A luciferase-fragment complementation assay to detect lipid droplet-associated protein-protein interactions

The regulation of organismic lipid storage amounts is a central goal of all organisms. Proteins associated with the dedicated lipid storage organelles, the so-called lipid droplets (LDs), are instrumental to this regulation. LD proteomes of diverse organisms have been characterized by mass spectrometry. Yet, the functional characterization of most of these proteins is still lagging behind. Moreover, knowledge of interactions among LD-associated proteins is sparse and mostly limited to proteins involved in mammalian lipolysis regulation. This process crucially depends on an orchestrated set of protein-protein interactions which limit the accessibility, and thus remobilization, of the lipid stores. Thus, a better knowledge of the LD-associated protein interactome likely reveals entry points to deeper understand lipid storage regulation on a mechanistic level. Here, we utilize a split-luciferase based protein-protein interaction assay to identify interactions among 46 LD-associated proteins and protein variants of which 40 are Drosophila and 6 mouse proteins. In 1531 complementation tests, we assayed for interactions among 487 luciferase fragment fusion protein pairs (covered by 830 different construct combinations) of which 85 (17.45 %) resulted in a significant luciferase complementation. These results suggest, that a prominent fraction of the LD-associated proteome participates in protein-protein interactions, and provide a basis for further functional studies. Besides confirming previously described interactions, we identified several new protein interaction pairs. Of these, we characterize the following three in greater detail: (i) Jabba and CG9186, (ii) Jabba and Histones and (iii) Ubiquitin and CG9186. The interaction among Jabba and Histones is of physiological significance and we provide data suggesting that Jabba alone is sufficient to recruit Histones to LDs. Further, we identified a positively charged amino acid stretch of 16 amino acids within the Jabba protein which is necessary for the Jabba-Histone interaction. CG9186 is an annotated esterase which is involved in lipid storage amount regulation and positioning of LDs. We present data supporting the ubiquitination of at least two lysine residues of CG9186 and demonstrate a role of CG9186 ubiquitination in the induction of LD clusters linked to the overexpression of CG9186.