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

Ning Ma,Ushio Kikkawa,Yan Ma,Akio Nakashima,Tomoyuki Furuyashiki,Reiko Sugiura

doi:10.1371/journal.pone.0156239

Abstract

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.

Highlights

Target of rapamycin (TOR) is a serine/threonine kinase, and plays fundamental roles in regulating cell growth and metabolism by coordinating diverse cellular processes including transcription, translation and autophagy [1, 2]
We found that the loss of Lam2 or Gtr1-Gtr2 increases TORC1 activity and phenocopies the loss of Npr2-Npr3 in multiple cellular functions
In Δlam2 cells, Δnpr2 cells and Δnpr3 cells, vacuoles were smaller, and the intensities of Gtr1-GFP and Gtr2-GFP at vacuolar membranes were decreased, compared with wild-type cells (Fig 8A and 8B). These findings suggest that Lam2 and Npr2-Npr3 promote the vacuolar localization of Gtr1 and Gtr2 as a tether for Gtr1-Gtr2 at vacuolar membranes

Summary

Introduction

Target of rapamycin (TOR) is a serine/threonine kinase, and plays fundamental roles in regulating cell growth and metabolism by coordinating diverse cellular processes including transcription, translation and autophagy [1, 2]. Mammalian cells express a single TOR isoform mTOR, which forms two types of protein complexes named mTORC1 and mTORC2 [1, 2]. There are two TOR isoforms Tor and Tor, each of which is included in TORC1 or TORC2 [3, 4]. TORC1 is activated by the GTP-bound form of Rheb small GTPase. Growth factors, energy status and oxygen levels increase the GTP-bound form of Rheb by inhibiting TSC2, a GTPase activing protein (GAP) for Rheb, and activate mTORC1. TSC2 ortholog is expressed in fission yeast, but not in budding yeast

Methods

Results

Conclusion