Eukaryotic Cell Signaling: Modulation of Molecular Microenvironments through the Scaffold Protein Asc1/RACK1

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In eukaryotes, the scaffold protein Asc1/RACK1 connects ribosomes with the network of signal transduction. Cell proliferation and differentiation are significantly affected by Asc1/RACK1. Both the ribosomal mRNA translation rate and the homeostasis of ribosomes are influenced by the WD40 repeat propeller protein. Asc1/RACK1 locates to the regulatory head region of the ribosomal 40S subunit and is substrate for phosphorylation. This project aims at the identification of kinases phosphorylating Asc1 in Saccharomyces cerevisiae, and the impact of stress/signal-induced Asc1 phosphorylation on its molecular proximity. The composition of protein modules at the head region of the ribosomal 40S subunit will be analyzed with the Biotin-dependent IDentification (BioID) approach, e.g. from the perspective of ribosomal neighbors (i.e. Rps2). Using the BioID approach with hTERT-RPE1 cells the modular composition of the RACK1 microenvironment in human cells will be compared to that of Asc1 in S. cerevisiae. The impact of Epidermal Growth Factor (EGF) and EGF receptor-inhibitors on the molecular proximity of RACK1 will be also studied in hTERT-RPE1 cells. In yeast, also context-specific sub-complexes of Asc1 with already identified proximal proteins (e.g. Bre5-Ubp3, Hel2, Stm1, Def1) will be characterized using Split-BioID and Biomolecular Fluorescence Complementation (BiFC). Moreover, signals for Asc1-homodimer formation and its molecular consequences will be studied. The impact of Asc1 on the ubiquitylation of proximal but also non-proximal proteins will be analyzed using a global ubiquitylome approach. The results of this project will lead to a substantial improvement of the general understanding of the molecular action of beta-propeller proteins during signal transduction and of Asc1/RACK1-specific ribosomal regulation of gene and protein expression.