Domains of Gln3p Interacting with Karyopherins, Ure2p, and the Target of Rapamycin Protein

Abstract

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)

Nuclear translocation of Gln3 in response to nutrient signals requires Golgi-to-endosome trafficking in Saccharomyces cerevisiae

“…Our results were in accord with the observed localization of these Gln3 mutants reported previously (27). Gln3 lacking the initial 200 amino acids (except construct 5) predominantly localized to the nucleus (27). Accordingly, these Gln3 mutants were recovered in the P13 and P100 membrane fractions (Fig.…”

“…Structure-function analyses of Gln3 have identified specific sequences for nuclear localization, nuclear export, and interaction with Ure2 and Tor1 (26,27). We find that a fraction of Ure2, expressed from its endogenous locus or from a low-copy centromeric plasmid to improve detection, also is tightly associated with light membranes and resistant to salt extraction (Fig.…”

“…However, deletion of the Ure2-binding domain of Gln3 did not affect Gln3 interaction with these membranes. Similarly, we excluded the possibility that the Tor1-Gln3 interaction previously characterized mediates Gln3 light membrane association (27).…”

“…Mutant yeast strains were constructed by PCR-mediated gene disruption as described previously (36,37), and gene deletions were PCR confirmed. The pRS315-GLN3-Myc 9 deletion alleles and the centromeric plasmid pVTG20 for Ure2 expression were obtained from Steven Zheng (27) and Reed Wickner (38).…”

“…Tor1 regulates Gln3 by promoting its association with Ure2 (3, 4). In addition, an interaction of Tor1 with Gln3 has been reported (27). Thus, it was important to test whether the Ure2-and Tor1-binding domains mediate Gln3 interaction with light membranes.…”