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      An in vitro TORC1 kinase assay that recapitulates the Gtr-independent glutamine-responsive TORC1 activation mechanism on yeast vacuoles.

      1 , 2
      Molecular and cellular biology
      American Society for Microbiology

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          Abstract

          Evolutionarily-conserved TOR complex 1 (TORC1) responds to nutrients, especially amino acids, to promote cell growth. In yeast Saccharomyces cerevisiae, various nitrogen sources activate TORC1 with different efficiencies although the mechanism remains elusive. Leucine, and perhaps other amino acids, was reported to activate TORC1 via the heterodimeric small GTPases Gtr1-Gtr2, the orthologs of the mammalian Rag GTPases. More recently, an alternative Gtr-independent TORC1 activation mechanism that may respond to glutamine was reported, although its molecular detail is not clear. In studying the nutrient-responsive TORC1 activation mechanism, the lack of an in vitro assay hinders associating particular nutrient compounds with the TORC1 activation status, whereas no in vitro assay that shows nutrient responsiveness has been reported. In this study, we have developed a new in vitro TORC1 kinase assay that reproduces, for the first time, the nutrient-responsive TORC1 activation. This in vitro TORC1 assay recapitulates the previously predicted Gtr-independent glutamine-responsive TORC1 activation mechanism. Using this system, we found that this mechanism specifically responds to L-glutamine, resides on the vacuolar membranes, and involves a previously uncharacterized Vps34-Vps15 phosphatidylinositol 3-kinase complex and the PI (3)P-binding FYVE domain-containing vacuolar protein Pib2. Thus, this system was proved to be useful for dissecting the glutamine-responsive TORC1 activation mechanism.

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          Author and article information

          Journal
          Mol. Cell. Biol.
          Molecular and cellular biology
          American Society for Microbiology
          1098-5549
          0270-7306
          May 08 2017
          Affiliations
          [1 ] Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan.
          [2 ] Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan maeda@iam.u-tokyo.ac.jp.
          Article
          MCB.00075-17
          10.1128/MCB.00075-17
          28483912
          62612f5a-f32c-463c-8753-66039744cc34
          History

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