Systematic VCP-UBXD Adaptor Network Proteomics Identifies a Role for UBXN10 in Regulating Ciliogenesis

The VCP AAA-ATPase, an evolutionarily conserved ATP-driven “segregase”, is a central regulator of protein quality control and ubiquitin-mediated signaling1. VCP functions in diverse processes such as ER associated degradation, macro and selective autophagy and DNA damage responses (DDR)2,3. ATP hydrolysis by VCP remodels protein complexes, unfolds ubiquitylated substrates for degradation, and extracts proteins embedded in membranes or chromatin. The prevailing model suggests that specific adaptor proteins target VCP to cellular structures and substrates where VCP activity is required3. The largest family of adaptors contains the ubiquitin X domain (UBXD), which adopts a ubiquitin fold and associates with the N-terminus of VCP (Fig. 1a)4. A subset of UBXD proteins also contain ubiquitin associated domains (UBA), to recognize polyubiquitin chains on substrates5. Other cofactors utilize shorter motifs to associate with VCP6. Some studies have suggested the involvement of distinct adaptors in specific processes, however UFD1L-NPLOC4 association with VCP is required in some cases for adaptor binding7. Figure 1 Proteomic Analysis of the VCP-UBXD adaptor interaction network Our understanding of how VCP is targeted to substrates is limited and only a subset of adaptors have been studied in detail. Even adaptors such as UBXN7 whose role in Cullin RING Ligase (CRL) function has been reported, the full repertoire of targets and biological pathways remain unknown. VCP is mutated in human neurodegenerative disorders including Inclusion Body Myopathy, Paget's disease of the bone and Frontotemporal Dementia (IBMPFD) and Amyotrophic Lateral Sclerosis (ALS)8,9. A significant fraction of disease-specific mutations cluster to the N-terminus where adaptors bind, suggesting that alterations in the repertoire of adaptors associated with VCP could contribute to disease10,11. Thus, a more complete understanding of adaptors and their targets is needed to fully understand VCP function. Here, we employed a previously described interaction proteomics platform12,13 to analyze the VCP-UBXD interaction landscape, including several adaptors that are hitherto unstudied. The VCP-adaptor network is linked with a vast array of biological processes and we explore a interaction between the unstudied adaptor UBXN10 and the intraflagellar transport B (IFT-B) complex involved in cilia biogenesis. Cilia are microtubule-based structures that are required for proper signaling in virtually every cell of the human body14. Defects in cilia formation or function cause a host of multi-system disorders referred to as ciliopathies. Cilia are assembled and disassembled by distinct IFT complexes that regulate anterograde (IFT-B) and retrograde (IFT-A) trafficking of ciliary components15,16 in a Bardet–Biedl syndrome (BBSome)17 complex-dependent manner. While hundreds of candidate ciliary proteins have been identified, their functions remain largely uncharacterized18–20. Through biochemical studies coupled with genetic and pharmacological disruption of VCP-UBXN10 function, we reveal a role for this complex in promoting ciliogenesis in mammalian cells in culture and in zebrafish. The VCP-UBXD network elaborated here provides a resource for further elucidation of the targeting mechanisms and biological functions of VCP.