title: A Luciferase-fragment Complementation Assay to Detect Lipid Droplet-associated Protein-Protein Interactions.
abstract: A critical challenge for all organisms is to carefully control the amount of lipids they store. An important node for this regulation is the protein coat present at the surface of lipid droplets (LDs), the intracellular organelles dedicated to lipid storage. Only limited aspects of this regulation are understood so far. For the probably best characterized case, the regulation of lipolysis in mammals, some of the major protein players have been identified, and it has been established that this process crucially depends on an orchestrated set of protein-protein interactions. Proteomic analysis has revealed that LDs are associated with dozens, if not hundreds, of different proteins, most of them poorly characterized, with even fewer data regarding which of them might physically interact. To comprehensively understand the mechanism of lipid storage regulation, it will likely be essential to define the interactome of LD-associated proteins.Previous studies of such interactions were hampered by technical limitations. Therefore, we have developed a split-luciferase based protein-protein interaction assay and test for interactions among 47 proteins from Drosophila and from mouse. We confirmed previously described interactions and identified many new ones. In 1561 complementation tests, we assayed for interactions among 487 protein pairs of which 92 (19%) resulted in a successful luciferase complementation. These results suggest that a prominent fraction of the LD-associated proteome participates in protein-protein interactions.In targeted experiments, we analyzed the two proteins Jabba and CG9186 in greater detail. Jabba mediates the sequestration of histones to LDs. We successfully applied our split luciferase complementation assay to learn more about this function as we weree.g.able to map the interaction between Jabba and histones. For CG9186, expression levels affect the positioning of LDs. Here, we reveal the ubiquitination of CG9186, and link this posttranslational modification to LD cluster induction.
authors.affiliation: From the ‡Institute for Mathematical Modeling of Biological Systems, Heinrich Heine University, Duesseldorf, Germany.
authors.forename: Petra
authors.initials: P
authors.lastname: Kolkhof
authors.affiliation: From the ‡Institute for Mathematical Modeling of Biological Systems, Heinrich Heine University, Duesseldorf, Germany.|§Systems Biology of Lipid metabolism, Heinrich Heine University, Duesseldorf, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Werthebach
authors.affiliation: From the ‡Institute for Mathematical Modeling of Biological Systems, Heinrich Heine University, Duesseldorf, Germany.|§Systems Biology of Lipid metabolism, Heinrich Heine University, Duesseldorf, Germany.
authors.forename: Anna
authors.initials: A
authors.lastname: van de Venn
authors.affiliation: ¶Molecular Proteomics Laboratory, Institute for Molecular Medicine, Heinrich Heine University, Duesseldorf, Germany.|‖Biomedical Research Center (BMFZ), Heinrich Heine University, Duesseldorf, Germany.
authors.forename: Gereon
authors.initials: G
authors.lastname: Poschmann
authors.affiliation: **Department of Biology, University of Rochester, Rochester, New York.
authors.forename: Lili
authors.initials: L
authors.lastname: Chen
authors.affiliation: **Department of Biology, University of Rochester, Rochester, New York.
authors.forename: Michael
authors.initials: M
authors.lastname: Welte
authors.affiliation: ¶Molecular Proteomics Laboratory, Institute for Molecular Medicine, Heinrich Heine University, Duesseldorf, Germany.|‖Biomedical Research Center (BMFZ), Heinrich Heine University, Duesseldorf, Germany.
authors.forename: Kai
authors.initials: K
authors.lastname: Stühler
authors.affiliation: From the ‡Institute for Mathematical Modeling of Biological Systems, Heinrich Heine University, Duesseldorf, Germany; mathias.beller@hhu.de.|§Systems Biology of Lipid metabolism, Heinrich Heine University, Duesseldorf, Germany.
authors.forename: Mathias
authors.initials: M
authors.lastname: Beller
mesh_terms: D000818:Animals; D002265:Carboxylic Ester Hydrolases; D002352:Carrier Proteins / Q000378:metabolism*; D029721:Drosophila Proteins / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000378:metabolism*; D006657:Histones / Q000378:metabolism; D066292:Lipid Droplets / Q000378:metabolism*; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D025941:Protein Interaction Mapping / Q000379:methods*; D060066:Protein Interaction Maps; D054875:Ubiquitination
chemical_list: D002352:Carrier Proteins; D029721:Drosophila Proteins; D006657:Histones; C580936:jabba protein, Drosophila; D008156:Luciferases; D002265:Carboxylic Ester Hydrolases; C585047:sturkopf protein, Drosophila