Toyoda, Y. | bioRxiv Science

bioRxiv2025

Compromised ESCRT signalling is sufficient for resistance to the Target of Rapamycin Complex inhibitor Torin1 in fission yeast

BackgroundFission yeast cells defective in Golgi-endosomal sorting display high resistance to Torin1, a pan-Target of Rapamycin (TOR) inhibitor. TOR complexes regulate the ESCRT system to integrate nutrient availability with cell division. TOR activity is frequently deregulated in cancer, making it an attractive therapeutic target. Deregulated ESCRT activity has also been associated with cancer, but its role in mediating drug resistance is not fully understood. Herein, we have investigated the role of the ESCRT system in regulating sensitivity to Torin1. MethodsGrowth assays were used to monitor the growth of yeast cells. The effect of Torin1 on protein expression was monitored by immunoblotting. Fluorescence microscopy was used to investigate the action of Torin1 on protein localization. ResultsThe ESCRT system mediates Torin1-induced degradation of amino acid and glucose transporters. The expression of these transporters at the plasma membrane is not abolished in ESCRT mutants. Mutants unable to effectively ubiquitylate these transporters are also resistant to Torin1. Impaired ESCRT-mediated protein degradation is associated with strong resistance to Torin1. ConclusionsMutations in genes encoding ESCRT components have been reported in cancer. We present evidence that compromised ESCRT signalling is sufficient for resistance to Torin1. Cells defective in ESCRT signalling or ubiquitin homeostasis are highly resistant to Torin1. Our studies demonstrate that defective ESCRT-mediated proteolysis can suppress sensitivity to Torin1.

cell biology↗

bioRxiv2024

Phosphorylation of Aly3 C-terminus impedes aberrant endocytosis of S. pombe hexose transporter Ght5

In fission yeast, Schizosaccharomyces pombe, transcriptional upregulation and cell-surface localization of the hexose transporter, Ght5, are required for cell proliferation in low glucose. As the target of rapamycin complex 2 (TORC2) signaling pathway inhibits -arrestin Aly3-dependent endocytosis of Ght5, we hypothesized that this endocytosis was inhibited by phosphorylation. To identify phosphorylation sites required for cell proliferation in low glucose, serine and threonine residues of Aly3 and Ght5 reportedly phosphorylated were replaced with alanine. We found that C-terminal serine residues of Aly3, but not Ght5, are necessary for proliferation in low glucose. Expression of Aly3 protein unphosphorylated at the C-terminus led to increased ubiquitination and vacuolar accumulation of Ght5 in low glucose, but reversion of one of the alanine residues to serine reduced ubiquitination and vacuolar accumulation of Ght5. Also, Aly3 physically interacted with the HECT-type ubiquitin ligases Pub1 and Pub3, and these interactions were required for surface localization of Ght5 and proliferation in low glucose. This study reveals mechanisms by which Aly3 is regulated so that fission yeast can adapt to nutritional stress.

cell biology↗