<p>In an attempt to gain more insight on functional evolution of cAMP-PKA pathway I have taken a comparative approach and examined functional interactions of cAMP-PKA signaling in well-studied yeast developmental programs and closely related <italic>Saccharomyces sensu stricto<italic/>. species. I have shown that variation in cAMP-PKA signaling contributes significantly to variation in developmental responses in <italic>S cerevisiae. Variation in pseudohyphal growth and sporulation, two inversely correlated developmental strategies to nutrient limitation in yeast, proportional to variation in intracellular cAMP levels. <italic>S. cerevisiae strains proficient in pseudohyphal growth have higher intracellular cAMP concentrations relative to strains that sporulate efficiently. Phenotypic, genetic and signaling data presented here suggest that the cAMP-PKA signaling underlies a phenotypic trade-off between sporulation and pseudohyphal growth in <italic>S. cerevisiae<italic/>.</p><p>Further investigation into the role of cAMP-PKA signaling in closely related <italic>S paradoxus<italic/> and <italic>S bayanus revealed an antagonistic function of cAMP-PKA signaling for developmental responses in <italic>S. bayanus. Unlike in <italic>S. cerevisiae, increased cAMP concentrations surprisingly inhibit pseudohyphal response in <italic>S. bayanus<italic/>. Another unanticipated finding in this work is that in <italic>S. bayanus<italic/>. Flo11, required for pseudohyphal differentiation in S. cerevisiae, is dispensable. Additionally, interactions of cAMP-PKA signaling and the general-stress response mechanism appear reversed in <italic>S. bayanus<italic/>. As shown by deletion mutation, gene expression and pharmacological treatment data, altered interactions and alternative targets downstream of cAMP-PKA could critically contribute to alternative regulation of nutrient-induced development in <italic>S. bayanus<italic/>.</p><p>Intracellular cAMP concentrations show decaying oscillations upon glucose replenishment in derepressed yeast cells. The quantitative characteristics of oscillations are distinct within and between Saccharomyces species. Given the tight regulation of cAMP levels and its critical role, the variation in cAMP oscillatory dynamics could be reflective of differential interactions of cAMP-PKA signaling that also underlie induction of developmental programs to changing environments. As such, intracellular cAMP levels and dynamics could potentially be used as molecular phenotypes.</p> / Dissertation

Prions are self-perpetuating and, in most cases, aggregation-prone protein isoforms that transmit neurodegenerative diseases in mammals and control heritable traits in yeast. Prion conversion requires a very high level of identity of the interacting protein sequences. Decreased transmission of the prion state between divergent proteins is termed "species barrier" and was thought to occur due to the inability of divergent prion proteins to co-aggregate. Species barrier can be overcome in cross-species infections, for example from "mad cows" to humans. We studied the counterparts of yeast prion protein Sup35, originated from three different species of the Saccharomyces sensu stricto group and exhibiting the range of prion domain divergence that overlaps with the range of divergence observed among distant mammalian species. Heterologous Sup35 proteins co-aggregated in S. cerevisiae cells. However, in vivo cross-species prion conversion was decreased and in vitro polymerization was cross-inhibited in at least some heterologous combinations, thus demonstrating the existence of prion species barrier. Our data suggests that species-specificity of prion transmission is controlled at the level of conformational transition rather than co-aggregation. We have shown the Sup35 prion domain is sufficient for the species barrier among the S. sensu stricto species, and constructed SUP35 chimeric prion domains, combining the subregions of various origins Our data demonstrated in different cross-species combinations, different modules of prion domain play a crucial role in the controlling of species-specificity of prion transmission. One essential amino acid position has been identified in S. cerevisiae and S. paradoxus system. Our data support a model suggesting that identity of the short amyloidogenic sequences is crucial for the species barrier. Sup35 originated from three different species of the S. sensu stricto group were capable of forming a prion in S. cerevisiae. However, it was not known whether they are capable of generating and maintaining the prion state in the homologous cell environment. We have constructed the S. paradoxus and S. bayanus strains with appropriate markers, and we were able to demonstrate de novo [PSI+] formation in S. paradoxus but not in S. bayanus. Our data show that [PSI+] formation is not a unique property of S. cerevisiae.