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

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 c...

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Main Authors: Aleksandra Bury, Klaas J. Hellingwerf
Format: Article
Language:English
Published: SpringerOpen 2018-04-01
Series:AMB Express
Subjects:
Online Access:http://link.springer.com/article/10.1186/s13568-018-0582-7
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author Aleksandra Bury
Klaas J. Hellingwerf
author_facet Aleksandra Bury
Klaas J. Hellingwerf
author_sort Aleksandra Bury
collection DOAJ
description 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.
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spelling doaj.art-9a1f6962f68e42fe897ed26782a296c62022-12-21T19:53:26ZengSpringerOpenAMB Express2191-08552018-04-018111110.1186/s13568-018-0582-7Design, characterization and in vivo functioning of a light-dependent histidine protein kinase in the yeast Saccharomyces cerevisiaeAleksandra Bury0Klaas J. Hellingwerf1Molecular Microbial Physiology Group, Swammerdam Institute for Life Sciences, University of AmsterdamMolecular Microbial Physiology Group, Swammerdam Institute for Life Sciences, University of AmsterdamAbstract 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.http://link.springer.com/article/10.1186/s13568-018-0582-7YtvACytoplasmic diffusionSln1Wall stressTwo-component regulation systemNuclear shuttling
spellingShingle Aleksandra Bury
Klaas J. Hellingwerf
Design, characterization and in vivo functioning of a light-dependent histidine protein kinase in the yeast Saccharomyces cerevisiae
AMB Express
YtvA
Cytoplasmic diffusion
Sln1
Wall stress
Two-component regulation system
Nuclear shuttling
title Design, characterization and in vivo functioning of a light-dependent histidine protein kinase in the yeast Saccharomyces cerevisiae
title_full Design, characterization and in vivo functioning of a light-dependent histidine protein kinase in the yeast Saccharomyces cerevisiae
title_fullStr Design, characterization and in vivo functioning of a light-dependent histidine protein kinase in the yeast Saccharomyces cerevisiae
title_full_unstemmed Design, characterization and in vivo functioning of a light-dependent histidine protein kinase in the yeast Saccharomyces cerevisiae
title_short Design, characterization and in vivo functioning of a light-dependent histidine protein kinase in the yeast Saccharomyces cerevisiae
title_sort design characterization and in vivo functioning of a light dependent histidine protein kinase in the yeast saccharomyces cerevisiae
topic YtvA
Cytoplasmic diffusion
Sln1
Wall stress
Two-component regulation system
Nuclear shuttling
url http://link.springer.com/article/10.1186/s13568-018-0582-7
work_keys_str_mv AT aleksandrabury designcharacterizationandinvivofunctioningofalightdependenthistidineproteinkinaseintheyeastsaccharomycescerevisiae
AT klaasjhellingwerf designcharacterizationandinvivofunctioningofalightdependenthistidineproteinkinaseintheyeastsaccharomycescerevisiae