The signalling pathway driven by TOR is highly conserved from yeast to mammals and plays a key role in the control of growth and proliferation in response to nutrients, growth factors and cellular energy. TOR contributes to the control of cell cycle progression, although currently not many molecular details are known. We aimed to understand how growth control regulates cell cycle events after anaphase. To synchronize cells in anaphase, we inactivated the Mitotic Exit Network (MEN) kinase Cdc15, using the temperature-sensitive mutant cdc15-2. After the release, we showed that cells continued their cell cycle as they were able to finish mitosis, cytokinesis, grow new buds and enter a new round of chromosome replication, but they failed to separate. Intriguingly, specific inactivation of TORC1 rescued cell separation defect associated to cdc15-2 cells. We found that both Cdc15 and TORC1 controlled cell separation through a novel molecular mechanism. TORC1 was able to interact with and modify the sole NDR/LATS family kinase in budding yeast, Cbk1, which is the downstream factor of the RAM pathway that drives cell separation. Using mass spectrometry analysis, we showed Cbk1 interaction landscape in anaphase and how changes in TORC1 function modulate specific phosphorylations in Cbk1 and Cbk1 interactors. In parallel, we found a conserved threonine that promoted cell separation defect in cdc15-2 cells, even in the absence of TORC1 activity, when modified to glutamic acid, as this version of Cbk1 lacked catalytic activity and was able to reproduce TORC1 inhibition over Cbk1. At the end of the cell cycle, cells drive secretory vesicles transporting hydrolases to their sites of division to promote cell separation as those hydrolases induce the degradation of the septum that joins mother and daughter cells. We found that fusion of secretory vesicle carrying hydrolases is defective when TORC1 is active after cells were released from anaphase arrest. We determined by mass spectrometry that Cbk1 interacted and phosphorylated components of the exocyst, which might explain how TORC1 controls cell separation. We proposed a model in which kinase Cdc15 promotes cell separation by releasing Cbk1 from TORC1 negative regulation, which coordinates cell cycle and cell growth.

Trabajo presentado en la XIII edición de la Red Española de Levaduras, celebrada en El Escorial (España) del 14 al 16 de diciembre de 2022.