The GATA transcription activator Gln3 in the budding yeast (Saccharomyces cerevisiae) activates transcription of nitrogen catabolite repression (NCR)-sensitive genes. In cells grown in the presence of preferred nitrogen sources, Gln3 is phosphorylated in a TOR-dependent manner and localizes in the cytoplasm. In cells grown in non-preferred nitrogen medium or treated with rapamycin, Gln3 is dephosphorylated and is transported from the cytoplasm to the nucleus, thereby activating the transcription of NCR-sensitive genes. Caffeine treatment also induces dephosphorylation of Gln3 and its translocation to the nucleus and transcription of NCR-sensitive genes. However, the details of the mechanism by which phosphorylation controls Gln3 localization and transcriptional activity are unknown. Here, we focused on two regions of Gln3 with nuclear localization signal properties (NLS-K, and NIS-C) and one with nuclear export signal (NES). We constructed various mutants for our analyses: gln3 containing point mutations in all potential phosphoacceptor sites (Thr-339, Ser-344, Ser-347, Ser-355, Ser-391) in the NLS and NES regions to produce non-phosphorylatable (alanine) or mimic-phosphorylatable (aspartic acid) residues; and deletion mutants. We found that phosphorylation of Gln3 was impaired in all of these mutations and that the aspartic acid substitution mutants showed drastic reduction of Gln3mediated transcriptional activity despite the fact that the mutations had no effect on nuclear localization of Gln3. Our observations suggest that these regions are required for transcription of target genes presumably through dephosphorylation. (C) 2014, The Society for Biotechnology, Japan. All rights reserved.