The Cul3 ubiquitin ligase engages Insomniac as an adaptor to impact sleep and synaptic homeostasis

Mutations of the Cullin-3 (Cul3) E3 ubiquitin ligase are associated with autism and schizophrenia, neurological disorders characterized by sleep disturbances and altered synaptic function. Cul3 engages dozens of adaptor proteins to recruit hundreds of substrates for ubiquitination, but the adaptors that impact sleep and synapses remain ill-defined. Here we implicate Insomniac (Inc), a conserved protein required for normal sleep and synaptic homeostasis in Drosophila , as a Cul3 adaptor. Inc binds Cul3 in vivo, and mutations within the N-terminal BTB domain of Inc that weaken Inc-Cul3 associations impair Inc activity, indicating that Inc function requires binding to the Cul3 complex. Deletion of the conserved C-terminus of Inc does not alter Cul3 binding but abolishes Inc activity in the context of sleep and synaptic homeostasis, suggesting that the C-terminal domain of Inc is a substrate recruitment domain. Mutation of a conserved, disease-associated arginine in the Inc C-terminus also abolishes Inc function, suggesting that this residue is vital for recruiting Inc targets. Inc levels are negatively regulated by Cul3 in neurons, consistent with Inc degradation by autocatalytic ubiquitination, a hallmark of Cullin adaptors. These findings link Inc and Cul3 in vivo and indicate that Cul3-Inc complexes are essential for normal sleep and synaptic function. Furthermore, these results indicate that dysregulation of conserved substrates of Cul3-Inc complexes may contribute to altered sleep and synaptic function in autism and schizophrenia associated with Cul3 mutations. Author Summary Cul3 is a highly conserved gene important for brain development and function. Cul3 mutations are a risk factor for autism and schizophrenia, neurological disorders associated with disturbed sleep and changes in neuronal synapses. A key challenge in understanding how Cul3 impacts brain function is elucidating the downstream molecular pathways. Cul3 is a ubiquitin ligase that assembles with dozens of adaptor proteins, which in turn recruit specific protein substrates for ubiquitination. Identifying and characterizing Cul3 adaptors in the nervous system is thus a critical step in understanding Cul3 function. Because Cul3 and its adaptors are conserved through evolution, simpler organisms including the fruit fly Drosophila provide powerful systems for identifying and characterizing Cul3 adaptors. We found that the Insomniac (Inc) protein has the properties of a Cul3 adaptor that impacts sleep and synaptic function in Drosophila . These results suggest that human proteins related to Inc may be relevant for changes in sleep and synaptic function in autism and schizophrenia associated with reduced Cul3 activity.