Lipid droplets as sites for protein sequestration

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DESCRIPTION (provided by applicant): Lipid droplets are intracellular organelles best known for their role in lipid storage and obesity. The goal of this proposal is to determine whether lipid droplets serve a general new function as sites of developmentally regulated protein sequestration. In Drosophila embryos, maternally provided histones appear to be stored on droplets until needed later in development. Various hints in the literature suggest that regulated sequestration - not only of histones but also of other proteins - to lipid droplets is wide spread in eukaryotes from yeast to mammals. Such sequestration could provide a novel mechanism of development, by releasing or inactivating proteins in a temporally or spatially controlled manner. It may also provide a general means by which cells protect themselves from harmful proteins. However, neither the generality nor the mechanisms of sequestration have been established, leaving the function and importance of sequestration unresolved. To determine whether proteins other than histones are sequestered in Drosophila embryos, two strategies will be pursued: it will be determined whether the candidate protein Importin alpha2 is sequestered to droplets in a developmentally regulated manner (by determining its intracellular distribution via immunolocalization and Western analysis of purified fractions) and whether proteotoxic stress induces sequestration of specific proteins (by proteomic analysis of isolated droplets). To uncover mechanisms of sequestration, it will be determined whether sequestered histones and Importin alpha2 carry signature post-translational modifications and which proteins on droplets the histones bind to. These studies will either identify existing mutants that disrupt sequestration or suggest strategies how to generate such mutants, thus leading to a functional test of the biological roles of sequestration, either immediately or in the near future.