Design, construction and testing of a photoactivatable and diffusive protein network in Saccharomyces cerevisiae

The primary goal of the work presented in this thesis was to provide a unique tool that may increase our understanding of the mechanism of cytoplasmic diffusion in signal transduction (networks) in eukaryotic cells, in the form of a chimeric, light-dependent, cytoplasmic signal transduction device in the yeast Saccharomyces cerevisiae.
We describe the design and functional in vitro and in vivo characterization of the intended chimera: A light-sensitive histidine protein kinase, derived from the Sln1 kinase of S. cerevisiae, translationally fused in a coiled-coil motif with the LOV domain of the stressosome component YtvA from Bacillus subtilis. In most chimeras exposure to blue light decreased the rate of autophosphorylation, but rational engineering also allowed the construction of a light-stimulated histidine protein fusion kinase. This orthogonal photo-transduction system can be used to both activate and repress gene expression in S. cerevisiae, depending on the specific promoter that is targeted. Furthermore, the device can also be used to initiate nuclear accumulation of a selected signal transduction protein with incident blue light.