Fission yeast mating pheromone triggers the RAS-MAPK signalling pathway essential for meiotic differentiation. It also induces a dramatic morphological change of the cells leading to mating, a cell fusion event between cells of opposite mating types. The system is ideal to dissect the mechanisms by which the RAS-MAPK signal activation is regulated and highlight basic regulatory concepts of RAS protein signalling. In this work we evaluated the role of Ras1 in coordinating both MAPK activation due to pheromone signalling and activation of the Cdc42 pathway responsible for actin reorganisation. We established a condition to induce highly synchronous mating of fission yeast cells and we also established an assay system to monitor the MAPK phosphorylation status with an anti-phospho ERK monoclonal antibody. In addition, the changes in cellular morphology during the time course of meiotic differentiation were monitored. These tools allowed us to carry out quantitative measurements of the MAPK phosphorylation status in cells harbouring various mutations in the signalling components. We confirmed that a constitutively active mutation of the MEK induces constitutive phosphorylation of the MAPK. Strikingly however, with a canonical oncogenic Ras mutation (ras1.val17), although the MAPK activation occurs acutely, it is rapidly down-regulated therefore identifying the role of an unidentified modulator of the pathway at the level of the MEKK or MEK. We have also identified that unlike Ras1 and its GEF, Ste6, the G-protein, Gpa1 and the adaptor protein Ste4 are essential for MAPK phosphorylation although Ras1 appears to be the main activator of MAPK phosphorylation exclusively through the MAPK cascade. We therefore conclude that Ras1 is activating both the MAPK branch - which leads to expression of meiotic genes - and the Cdc42 pathway for polarised growth in response to pheromone. Successful MAPK activation is required for a morphological response even in the presence of oncogenic Ras1 indicating that MAPK activation is likely to be essential for site selection for polarised growth and that preventing MAPK activation can successfully prevent the oncogenic Ras1 phenotype. Importantly, the oncogenic phenotype of ras1.val17 can be rescued by the overexpression of the Ras1GAP, Gap1, therefore highlighting the importance of Ras proteins as potential therapeutic targets.

The ubiquitin-proteasome pathway is a central mechanism for regulating protein stability in eukaryotic cells. It involves the attachment of ubiquitin to a target protein marking it for degradation by the 26S proteasome. E3 ubiquitin ligases are an essential part of this process and one of the largest and most variable E3 ligase families is that of the SCF complexes. In order to study the role of the SCF complex in fission yeast, the role of Pof1, an essential fission yeast F-box protein, has been characterised. Temperature-sensitive pof1 mutants were generated. At the restrictive temperature these display acute growth arrest with small cell size. Extragenic suppressor analysis identified Zip1, a bZIP transcription factor, as a target for Pof1. Zip1 is shown to be stabilised in pof1 mutants, Pof1 binds only phosphorylated forms of Zip1, and Zip1 is ubiquitylated in vivo, indicating that Zip1 is a substrate of SCFpof1. Genome-wide DNA microarray assay shows that many cadmium-induced genes are under the control of Zip1, suggesting Zip1 plays a role in the cadmium response. Consistently, zip1 mutants are hypersensitive to cadmium and unlike wild type, lose cell viability under this stress. Intriguingly, cadmium exposure results in upregulation of Zip1 levels and leads wild type cells to arrest growth with small cell size, reminiscent of pof1 phenotypes. Our results indicate that Zip1 mediates growth arrest in response to cadmium, which is essential to maintain viability. Normally growing cells prevent this response through constitutive ubiquitylation and degradation of Zip1 via SCFpof1.

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The staphylococcal superantigen-like proteins (SSLs) are a family of polymorphic paralogs encoded within the Staphylococcus aureus genome, whose function remains unknown. The ability of SSL7, and a closely related paralog SSL9, to interact with cells of the immune system was investigated. Within the populations of human white blood cells, both SSLs interact selectively with monocytes, via specific but separate binding sites, leading to rapid uptake of SSLs. In addition, SSLs are rapidly taken up by dendritic cells (DC), but not macrophages, and target the mannose-receptor dependent endosomal antigen processing pathway. The effect of these proteins on the functional capacity of antigen presenting cells to uptake and present antigens to T cells was also determined. Neither SSL was toxic to DCs and the presence of SSL protein did not inhibit the antigen presenting cell activity, in terms of stimulation of either allogeneic or recall T cell responses. The immunological response to the SSL proteins in the normal human population was investigated. More than 30% of healthy normal subjects tested showed T cell responses to both SSL7 and SSL9. Moreover, almost all individuals had specific non- cross reacting antibodies against this family of proteins. In order to identify the SSL receptor(s), affinity chromatography techniques were used to isolate and identify the receptor(s) from selected cell lines. A single specific protein band that may represent the putative SSL receptor was observed, and mass spectrometry identified a candidate binding protein as heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa, GRP78 (BiP)). The presence of eleven members of this protein family within the pathogenicity island SaPIn2 in almost all S. aureus strains tested so far, suggest that these proteins have important non-redundant biological functions as agents of host/pathogen interactions. The data presented in this thesis further suggests that this function may involve targeting the host antigen presenting cell.

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