ZDHHC5 interacts physically and functionally with DLG1 at primary cilia and regulates ciliary length and kidney morphology.

Introduction: Primary cilia are critical sensory organelles whose structure and composition are tightly regulated. The Scribble polarity complex protein DLG1 has recently been implicated in controlling both ciliary protein composition and length in mouse kidney epithelial cells. In addition, the palmitoyl transferase ZDHHC5 localizes to primary cilia and negatively regulates their length. However, the molecular mechanisms underlying DLG1-mediated ciliary regulation and its interaction network remain poorly defined. Methods: To identify cilium-associated DLG1 interactors, mouse inner medullary collecting duct (IMCD3) cells stably expressing DLG1-BioID2 were generated and subjected to proximity-dependent biotinylation proteomics in ciliated conditions. Candidate interactions were validated using immunoprecipitation, AlphaFold structural modeling, and quantitative immunofluorescence microscopy. Functional relevance was further assessed in vivo using zebrafish models with depletion of zdhhc5 paralogs. Results: Proximity labeling identified 46 high-confidence DLG1 interactors, of which 35 were previously associated with ciliary or centrosomal protein networks. Among these, ZDHHC5 was confirmed as a DLG1 interactor by immunoprecipitation, and structural modeling supported a potential direct interaction. Quantitative imaging demonstrated that ZDHHC5 localizes to primary cilia independently of DLG1. Additional analyses revealed that DLG1 also interacts with SCAMP3 and STXBP1 and is required for proper localization of STXBP1 at the ciliary base. In zebrafish, depletion of zdhhc5a and zdhhc5b resulted in ciliopathy-like phenotypes, including brain edema, curved body axis, and enlarged glomerular structures, accompanied by significantly elongated cilia in both the brain and pronephros. Discussion: These findings expand the DLG1 interactome and identify key cilia-associated partners, including ZDHHC5 and STXBP1, that may contribute to the regulation of ciliary structure and function. The data support a model in which DLG1 modulates ciliary length and signaling through specific protein-protein interactions, with disruption of this network leading to ciliopathy-related phenotypes. Together, this study provides new mechanistic insight into polarity complex-mediated control of primary cilia in kidney epithelial cells.