The roles and regulation of heat shock proteins in yeast

PhD ThesisHeat shock proteins (HSPs) such as the HSP70s Ssa1 and Ssa2 and the HSP40 Ydj1 in Saccharomyces cerevisiae are ubiquitous abundant proteins. With well characterised roles as protein chaperones, HSPs function in heat and oxidative stress responses to prevent aggregation and to aid degradation. The functions of HSPs are important in humans to prevent diseases such as cardiovascular disease and Parkinson’s disease. Interestingly, recent studies have also begun to identify key roles for specific HSPs in stress sensing and signal transduction and the regulation of the cell cycle. For example, work in S. cerevisiae revealed that Ssa1 and Ydj1 regulate cell cycle commitment in G1 phase by controlling the availability of the Cdc28-Cln3 cyclin dependent protein kinase complex. Moreover, Ssa1 activity and modification regulates the heat/oxidative stress-dependent activation of the conserved Hsf1 transcription factor. However, despite these yeast studies there is still much to learn about the conserved and organism-specific roles and regulation of HSP70 and HSP40 proteins in cellular processes. Here studies were initiated in the evolutionarily distant fission yeast Schizosaccharomyces pombe to investigate the roles and regulation of Ssa1 (HSP70), Ssa2 (HSP70) and Mas5 (HSP40), homologues of S. cerevisiae Ssa1, Ssa2 and Ydj1, respectively. Excitingly, these HSPs were found to play key roles in S. pombe in conserved processes such as mitosis, meiosis and autophagy. For example, Ssa2 and Mas5, but not Ssa1, were found to localise to the site of new cell growth suggesting roles in the cell cycle in addition to their roles in the regulation of Hsf1. Moreover, microtubule dynamics and cell size were found to be dependent on Ssa1 and Mas5. Interestingly, microtubule dynamics and cell size are also regulated by their homologues Ssa1 and Ydj1 in S. cerevisiae, suggesting conserved roles in cell cycle progression. 4 Investigations were also performed to identify any connections between Ssa1, Ssa2 and Mas5 in the responses of S. pombe cells to heat and/or oxidative stresses. Studies in other organisms have revealed the importance of HSP70 and HSP40 as repressors of the activation of Hsf1. Indeed, consistent with these results Mas5 was found to act as a repressor of the heat stress-induced nuclear accumulation of Hsf1. Previous work revealed that different signalling pathways/transcription factors determine the response of S. pombe cells to increasing concentrations of H2O2. For example, the thioredoxin system regulated AP-1-like transcription factor Pap1 is important for the response to low but not high concentrations of H2O2. Moreover, the oxidation status of thioredoxin system proteins determines the timing of oxidation, activation and nuclear accumulation of Pap1. Excitingly, the studies described here revealed novel insights into the roles and regulation of Hsf1 in response to H2O2 in S. pombe. For example, Hsf1 was found to accumulate in the nucleus in response to H2O2 in a concentration dependent manner like Pap1. Significantly, although this accumulation was found, like Pap1, to be linked to the thioredoxin system, Hsf1 accumulation appears to be regulated in a different manner to Pap1. Results also revealed that Ssa1 acts as an activator of Hsf1 in the H2O2 response while Mas5 acts as a repressor of Hsf1. In S. cerevisiae the potential relationship between Hsf1 and Yap1, the Pap1 homologue, in the response to oxidative stress have not been studied. Excitingly, the results presented here suggest that Mas5 is a new regulator of Pap1 activity, influencing Pap1 through the thioredoxin system. Moreover, data suggests cross talk between Hsf1/Pap1 transcriptional responses with Mas5 acting as a previously unidentified key regulator of both responses. A model is proposed where Mas5 regulation of Hsf1 influences Pap1 activation by regulating the expression of the tpx1+ gene encoding the 2-Cys peroxiredoxin Tpx1, a known regulator of 5 Pap1 that acts in the thioredoxin system. These results are intriguing given recent studies in S. cerevisiae suggesting that the thioredoxin system protein Tsa1, the homologue of Tpx1, replaces Ydj1 (Mas5 homologue) as a co-chaperone for HSP70 roles in aggregate recognition and clearance in response to oxidative stress and not heat stress. In conclusion, the studies presented in this thesis provide new insights into the regulation of Hsf1 and the roles of HSP70 and HSP40 HSPs in eukaryotes. Given their relationships with common human diseases it is important to understand the organism-specific and conserved roles of these proteins to allow the development of future medical interventions.BBSRC fundin