A remarkable characteristic of nutritional homeostatic mechanisms is the breadth of metabolite concentrations to which they respond and resolution of those responses; adequate but rarely excessive. Two general ways of achieving such exquisite control are known: stoichiometric mechanisms where increasing metabolite concentrations elicit proportionally increasing responses, and the actions of multiple independent metabolic signals that cumulatively generate appropriately measured responses. Intracellular localization of the nitrogen-responsive transcription activator, Gln3 responds to four distinct nitrogen environments: nitrogen limitation or short-term starvation, i.e. nitrogen catabolite repression (NCR), long-term starvation, glutamine starvation and rapamycin inhibition of mTorC1. We have previously identified unique sites in Gln3 required for rapamycin-responsiveness and Gln3-mTor1 interaction. Alteration of the latter results in loss of about 50% of cytoplasmic Gln3 sequestration. However, except for the Ure2-binding domain, no evidence exists for a Gln3 site responsible for the remaining cytoplasmic Gln3-Myc13 sequestration in nitrogen excess. Here we identify a serine/threonine-rich (Gln3477-493) region required for effective cytoplasmic Gln3-Myc13 sequestration in excess nitrogen. Substitutions of alanine but not aspartate for serines in this peptide partially abolish cytoplasmic Gln3 sequestration. Importantly, these alterations have no effect on Gln3-Myc13’s responses to rapamycin, methionine sulfoximine or limiting nitrogen. However, cytoplasmic Gln3-Myc13 sequestration is additively and almost completely abolished when mutations in the Gln3-Tor1 interaction site are combined with those in Gln3477-493 cytoplasmic sequestration site. These findings clearly demonstrate that multiple individual regulatory pathways cumulatively control cytoplasmic Gln3 sequestration. Supported by NIH grant GM-35642-26.