TY - JOUR T1 - Structural and behavioral analysis reveals that Insomniac impacts sleep by functioning as a Cul3 adaptor JF - bioRxiv DO - 10.1101/689471 SP - 689471 AU - Qiuling Li AU - Kayla Y. Y. Lim AU - Nicholas Stavropoulos Y1 - 2019/01/01 UR - http://biorxiv.org/content/early/2019/07/02/689471.abstract N2 - The insomniac (inc) gene is required for normal sleep in Drosophila and encodes a conserved BTB protein that is a putative adaptor for the Cullin-3 (Cul3) ubiquitin ligase. Here we test whether Inc serves as a Cul3 adaptor by generating mutant forms of Inc and assessing their biochemical properties and physiological activity in vivo. We show that the N-terminal BTB domain of Inc is necessary and sufficient for Inc self-association and interactions with Cul3. Inc point mutations that weaken interactions with Cul3 impair the ability of Inc to rescue the sleep deficits of inc mutants, indicating that Cul3-Inc binding is critical for Inc function in vivo. Deletions of the conserved Inc C-terminus preserve Inc-Inc and Inc-Cul3 interactions but abolish Inc activity in vivo, implicating the Inc C-terminus as an effector domain that recruits Inc substrates. Mutation of a conserved C-terminal arginine similarly abolishes Inc function, suggesting that this residue is vital for the recruitment or ubiquitination of Inc targets. Mutation of the same residue in the human Inc ortholog KCTD17 is associated with myoclonic dystonia, indicating its functional importance in Inc family members. Finally, we show that Inc assembles into multimeric Cul3-Inc complexes in vivo and that depleting Cul3 causes accumulation of Inc, suggesting that Inc is negatively regulated by Cul3-dependent autocatalytic ubiquitination, a hallmark of Cullin adaptors. Our findings implicate Inc as a Cul3 adaptor and provide tools to identify the targets of Inc family proteins that impact sleep and neurological disorders. ER -