IFT88 Knockout Cell Line (HEK293)

UFMylation, a post-translational modification involving the covalent conjugation of ubiquitin-fold modifier 1 (UFM1) to target proteins, has been implicated in a wide spectrum of human diseases. However, the underlying molecular mechanisms are poorly understood. Herein, we demonstrate that UFM1-specific ligase 1 (UFL1), the sole ligase for UFMylation, is indispensable for ciliary homeostasis. Genetic ablation of UFL1 in mice results in severe defects in ciliary structure and function in multiple tissues. Mechanistic investigation reveals that intraflagellar transport 88 (IFT88), a protein essential for ciliary assembly and maintenance, undergoes UFMylation at lysine 572. The UFMylation antagonizes IFT88 ubiquitination by Praja ring finger ubiquitin ligase 2 (PJA2), thereby preventing its proteasomal degradation. The lysine 572-to-arginine mutant of IFT88 exhibits increased stability and efficacy in rescuing ciliary defects induced by UFL1 depletion. Our findings identify a critical role for IFT88 UFMylation in ciliary homeostasis and offer novel insights into human ciliopathies.

Cilia sense and transduce extracellular signals via specific receptors. The intraflagellar transport (IFT) machinery mediates not only bidirectional protein trafficking within cilia but also the import/export of ciliary proteins across the ciliary gate. The IFT machinery is known to comprise two multisubunit complexes, namely, IFT-A and IFT-B; however, little is known about how the two complexes cooperate to mediate ciliary protein trafficking. We here show that IFT144-IFT122 from IFT-A and IFT88-IFT52 from IFT-B make major contributions to the interface between the two complexes. Exogenous expression of the IFT88(Δα) mutant, which has decreased binding to IFT-A, partially restores the ciliogenesis defect of -knockout (KO) cells. However, IFT88(Δα)-expressing -KO cells demonstrate a defect in IFT-A entry into cilia, aberrant accumulation of IFT-B proteins at the bulged ciliary tips, and impaired import of ciliary G protein-coupled receptors (GPCRs). Furthermore, overaccumulated IFT proteins at the bulged tips appeared to be released as extracellular vesicles. These phenotypes of IFT88(Δα)-expressing -KO cells resembled those of -KO cells. These observations together indicate that the IFT-A complex cooperates with the IFT-B complex to mediate the ciliary entry of GPCRs as well as retrograde trafficking of the IFT machinery from the ciliary tip.