Five conditions commonly used to down-regulate tor complex 1 generate different physiological situations exhibiting distinct requirements and outcomes.
Five different physiological conditions have been used interchangeably to establish the sequence of molecular events needed to achieve nitrogen-responsive down-regulation of TorC1 and its subsequent regulation of downstream reporters: nitrogen starvation, methionine sulfoximine (Msx) addition, nitrogen limitation, rapamycin addition, and leucine starvation. Therefore, we tested a specific underlying assumption upon which the interpretation of data generated by these five experimental perturbations is premised. It is that they generate physiologically equivalent outcomes with respect to TorC1, i.e. its down-regulation as reflected by TorC1 reporter responses. We tested this assumption by performing head-to-head comparisons of the requirements for each condition to achieve a common outcome for a downstream proxy of TorC1 inactivation, nuclear Gln3 localization. We demonstrate that the five conditions for down-regulating TorC1 do not elicit physiologically equivalent outcomes. Four of the methods exhibit hierarchical Sit4 and PP2A phosphatase requirements to elicit nuclear Gln3-Myc(13) localization. Rapamycin treatment required Sit4 and PP2A. Nitrogen limitation and short-term nitrogen starvation required only Sit4. G1 arrest-correlated, long-term nitrogen starvation and Msx treatment required neither PP2A nor Sit4. Starving cells of leucine or treating them with leucyl-tRNA synthetase inhibitors did not elicit nuclear Gln3-Myc(13) localization. These data indicate that the five commonly used nitrogen-related conditions of down-regulating TorC1 are not physiologically equivalent and minimally involve partially differing regulatory mechanisms. Further, identical requirements for Msx treatment and long-term nitrogen starvation raise the possibility that their effects are achieved through a common regulatory pathway with glutamine, a glutamate or glutamine metabolite level as the sensed metabolic signal.
Department of Microbiology, Immunology, and Biochemistry, University of Tennessee Health Science Center, Memphis, Tennessee 38163.
Jennifer J
JJ
Tate
Department of Microbiology, Immunology, and Biochemistry, University of Tennessee Health Science Center, Memphis, Tennessee 38163. Electronic address: tcooper@uthsc.edu.
Terrance G
TG
Cooper
D000935:Antifungal Agents / Q000494:pharmacology; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology*; D059585:G1 Phase Cell Cycle Checkpoints / Q000187:drug effects / Q000502:physiology; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects / Q000502:physiology*; D000076222:Mechanistic Target of Rapamycin Complex 1; D046912:Multiprotein Complexes / Q000096:biosynthesis* / Q000235:genetics; D054648:Protein Phosphatase 2 / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases / Q000096:biosynthesis* / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D000935:Antifungal Agents; C071664:GLN3 protein, S cerevisiae; D046912:Multiprotein Complexes; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D000076222:Mechanistic Target of Rapamycin Complex 1; D058570:TOR Serine-Threonine Kinases; D054648:Protein Phosphatase 2; C104343:SIT4 protein, S cerevisiae; D020123:Sirolimus
Amino Acid; Cell Signaling; GATA; Gating; Gln3; Glutamine Synthase; Methionine Sulfoximine; Nitrogen Metabolism; Rapamycin; TorC1