Research Article: Design, characterization and in vivo functioning of a light-dependent histidine protein kinase in the yeast Saccharomyces cerevisiae

Date Published: April 2, 2018

Publisher: Springer Berlin Heidelberg

Author(s): Aleksandra Bury, Klaas J. Hellingwerf.


Helical alignment of the α-helical linker of the LOV (light-oxygen-voltage) domain of YtvA from Bacillus subtilis with the α-helical linker of the histidine-protein kinase domain of the Sln1 kinase of the phospho-relay system for osmoregulation of Saccharomyces cerevisiae has been used to construct a light-modulatable histidine protein kinase. In vitro, illumination with blue light inhibits both the ATP-dependent phosphorylation of this hybrid kinase, as well as the phosphoryl transfer to Ypd1, the phosphoryl transfer domain of the Sln1 system. The helical alignment was carried out with conservation of the complete Jα helix of YtvA, as well as of the phosphorylatable histidine residue of the Sln1 kinase, with conservation of the hepta-helical motive of coiled-coil structures, recognizable in the helices of the two separate, constituent, proteins. Introduction of the gene encoding this hybrid histidine protein kinase into cells of S. cerevisiae in which the endogenous Sln1 kinase had been deleted, allowed us to modulate gene expression in the yeast cells with (blue) light. This was first demonstrated via the light-induced alteration of the expression level of the mannosyl-transferase OCH1, via a translational-fusion approach. As expected, illumination decreased the expression level of OCH1; the steady state decrease in saturating levels of blue light was about 40%. To visualize the in vivo functionality of this light-dependent regulation system, we fused the green fluorescent protein (GFP) to another regulatory protein, HOG1, which is also responsive to the Sln1 kinase. HOG1 is phosphorylated by the MAP-kinase-kinase Pbs2, which in turn is under control of the Sln1 kinase, via the phosphoryl transfer domain Ypd1. Fluorescence microscopy was used to show that illumination of cells that contained the combination of the hybrid kinase and the HOG1::GFP fusion protein, led to a persistent increase in the level of nuclear accumulation of HOG1, in contrast to salt stress, which—as expected—showed the well-characterized transient response. The system described in this study will be valuable in future studies on the role of cytoplasmic diffusion in signal transduction in eukaryotic cells.

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During the last decade of the previous century, progress in the dynamic resolution of protein structure, in the availability of genomic DNA sequence information, and in the synthetic biology of the heterologous production of complex holo-proteins, have brought our understanding of the molecular basis of cellular signal transduction networks down to the atomic level (see e.g. (Ridge et al. 2003)). This development was aided by the modular nature of many signal transduction proteins, which is particularly notable in the dominant type of prokaryotic signal transduction network, the ‘two-component regulatory system’, including its more complex variant, the ‘phosphorelay system’ (Nixon et al. 1986; Burbulys et al. 1991). In this development photosensory receptor proteins did play an important role because of the ease and accuracy with which these proteins can be (de)activated (for review see e.g. (van der Horst and Hellingwerf 2004; Hoff et al. 1997)). Understanding of the atomic basis of the structural and dynamic aspects of the transitions between the receptor- and the signalling state of signal transduction proteins then led to the development of rational and intuitive guidelines to combine functional (input/output) domains into new functional chimera’s, as could be concluded from analyses of their performance both in vitro and in vivo (Levskaya et al. 2005; Wu et al. 2009; Möglich et al. 2009).




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