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 were 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.

Lipid droplet functions beyond energy storage

“…Curiously, the core histones H3 and H4 are similarly overabundant, but present in a different, as yet uncharacterized location. LD localization of histones is mediated by the novel protein Jabba, which likely acts as histone anchor: Jabba physically interacts with these histones, histones are absent from embryonic LDs in Jabba mutants, and the expression of Jabba in cultured Drosophila cells is sufficient to induce recruitment of histones to LDs [156, 157]. …”

“…Restriction of both organelles to the same 1D tracks may promote enhanced interactions between them. Clustering of LDs is also frequently observed when certain droplet-localized proteins are up- or downregulated or posttranslationally modified, such as PLIN1 [212, 226], ancient ubiquitous protein 1 (AUP1 [227]), and CG9186 [157, 228]. To what extent such clustering involves altered droplet motility has yet to be examined but, for U2OS cells, it was recently shown that dissociation of clustered droplets involves actin and myosin [229].…”

Establishing the lipid droplet proteome: Mechanisms of lipid droplet protein targeting and degradation

“…The clustering phenotype was dependent on the ubiquitination status of AUP1, since overexpression of a CUE domain mutant that was not ubiquitinated did not induce clustering, but fusion of this mutant to ubiquitin at its C-terminus rescued AUP1-induced clustering [136]. Similarly, ubiquitination of Drosophila LD protein CG9186 in Kc167 cells was required for LD clustering induced by CG9186 overexpression [137,138]. A model that explains the clustering phenotype observed in these studies is that ubiquitinated AUP1 or CG9186 promotes the formation of ubiquitin-dependent homo- or hetero-dimers that bridge adjacent LDs.…”

Arabidopsis lipid droplet‐associated protein (LDAP) – interacting protein (LDIP) influences lipid droplet size and neutral lipid homeostasis in both leaves and seeds

“…Given that LDAPs are involved in multiple aspects of LD biology, and that LD coat proteins are known to function in general as part of highly regulated protein-protein interaction networks in other organisms (Tsai et al, 2015;Kolkhof et al, 2017), we employed Arabidopsis LDAP3 as 'bait' in a yeast two-hybrid (Y2H) assay screen to identify new proteins involved in biogenesis and/or function of plant LDs. We chose LDAP3 as the specific 'bait' since it is the most highly and ubiquitously expressed LDAP gene in Arabidopsis, including in seeds (Gidda et al, 2016).…”