The Loss of Lam2 and Npr2-Npr3 Diminishes the Vacuolar Localization of Gtr1-Gtr2 and Disinhibits TORC1 Activity in Fission Yeast

In mammalian cells, mTORC1 activity is regulated by Rag GTPases. It is thought that the Ragulator complex and the GATOR (GAP activity towards Rags) complex regulate RagA/B as its GDP/GTP exchange factor (GEF) and GTPase-activating protein (GAP), respectively. However, the functions of components in these complexes remain elusive. Using fission yeast as a model organism, here we found that the loss of Lam2 (SPBC1778.05c), a homolog of a Ragulator component LAMTOR2, as well as the loss of Gtr1 or Gtr2 phenocopies the loss of Npr2 or Npr3, homologs of GATOR components Nprl2 or Nprl3, respectively. These phenotypes were rescued by TORC1 inhibition using pharmacological or genetic means, and the loss of Lam2, Gtr1, Gtr2, Npr2 or Npr3 disinhibited TORC1 activity under nitrogen depletion, as measured by Rps6 phosphorylation. Consistently, overexpression of GDP-locked Gtr1S20L or GTP-locked Gtr2Q60L, which suppress TORC1 activity in budding yeast, rescued the growth defect of Δgtr1 cells or Δgtr2 cells, respectively, and the loss of Lam2, Npr2 or Npr3 similarly diminished the vacuolar localization and the protein levels of Gtr1 and Gtr2. Furthermore, Lam2 physically interacted with Npr2 and Gtr1. These findings suggest that Lam2 and Npr2-Npr3 function together as a tether for GDP-bound Gtr1 to the vacuolar membrane, thereby suppressing TORC1 activity for multiple cellular functions.

在哺乳动物细胞中，雷帕霉素靶蛋白复合物1（mTORC1）的活性受Rag鸟苷三磷酸酶（Rag GTPases）调控。学界普遍认为，Ragulator复合物（Ragulator complex）与GATOR复合物（GAP activity towards Rags）分别作为鸟苷二磷酸/鸟苷三磷酸交换因子（GEF）与GTP酶激活蛋白（GAP）调控RagA/B。然而，上述复合物各组分的具体功能仍有待阐明。本研究以裂殖酵母（fission yeast）为模式生物，发现Ragulator组分LAMTOR2的同源蛋白Lam2（SPBC1778.05c）缺失，以及Gtr1或Gtr2缺失，分别重现了GATOR组分Nprl2与Nprl3的同源蛋白Npr2或Npr3缺失所产生的表型。通过药理学或遗传学手段抑制TORC1可挽救上述表型；在氮饥饿条件下，Lam2、Gtr1、Gtr2、Npr2或Npr3的缺失会解除对TORC1活性的抑制，这一结果可通过Rps6磷酸化（Rps6 phosphorylation）水平得以验证。一致的是，在出芽酵母（budding yeast）中可抑制TORC1活性的GDP锁定型Gtr1S20L与GTP锁定型Gtr2Q60L的过表达，可分别挽救gtr1缺失型细胞与gtr2缺失型细胞的生长缺陷；而Lam2、Npr2或Npr3的缺失，同样会降低Gtr1与Gtr2的液泡定位（vacuolar localization）水平及其蛋白表达量。此外，Lam2可与Npr2及Gtr1发生物理相互作用。本研究结果表明，Lam2与Npr2-Npr3复合物共同作为GDP结合型Gtr1的锚定因子，将其锚定至液泡膜，从而在多种细胞功能中抑制TORC1的活性。