Systematic screen for mutants resistant to TORC1 inhibition in fission yeast reveals genes involved in cellular ageing and growth

Abstract

SummaryTarget of rapamycin complex 1 (TORC1), which controls growth in response to nutrients, promotes ageing in multiple organisms. The fission yeast Schizosaccharomyces pombe emerges as a valuable genetic model system to study TORC1 function and cellular ageing. Here we exploited the combinatorial action of rapamycin and caffeine, which inhibit fission yeast growth in a TORC1-dependent manner. We screened a deletion library, comprising ∼84% of all non-essential fission yeast genes, for drug-resistant mutants. This screen identified 33 genes encoding functions such as transcription, kinases, mitochondrial respiration, biosynthesis, intra-cellular trafficking, and stress response. Among the corresponding mutants, 5 showed shortened and 21 showed increased maximal chronological lifespans; 15 of the latter mutants showed no further lifespan increase with rapamycin and might thus represent key targets downstream of TORC1. We pursued the long-lived sck2 mutant with additional functional analyses, revealing that the Sck2p kinase functions within the TORC1 network and is required for normal cell growth, global protein translation, and ribosomal S6 protein phosphorylation in a nutrient-dependent manner. Notably, slow cell growth was associated with all long-lived mutants while oxidative-stress resistance was not.

Genetic interactions and functional analyses of the fission yeast gsk3 and amk2 single and double mutants defective in TORC1-dependent processes

“…1a)15. To gain insight into functional relationships of Gsk3 within the TOR signalling network, we examined how Gsk3 affects phenotypes related to TORC1 inhibition.…”

“…Query strains were the following: h − gsk3::hphMX6, h − amk2::natMX6, h − gsk3::hphMX6 amk2::natMX6 and control query h − ade6::natMX6 15. Colony size was used as a proxy for double/triple mutant fitness.…”

“…However, its role in unicellular eukaryotes remains poorly characterized. We have shown that gsk3- mutant cells are resistant to TORC1 inhibition by caffeine and rapamycin and feature a shortened CLS when glucose is depleted15. Human GSK3 inhibits AMPK by interacting with the AMPK β subunit while phosphorylating and inactivating the α subunit.…”

Chemical genomics approach to identify genes associated with sensitivity to rapamycin in the fission yeast Schizosaccharomyces pombe

“…), would enable the integration of chemical genetics and genomic analyses and thus greatly increase our understanding of the molecular functions and dynamic regulation of the TOR kinase in fission yeast (Rallis et al . ).…”

“…Because in budding yeast, information on mutants showing altered sensitivity to rapamycin is also available (Xie et al 2005), comparison of a list of genes related to rapamycin sensitivity between species would also be intriguing. Furthermore, genomewide tools and resources, such as the genome-deletion library and the ORFeome overexpression collections available in fission yeast (Matsuyama et al 2006;Kim et al 2010;Takeda et al 2011), would enable the integration of chemical genetics and genomic analyses and thus greatly increase our understanding of the molecular functions and dynamic regulation of the TOR kinase in fission yeast (Rallis et al 2014).…”