletion mutants generated by the Saccharomyces Genome Deletion Project was systematically measured. Our results provide a global view of the rapamycin-sensitive functions of TOR. In contrast to conventional genetic analysis, this approach offers a simple and thorough analysis of genetic interaction on a genomic scale and measures genetic interaction at different possible levels. It can be used to study the functions of other drug targets and to identify novel protein components of a conserved core biological process such as DNA damage checkpoint͞repair that is interfered with by a cell-permeable chemical compound.

Carbon and nitrogen are two basic nutrient sources for cellular organisms. They supply precursors for energy metabolism and metabolic biosynthesis. In the yeast Saccharomyces cerevisiae, distinct sensing and signaling pathways have been described that regulate gene expression in response to the quality of carbon and nitrogen sources, respectively. Gln3 is a GATA-type transcription factor of nitrogen catabolite-repressible (NCR) genes. Previous observations indicate that the quality of nitrogen sources controls the phosphorylation and cytoplasmic retention of Gln3 via the target of rapamycin (TOR) protein. In this study, we show that glucose also regulates Gln3 phosphorylation and subcellular localization, which is mediated by Snf1, the yeast homolog of AMP-dependent protein kinase and a cytoplasmic glucose sensor. Our data show that glucose and nitrogen signaling pathways converge onto Gln3, which may be critical for both nutrient sensing and starvation responses.Carbon and nitrogen are the two most basic nutrient sources for cellular organisms. They are used to produce energy and synthesize a wide range of biomolecules. Energy metabolism and metabolic biosynthesis are carried out by some 500 individual chemical reactions, which are well organized along the centrally placed glycolysis and tricarboxylic acid (TCA) cycle (1). Individual reactions are well calibrated by a feedback regulation, which fine-tunes the flux of metabolites through a particular pathway by temporarily increasing or decreasing the activity of crucial enzymes. In response to the quality of carbon and nitrogen, cells can also regulate the expression of genes involved in different metabolic pathways, particularly those involved in utilization and transport of the available nutrients.

Gln3p is a GATA-type transcription factor responsive to the quality of nitrogen and carbon. In preferred nitrogen such as glutamine, Gln3p is phosphorylated and sequestered in the cytoplasm in a manner that is dependent on the target of rapamycin (TOR) protein and Ure2p. In nonpreferred nitrogen or nitrogen starvation, Gln3p is dephosphorylated and imported into the nucleus via karyopherin ␣/Srp1p. Upon reintroduction of preferred nitrogen, Gln3p is exported from the nucleus by Crm1p/Xpo1p. Although recent work has provided insights into Gln3p, a more detailed understanding is needed to elucidate the mechanism of its localization and function. In this study, we show that Gln3p contains canonical nuclear localization signal and nuclear export signal sequences necessary for its localization and interaction with its relevant karyopherins. In addition, we identify an N-terminal domain of Gln3p interacting with Ure2p and a C-terminal region for binding to TOR. Finally, we find a lysine/arginine-rich domain essential for the rapamycin-sensitive function, but dispensable for its localization. Our results reveal key domains of Gln3p important for its function and regulation.Carbon and nitrogen are two basic nutrient sources used by eukaryotic cells to produce energy and synthesize a wide range of biomolecules important for cell growth and proliferation. In response to the quality of carbon and nitrogen, cells can regulate the expression of genes involved in different metabolic pathways. A classic example is nitrogen catabolite repression (NCR)

CLIP-170/Restin belongs to a family of conserved microtubule (MT)-