Investigation of the functions of sumo in conserved biological processes in Saccharomyces cerevisiae

Abstract

The small ubiquitin-like modifier (sumo) is a conserved post-translational modification found throughout eukaryotes. Over the last 25 years a large range of studies have investigated the role of sumoylation, identifying hundreds of substrates and linking sumo to a diverse range of key cellular processes, including stress responses, the response to DNA damage and cell cycle progression. Although sumoylation has also been shown to be essential in a number of eukaryotes, including the model yeast Saccharomyces cerevisiae, the fundamental role(s) of sumoylation remains unclear. Sumo dysregulation is associated with a number of human diseases, including cancer, hence it is important to understand and characterise the role(s) of sumo pathways within these diseases. In an attempt to identify the important functions of sumoylation, a recent SGA screen carried out in our lab used a S. cerevisiae strain with reduced sumo (Smt3) function to identify a number of suppressor proteins which were able to suppress the growth defects of this smt3 mutant. Excitingly, several novel cytoskeletalrelated suppressors proteins were identified which rescued the smt3 growth phenotype, including subunits of the CCT chaperonin complex, b-tubulin and branched F-actin. Hence, the aim of this thesis was to further characterise the phenotypes associated with the smt3 mutant and to investigate the relationship of other proteins in the sumo conjugation/deconjugation pathways, the role of polysumoylation and the effect of different S. cerevisiae strain backgrounds within stress responses including exposure to cold temperature, responses to oxidative stress and cell cycle progression. In addition, another aspect of this study was to investigate the relationship of the smt3 mutant with the novel suppressor proteins including subunits of the CCT complex, b-tubulin and F-actin. Excitingly, this study has revealed novel and strain specific roles for sumoylation and polysumoylation within S. cerevisiae stress responses, cell cycle progression and chromosome dynamics, including a novel, strain specific role for sumoylation during S phase. In addition, data in this study also revealed that enzymes within the sumo conjugation and deconjugation pathways respond differently when presented with different stresses. Interestingly, our studies of the relationships between smt3 and the novel cytoskeletal suppressor proteins iv revealed that although these suppressors partially suppress the smt3 growth defects, the smt3 strain also suppresses several phenotypes associated with the cytoskeletal suppressors. Furthermore, our data suggests that b-tubulin is a substrate of sumoylation in S. cerevisiae cells. Thus, these results are consistent with a model in which sumoylation is functionally linked to the cytoskeleton by the interaction of sumo with microtubules and F-actin. Given that dysregulation of sumo, the CCT complex, F-actin and microtubules are common in many human diseases, this study provides novel insights into the relationship between the mutations in these complexes, potentially identifying new routes for the development of therapeutic treatments for human diseases.