A domain in the transcription activator Gln3 specifically required for rapamycin responsiveness.

Nitrogen-responsive control of Gln3 localization is implemented through TorC1-dependent (rapamycin-responsive) and TorC1-independent (nitrogen catabolite repression-sensitive and methionine sulfoximine (Msx)-responsive) regulatory pathways. We previously demonstrated amino acid substitutions in a putative Gln3 α-helix(656-666), which are required for a two-hybrid Gln3-Tor1 interaction, also abolished rapamycin responsiveness of Gln3 localization and partially abrogated cytoplasmic Gln3 sequestration in cells cultured under nitrogen-repressive conditions. Here, we demonstrate these three characteristics are not inextricably linked together. A second distinct Gln3 region (Gln3(510-589)) is specifically required for rapamycin responsiveness of Gln3 localization, but not for cytoplasmic Gln3 sequestration under repressive growth conditions or relocation to the nucleus following Msx addition. Aspartate or alanine substitution mutations throughout this region uniformly abolish rapamycin responsiveness. Contained within this region is a sequence with a predicted propensity to form an α-helix(583-591), one side of which consists of three hydrophobic amino acids flanked by serine residues. Substitution of aspartate for even one of these serines abolishes rapamycin responsiveness and increases rapamycin resistance without affecting either of the other two Gln3 localization responses. In contrast, alanine substitutions decrease rapamycin resistance. Together, these data suggest that targets in the C-terminal portion of Gln3 required for the Gln3-Tor1 interaction, cytoplasmic Gln3 sequestration, and Gln3 responsiveness to Msx addition and growth in poor nitrogen sources are distinct from those needed for rapamycin responsiveness.