Regulation of nuclear tRNA export in response to nutrient stress is not evolutionarily conserved and requires the TORC1 and PKA signaling pathways in Saccharomyces cerevisiae

REGULATION OF NUCLEAR tRNA EXPORT IN RESPONSE TO NUTRIENT STRESS IS NOT EVOLUTIONARILY CONSERVED AND REQUIRES THE TORC1 AND PKA SIGNALING PATHWAYS IN SACCHAROMYCES CEREVISIAE Jacqueline Barbara Pierce, B.Sc. (Hons) Advisor: University of Guelph, 2012 Dr. D. Mangroo Dr. G. van der Merwe Saccharomyces cerevisiae are unicellular organisms that are highly adaptable to acute changes in nutrient availability. The two main signaling pathways that allow S. cerevisiae to sense and respond to changes in glucose availability in the environment are the conserved cAMP/PKA and AMPK/Snf1 kinase-dependent pathways. The conserved TORC1 pathway is primarily responsible for allowing cells to respond to the availability of nitrogen. Studies have shown that S. cerevisiae, but not mammalian and plant cells, regulate nuclear tRNA trafficking in response to nutrient stress. Here, we show that the yeast species of the Saccharomyces genus, but not Schizosaccharomyces pombe and Kluyveromyces lactis specifically regulate nuclear tRNA export in response to nutrient stress, providing further evidence that regulation of nuclear tRNA export in response to nutrient availability is not evolutionarily conserved. We also established that amino acid and nitrogen starvation affects nuclear export of a subset of tRNAs in S. cerevisiae. Inhibition of TORC1 signaling by rapamycin treatment, which simulates nitrogen starvation, also affects nuclear export of the same subset of tRNAs, suggesting that the TORC1 signaling pathway plays a role in regulating nuclear export of the tRNAs in response to nitrogen level. Regulation of nuclear export of these tRNAs by nitrogen deprivation is most likely due to an effect on the function of the nuclear tRNA export receptors, as overexpression of the tRNA export receptor, Los1p, restores export of the tRNAs during nitrogen starvation. These findings suggest that the TORC1 signaling pathway may, in part, regulate nuclear export of the tRNAs by affecting the function of the tRNA export receptors. In contrast to amino acid and nitrogen starvation, glucose depletion affects nuclear export of all tRNA species in S. cerevisiae. Evidence obtained suggests that nuclear retention of tRNA in cells deprived of glucose is due to a block in nuclear re-import of the nuclear tRNA export receptors. Retention of the receptors in the cytoplasm is not caused by activation of Snf1p, but by the inactivation of PKA during glucose deprivation. Furthermore, regulation of nuclear re-import of the receptors is not due to phosphorylation of the tRNA export receptors by PKA. However, PKA phosphorylates known components of the tRNA export machinery. A model that is consistent with the data is that PKA and an unknown mechanism regulate the activity of these components or an unidentified protein(s) to control nuclear re-import of the receptors in response to glucose availability. Acknowledgements I would like to thank my supervisor, Dr. Dev Mangroo, for the opportunity to pursue my graduate school education in his laboratory and for providing motivation and guidance during the course of my studies. I would also like to thank Dr. George van der Merwe for his support and encouragement during the final stages of completing my degree. I must also thank my advisory committee, Dr. Marc Coppolino, Dr. Robert Mullen and Dr. Nina Jones for their continued support during the completion of this thesis. I would like to thank NSERC and OGS for their financial support throughout my studies. There are so many people that I had the pleasure of knowing during my time at the University of Guelph. I would like to thank all of my friends who provided encouragement, support and friendship for the past 6 years. I especially thank my friends Sarah Moulaison, Adam McCarthy, Chris Stephens, Ashleigh Grech and John Jonkman for always keeping me grounded outside of the lab. I must extend a very special thanks to the members of my past and current lab Dr. Manoja Eswara, Dr. Shawn Chafe, Dr. Andy McGuire, Dr. Chris Snowdon, Ben Strub, Aaron Johnstone, and Ashley Clayton who provided guidance, motivation, company and good times during the many hours and late nights in the lab. I also must thank all the other members of the 2 floor especially Marissa Dahari, Dr. Danielle Visschedyk, Ashley Hillier, David Marom, Anita Agblor, Dr. Alex Perieteanu, Karla Williams, Paul McRorie and Ryan Demers for their support and friendship. Finally, I would like to show my sincerest gratitude to my family especially my parents John and Barbara Pierce, my sisters Kathleen and Mandy and my brother Matt for their unwavering support and reassurance over the years. I could never have achieved this goal without them.