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

Aleksandra Bury,Klaas J Hellingwerf

doi:10.1186/s13568-018-0582-7

Abstract

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.

Highlights

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 productionBury and Hellingwerf AMB Expr (2018) 8:53 and accuracy with which these proteins can beactivated (for review see e.g.)
In vitro phosphorylation assays The helical linker regions of YtvA and Sln1 were aligned according to the hepta-helical pattern of the coiled-coil structure that presumably is present in both of them, and joined in several different ways (Additional file 1: Table S1 and Fig. 2a), i.e. with preservation of the (Jα-) helix from either protein completely, or partially; with or without insertion of extra amino acids to translationally shift the hepta-helical pattern and with or without conservation of the position of the crucial phosphorylatable histidine of the Sln1 kinase domain
The resulting hybrid kinases, with the truncated Sln1 kinase domain as a reference, were assayed for kinase activity in the dark with the classical kinase assay based on the use of 32P[ATP]

Summary

Introduction

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 productionBury and Hellingwerf AMB Expr (2018) 8:53 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 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) These technical developments, and the derived improved insight, have led to the emergence of the interdisciplinary research field of ‘optogenetics’ (Miller 2006; Ernst et al 2008; Zhang et al 2010). This latter aspect is dictated by association/dissociation kinetics of the underlying physicochemical signals (e.g. an electric field or osmotic pressure), signaling molecules and signal-transmission- and output proteins, and by the processes of classical- and/or anomalous diffusion of all these components, either in the cytoplasm or in the cytoplasmic membrane, with possibly additional effects of molecular crowding

Methods

Results

Conclusion