Kingsbury Joanne M, Sen Neelam D, Maeda Tatsuya, Heitman Joseph, Cardenas Maria E
Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 27710.
Genetics. 2014 Apr;196(4):1077-89. doi: 10.1534/genetics.114.161646. Epub 2014 Feb 10.
The rapamycin-sensitive and endomembrane-associated TORC1 pathway controls cell growth in response to nutrients in eukaryotes. Mutations in class C Vps (Vps-C) complexes are synthetically lethal with tor1 mutations and confer rapamycin hypersensitivity in Saccharomyces cerevisiae, suggesting a role for these complexes in TORC1 signaling. Vps-C complexes are required for vesicular trafficking and fusion and comprise four distinct complexes: HOPS and CORVET and their minor intermediaries (i)-CORVET and i-HOPS. We show that at least one Vps-C complex is required to promote TORC1 activity, with the HOPS complex having the greatest input. The vps-c mutants fail to recover from rapamycin-induced growth arrest and show low levels of TORC1 activity. TORC1 promotes cell growth via Sch9, a p70(S6) kinase ortholog. Constitutively active SCH9 or hyperactive TOR1 alleles restored rapamycin recovery and TORC1 activity of vps-c mutants, supporting a role for the Vps-C complexes upstream of TORC1. The EGO GTPase complex Exit from G0 Complex (EGOC) and its homologous Rag-GTPase complex convey amino acid signals to TORC1 in yeast and mammals, respectively. Expression of the activated EGOC GTPase subunits Gtr1(GTP) and Gtr2(GDP) partially suppressed vps-c mutant rapamycin recovery defects, and this suppression was enhanced by increased amino acid concentrations. Moreover, vps-c mutations disrupted EGOC-TORC1 interactions. TORC1 defects were more severe for vps-c mutants than those observed in EGOC mutants. Taken together, our results support a model in which distinct endolysosomal trafficking Vps-C complexes promote rapamycin-sensitive TORC1 activity via multiple inputs, one of which involves maintenance of amino acid homeostasis that is sensed and transmitted to TORC1 via interactions with EGOC.
雷帕霉素敏感且与内膜相关的TORC1途径控制真核生物中细胞对营养物质的生长反应。C类Vps（Vps-C）复合物中的突变与tor1突变具有合成致死性，并在酿酒酵母中导致雷帕霉素超敏性，表明这些复合物在TORC1信号传导中起作用。Vps-C复合物是囊泡运输和融合所必需的，由四种不同的复合物组成：HOPS和CORVET及其次要中间体（i）-CORVET和i-HOPS。我们表明，至少需要一种Vps-C复合物来促进TORC1活性，其中HOPS复合物的作用最大。vps-c突变体无法从雷帕霉素诱导的生长停滞中恢复，并且显示出低水平的TORC1活性。TORC1通过Sch9（一种p70(S6)激酶直系同源物）促进细胞生长。组成型活性SCH9或超活性TOR1等位基因恢复了vps-c突变体的雷帕霉素恢复能力和TORC1活性，支持Vps-C复合物在TORC1上游的作用。EGO GTP酶复合物退出G0复合物（EGOC）及其同源的Rag-GTP酶复合物分别在酵母和哺乳动物中将氨基酸信号传递给TORC1。活化的EGOC GTP酶亚基Gtr1(GTP)和Gtr2(GDP)的表达部分抑制了vps-c突变体的雷帕霉素恢复缺陷，并且这种抑制作用随着氨基酸浓度的增加而增强。此外，vps-c突变破坏了EGOC-TORC1相互作用。vps-c突变体的TORC1缺陷比在EGOC突变体中观察到的更严重。综上所述，我们的结果支持一个模型，即不同的内溶酶体运输Vps-C复合物通过多种输入促进雷帕霉素敏感的TORC1活性，其中之一涉及维持氨基酸稳态，该稳态通过与EGOC的相互作用被感知并传递给TORC1。
