Lipid droplets as protein sequestration sites

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Project summary Lipid droplets are the intracellular sites for fat storage, with essential functions in lipid metabolism and energy storage. They also have a second major role in controlling the fate of specific proteins, namely mediating their maturation, transport, refolding, storage, and turnover. While there has been great progress in unraveling how droplets control lipid metabolism, studies of their protein-handling roles remain limited. The goal of this project is to dissect the mechanism, regulation, and physiological relevance of this protein handling function, by taking advantage of the best characterized example of this phenomenon, the sequestration of histone H2Av to lipid droplets in Drosophila ovaries and embryos. H2Av is dynamically stored, exchanging constantly between lipid droplets via the cytoplasm. Lipid droplet sequestration prevents H2Av degradation in oocytes and its premature import into nuclei in embryos. It is also developmentally regulated, being switched off in embryos at the mid- blastula transition. Progress in the last granting period identified the importins Impa2 and Ipo9 as critical factors mediating H2Av exchange, uncovered a correlation between the activity of the cell cycle kinase Cdk1, the phosphorylation state of Impa2, and the rate of H2Av exchange, and discovered that excess nuclear H2Av dramatically alters the transcriptome. These insights led to new models about the mechanism of H2Av exchange, its regulation, and its biological role, models that the current application proposes to test. It is hypothesized that Impa2 promotes H2Av exchange by physically interacting with Jabba and reducing its affinity to H2Av and that Ipo9 acts as cytoplasmic chaperone, accompanying H2Av on its journey between LDs. This model will be tested by determining intracellular distribution and dynamics of the two importins in ovaries and embryos and by analyzing how mutants in key functional domains affect H2Av exchange. To understand the developmental regulation, point mutants in Impa2 will be generated to prevent or mimic phosphorylation and Cdk1 activity will be inhibited. The consequences of these manipulations for H2Av exchange and the physical interactions between importins, the H2Av anchor Jabba, and H2Av will be determined. In nurse cells lacking Jabba, absence of sequestration results in increased levels of nuclear H2Av. Using RNA seq analysis, manipulation of H2Av dosage, and Jabba mutants unable to bind H2Av, it will be determined whether the altered H2Av levels result in changes to the transcriptome. If successful, these studies will test key predictions about the mechanism, regulation, and function of H2Av sequestration by lipid droplets and develop general paradigms for how protein handling by lipid droplets can control processes elsewhere in the cell. These studies will thus provide the groundwork for determining if and how protein sequestration by lipid droplets contributes to their prominent roles in health and disease.