Signalling is an important cellular process that requires fast and accurate protein interactions to efficiently transmit signals that ultimately lead to a cellular response, ensuring cell survival. Furthermore, the constant changes of environmental conditions raise the need for a cell to be able to transiently alter signal transduction and protein-protein interactions (PPIs). In this work we try to gain more insight into PPIs and the transient changes in signal transduction in the osmostress

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... hway of S. cerevisiae. The osmostress pathway is composed of two functionally redundant branches, and employs a mitogen activated protein kinase (MAPK) cascade, a central component of stress signalling. We particularly focussed our studies on transient protein-protein interactions occurring between membrane bound proteins and cytoplasmic proteins. Therefore, we developed a new protein interaction assay based on an enzymatic reaction between the two potential interaction partners. This method relies on a mammalian histone methyl transferase (HMT) and a 20 amino acid stretch of its substrate histone H3 protein. This system proved to be an in vivo protein interaction assay capable of catching transient changes of interaction rates of signalling proteins. Using this assay we discovered that the interaction of the membrane protein Sho1 and the MAPKKK Ste11 increases upon osmostress and that this interaction is enhanced in strains incapable of downstream signalling (Δpbs2, Δhog1). Furthermore this interaction depends on Ste50, an adaptor for Ste11, and the osmosensors Msb2 and Hkr1. However, this interaction was found to be independent of the membrane bound adaptor protein Opy2. Additionally, we discovered that the affinity of Sho1 and the MAPKK Pbs2 decreases upon pathway activation, which may reflect the dissociation of the Sho1-Pbs2 complex. We also investigated how pathway crosstalk is avoided, for the same proteins are shared in different signal transduction pathways. We confirmed that Fus1, a protein necessary for cell fus [...]