Protein-protein interactions (PPIs) drive virtually all biological processes, yet most PPIs have not been identified and even more remain structurally unresolved. We developed a two-step computational...

The temporospatial distribution of proteins within cilia is regulated by intraflagellar transport (IFT), wherein molecular trains shuttle between the cell body and cilium. Defects in this process impair various signal-transduction pathways and cause ciliopathies. Although K63-linked ubiquitination appears to trigger protein export from cilia, the mechanisms coupling polyubiquitinated proteins to IFT remain unclear. Using a multidisciplinary approach, we demonstrate that a complex of CFAP36, a conserved ciliary protein of previously unknown function, and ARL3, a GTPase involved in ciliary import, binds polyubiquitinated proteins and links them to retrograde IFT trains. CFAP36 uses a coincidence detection mechanism to simultaneously bind two IFT subunits accessible only in retrograde trains. Depleting CFAP36 accumulates K63-linked ubiquitin in cilia and disrupts Hedgehog signaling, a pathway reliant on the retrieval of ubiquitinated receptors. These findings advance our understanding of ubiquitin-mediated protein transport and ciliary homeostasis, and demonstrate how structural changes in IFT trains achieve cargo selectivity. ### Competing Interest Statement The authors have declared no competing interest. Sara Elizabeth O'Brien Trust Postdoctoral Fellowship awarded through the Charles A. King Trust Postdoctoral Research Fellowship Program, , 8460873-01 Richard and Susan Smith Family Foundation, https://ror.org/05j95n956, National Institute of General Medical Sciences (NIGMS), , R01GM141109, R01GM143183

The movement and pathogenicity of trypanosomatid species, the causative agents of trypanosomiasis and leishmaniasis, are dependent on a flagellum that contains an axoneme of dynein-bound doublet micro...