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

Abstract

The Target of Rapamycin (TOR) signalling network plays important roles in aging and disease. The AMP-activated protein kinase (AMPK) and the Gsk3 kinase inhibit TOR during stress. We performed genetic interaction screens using synthetic genetic arrays (SGA) with gsk3 and amk2 as query mutants, the latter encoding the regulatory subunit of AMPK. We identified 69 negative and 82 positive common genetic interactors, with functions related to cellular growth and stress. The 120 gsk3-specific negative interactors included genes functioning in translation and ribosomes. The 215 amk2-specific negative interactors included genes functioning in chromatin silencing and DNA damage repair. Both amk2- and gsk3-specific interactors were enriched in phenotype categories related to abnormal cell size and shape. We also performed SGA screen with the amk2 gsk3 double mutant as a query. Mutants sensitive to 5-fluorouracil, an anticancer drug are under-represented within the 305 positive interactors specific for the amk2 gsk3 query. The triple-mutant SGA screen showed higher number of negative interactions than the double mutant SGA screens and uncovered additional genetic network information. These results reveal common and specialized roles of AMPK and Gsk3 in mediating TOR-dependent processes, indicating that AMPK and Gsk3 act in parallel to inhibit TOR function in fission yeast.

Iron regulates hexose transporters in Schizosaccharomyces pombe

“…Our results support recently findings related to a cross‐regulatory relationship between CaMK and TOR pathways . And, significantly reduced transcription of the ght5 gene by increased iron stress has been thought that Ght5 protein might play a central role in complicated cross‐regulatory relationship.…”

Mapping a diversity of genetic interactions in yeast

“…In such cases, inactivation of both genes allows for the identification of trigenic interactions that would not have been identified by digenic interaction screens [41]. Genome-wide genetic interaction screens have been performed for a few double mutant pairs in both baker’s and fission yeast, often providing new insights into the functional connections between the genes [41–43]. For example, the genetic interaction profile of a double mutant deleted for CLB5 and CLB6 , both encoding cyclins that regulate DNA replication, showed a strong resemblance to that of single mutants involved in sister chromatid cohesion, establishing a role of Clb5 and Clb6 in chromosome segregation [42].…”

The Target of Rapamycin Signalling Pathway in Ageing and Lifespan Regulation

“…Secondly, through regulation of transcription (dependent on all three RNA polymerases) of genes coding for ribosomal proteins [ 1 , 9 , 14 ], ribosome biogenesis factors [ 15 ] and tRNAs [ 16 , 17 , 18 , 19 , 20 ]. Apart from translation-related genes TORC1 has major positive impact on gene expression related to growth and metabolism [ 2 , 18 ] and a negative action on genes coding for proteins related to cellular stress [ 21 , 22 , 23 ]. Genetic or pharmacological inhibition of mTOR activity stimulates autophagy, a conserved lysosomal catabolic pathway controlling the degradation and turnover of macromolecules and organelles and maintaining metabolic homeostasis at both the cellular and whole-organism level [ 24 , 25 ].…”