Shedding light on the link between the actin cytoskeleton and stress response in Saccharomyces cerevisiae : a thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry, Massey University, Auckland, New Zealand

Abstract

Understanding how eukaryotic cells adapt to stress remains a fundamental question in biology. One important stress which affects all cells is amino acid (AA) starvation. Upon perception of AA starvation the Gcn2 protein binds an effector protein complex consisting of Gcn1 and Gcn20. Subsequently, Gcn2 becomes activated and phosphorylates the eukaryotic translation initiation factor 2. This leads to a reduced global protein synthesis and simultaneously to an increased translation of Gcn4, a transcriptional activator of genes necessary for overcoming stress. This signaling pathway is called general amino acid control (GAAC) in yeast. Failing to activate this signaling cascade impairs the starvation response and cellular growth. Yih1 inhibits Gcn2 by competing with Gcn2 for Gcn1 binding, consequently leading to an impaired stress response. However, Yih1 is not a general inhibitor of Gcn2 but only impedes Gcn2 activation upon release from the cytoskeleton protein actin. Our understanding of the role of actin in Gcn2 signaling and Yih1 itself is limited. Also, the circumstances under which Yih1 is released from actin are unknown. Thus, the scope of this study is to elucidate the link between the actin cytoskeleton and stress response. To achieve this goal, actin mutants were screened for an impaired ability to overcome starvation. Out of 24 mutant strains five exhibited an impaired stress response as indicated by sensitivity to AA analogues, which could be reverted upon Gcn4 induction, and sensitivity to AA imbalance. One of this actin mutations has been proposed to affect the GAAC via an impaired Yih1-actin binding. Another mutation appears to weaken the actin-eEF1A interaction therefore promoting eEF1A mediated Gcn2 inhibition. For the remaining 3 actin mutations the mechanism might be at the transcriptional or translational level. These findings show that actin mutations do affect the GAAC at multiple levels. In addition, the cyclin dependent kinase Cdc28 has been identified as a novel interaction partner of Yih1 and it has been speculated that a link between Yih1 and bud emergence might exist. The results from this study achieved to shed light on the link between Yih1, the GAAC and the actin cytoskeleton. Yih1 has been placed in the midst of highly regulated and diverse cellular processes, which emphasizes the interconnection that exist between cellular pathways and the likely importance of Yih1 in the cell.