A Luciferase-fragment Complementation Assay to Detect Lipid Droplet-associated Protein-Protein Interactions

Petra Kolkhof,Michael Werthebach,Anna Van De Venn,Gereon Poschmann,Lili Chen,Michael Welte,Kai Stühler,Mathias Beller

doi:10.1074/mcp.m116.061499

Petra Kolkhof, Michael Werthebach + Show 6 more

Open Access

https://doi.org/10.1074/mcp.m116.061499

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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 were e.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.

Highlights

Previous studies of such interactions were hampered by technical limitations
Bmm:GFP positive cells showed no or only very little Lipid droplets (LDs) deposits, suggesting that overexpression of the active lipase depleted the lipid stores. Consistent with this hypothesis, we found a clear-cut localization of Brummer to LDs when we treated the cells with a short pulse of high oleic acid (OA) amounts prior to fixation of the cells (Fig. 1C)
We report the utilization of a Gaussia princeps split luciferase complementation based protein-protein interaction (PPI) assay to study interactions of LD-associated proteins

Summary

Introduction

Previous studies of such interactions were hampered by technical limitations. we have developed a split-luciferase based protein-protein interaction assay and test for interactions among 47 proteins from Drosophila and from mouse. We performed more than 1500 PPI assays testing for interactions among 487 luciferase fragment fusion protein pairs (covered by 830 different construct combinations). Each plasmid combination was tested in triplicate in each assay plate, which always included controls in the form of [1] untransfected cells, [2] the published Gcn4 leucine zipper interaction [16] for thresholding purposes, and [3] the robust interaction between the LD-associated proteins Jabba [21] and CG9186 [22].

Results

Conclusion