Considering the Role of Disorder in Protein Interactions and Localization




Loe, Martin

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Though it is clear that a protein’s structure is intimately linked to its function, the role of disorder in protein function is increasingly becoming a focus in molecular biology. Liquid-liquid phase separation (LLPS), a process wherein proteins spontaneously condense into membraneless liquid droplets, is driven by disordered regions found within proteins. The histone chaperone protein Fpr4, found in Saccharomyces Cerevisiae, is predicted to undergo LLPS courtesy of its disordered loops, known as A1/B1, A2 and B2. There are a number of Fpr4 orthologues found in higher eukaryotes including humans. Hence, understanding the molecular complexities of Fpr4 LLPS in Saccharomyces Cerevisiae can have direct implications in understanding human molecular biology. Currently, it is unknown what proteins, if any, join these LLPS foci with Fpr4 and if these foci regulate the localization of Fpr4. A proximity-based labelling system called TurboID-3Myc was fused to the C-terminus of Fpr4 and a set of control proteins to investigate these questions. TurboID is a promiscuous biotin ligase that will biotinylate proximal proteins. Cellular fractionation in tandem with western blotting revealed no clear dependence on the disordered loops for Fpr4 localization. A streptavidin probe of the chromatin fraction suggested that a set of Fpr4 mutants have changed set of proximal proteins. Streptavidin bead capture and western blotting showed that TurboID-3Myc tagged Fpr1, Fpr3, and Fpr4, Sir2 and Heh1 each have a unique set of biotinylated proteins. Together, these suggest interacting proteins can be reliably labelled by TurboID and that upon mutation of Fpr4, biologically relevant changes occur to the interactome.



protein interactions, liquid-liquid phase separation, LLPS, protein disorder, yeast, Saccharomyces Cerevisiae, TurboID, Fpr4