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Biochemical and structural analysis of a eukaryotic "two-component" signal transduction pathway
In prokaryotic organisms, adaptive responses to environmental changes such as nutrient limitation, oxygen deprivation, and osmotic shock are regulated by so-called "two-component" signal transduction pathways. These systems have in common a biochemical strategy involving phosphoryl transfer between two protein components: a histidine protein kinase and a response regulator protein. The histidine kinase is commonly a transmembrane receptor that upon ligand stimulation undergoes autophosphorylation of a specific histidine residue within the cytoplasmic signaling domain. The phosphoryl group is then transferred to a specific aspartic acid residue on the response regulator protein. Phosphorylation of the response regulator results in the activation of either an associated or downstream effector function.
Two-component signal transduction pathways were long thought to be restricted to the bacterial kingdom. However, it is now recognized that several eukaryotic organisms, such as plants and fungi, have proteins that are homologous to the bacterial histidine kinases and response regulators. Research in our laboratory focuses on the investigation of a two-component signaling pathway in yeast, Saccharomyces cerevisiae, that regulates cellular responses to osmolarity changes in the environment. With higher complexity than typical two-component signaling pathways, osmoregulation in yeast is mediated by a multi-step phosphorelay mechanism involving three proteins, SLN1, YPD1, and SSK1. 
The histidine kinase, SLN1, is a transmembrane receptor that senses changes in osmotic pressure across the plasma membrane. SLN1 is a hybrid protein composed of an N-terminal region that shares sequence homology to the bacterial histidine kinase family and a C-terminal region that has all the conserved features of the regulatory domain of bacterial response regulator proteins. SSK1 is a response regulator protein consisting of a C-terminal regulatory domain that is subject to phosphorylation and an N-terminal domain(s) of unknown function. The YPD1 protein, also referred to as a histidine-containing phosphotransfer or HPt protein, acts a phosphoprotein intermediate in the transfer of a phosphoryl group from SLN1 to two downstream response regulators, SKN7 and SSK1. The SKN7 protein is a transcription factor that is involved in several other important cellular processes, including environmental stress responses and cell cycle regulation.
The yeast osmoregulation pathway represents a useful model system for investigating, at a biochemical and structural level, the role of phosphorylation and dephosphorylation in regulating protein function. The novelty of this system is that it combines the bacterial two-component paradigm of histidine to aspartate phosphoryl transfer (the SLN1-YPD1-SSK1/SKN7 phosphorelay) with serine/threonine/tyrosine phosphorylation of the proteins of the MAP kinase cascade which is more commonly observed in eukaryotic organisms. Experiments are directed at determining the functional importance of each of the domains of SLN1, YPD1, SSK1, and SKN7 by molecular genetics, biochemical analysis of the phosphoryl transfer reactions, and protein structure analysis by X-ray crystallography. For further information, please contact Dr. West Full List of Publications: view or download |