Analysis of the spatial distribution of the key components of the HOG signaling pathway, TcsC and Ypd1, and its impact on their function in the pathogenic mold Aspergillus fumigatus.

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The High Osmolarity Glycerol (HOG) pathway is particular interesting, firstly, as a paradigm for a fungal signaling cascade and secondly, because its sensing kinase TcsC represents an attractive target structure for new antifungal therapies. Our aim is to study the spatial organization of the initial part of the HOG pathway, the so-called two-component phosphorelay, and to define its impact on the function of the respective proteins, e.g. their ability to organize an adaptive response to hyperosmotic stress. Our preliminary results show novel and unexpected localization patterns for two key components of HOG two-component phosphorelay in A. fumigatus, TcsC and Ypd1. This led us to a new hypothesis, and the proposed project aims to verify our concept:The fact that the sensor kinase TcsC resides in the nucleus at ambient conditions and translocates to the cytoplasm upon activation provides a strong hint that Aspergillus senses hyperosmotic stress in the nucleus. We hypothesize that this takes place at the nuclear membrane and involves a yet uncharacterized A. fumigatus SUN-domain protein that in turn is assumed to be part of a structure that connects the nuclear membrane to the cytoskeleton. If correct, this would establish a completely new model of how filamentous fungi sense hyperosmotic stress. In this project, we will also define the impact of a potential TcsC multimerization on its function and shuttling between the nucleus and the cytoplasm. Finally, we will determine the functional relevance of the observed translocation of activated Ypd1 from the cytoplasm to the nucleus and define the relevant domains/motifs of Ypd1.