Interplay between the exocyst complex and TOR signal transduction in the fission yeast Schizosaccharomyces pombe

Abstract

Adaptation to environmental changes is required for organism survival. Single celled organisms need to maintain a good surface area to volume ratio in order to take up enough nutrients without reaching an unsustainable size. Bending and shaping the plasma membrane through interplay between lipid domains and the actin cytoskeleton is required to localise permeases and sensors that detect environmental nutrients. This relies on a fine balance of exo- and endocytosis of plasma membrane components. The molecular mechanisms controlling nutrient sensing and uptake are not completely understood. The conserved exocyst complex has been implicated in both secretion and endocytosis making it a good candidate for regulating these membrane processes. Here, I use a combination of yeast genetics, biochemistry, and imaging to investigate how the exocyst member Sec3 influences nutrient sensing and uptake in the fission yeast Schizosaccharomyces pombe. I find that growth of temperature sensitive (ts) sec3 mutants is compromised in a leucine-rich environment via a mechanism independent of amino acid import. Because leucine regulates the target of rapamycin (TOR) growth signalling pathway, I examined the relationship between Sec3 and the TOR signalling axis. I found that inhibition of TOR by the macrolide rapamycin rescues the ts phenotype of sec3 mutants. Surprisingly, this was via the plasma membrane-associated TORC2 complex, which has been regarded as rapamycin insensitive. I asked if Sec3 cooperates with TORC2 to regulate membrane tension, and found that both sec3 and tor1 mutants had depolarised membrane sterols. Proteomic analyses of Sec3 binding partners indicated that a direct interaction between TOR and Sec3 is unlikely. Instead, Sec3 interacted with small GTPases that regulate TORC2 signalling and endocytic actin patch components. As both TORC2 and actin patches influence membrane tension, I hypothesised that Sec3 and TORC2 may intersect at the actin patch. I found that deletion of the endocytic myosin MyoI rescues the temperature sensitivity of sec3 mutants. I propose that Sec3 and TORC2 differentially regulate MyoI to control sterol polarisation and membrane tension. In conclusion, this thesis provides new insight into how fission yeast regulates membrane content and tension through a novel interaction between the exocyst complex and TORC2 signalling.