Interactions between TULP3 tubby domain and ARL13B amphipathic helix promote lipidated protein transport to cilia

Abstract

The primary cilium is a nexus for cell signaling and relies on specific protein trafficking for function. The tubby family protein–TULP3 transports integral membrane proteins into cilia through interactions with the intraflagellar transport complex-A (IFT-A) and phosphoinositides. We previously showed that short motifs called ciliary localization sequences (CLSs) are necessary and sufficient for TULP3-dependent ciliary trafficking of transmembrane cargoes. However, the mechanisms by which TULP3 regulates ciliary compartmentalization of non-integral, membrane-associated proteins, and if such trafficking requires TULP3-dependent CLSs is unknown. Here we show that TULP3 is required for ciliary transport of the Joubert syndrome-linked palmitoylated GTPase–ARL13B through a CLS. An N-terminal amphipathic helix, preceding the GTPase domain of ARL13B, couples with the TULP3 tubby domain for ciliary trafficking, irrespective of palmitoylation. ARL13B transport requires TULP3 binding to IFT-A but not to phosphoinositides, indicating strong membrane-proximate interactions, unlike transmembrane cargo transport requiring both properties of TULP3. TULP3-mediated trafficking of ARL13B also regulates ciliary enrichment of farnesylated and myristoylated downstream effectors of ARL13B. The lipidated cargoes show distinctive depletion kinetics from kidney epithelial cilia with relation to Tulp3 deletion-induced renal cystogenesis. Overall, these findings indicate an expanded role of the tubby domain in capturing analogous helical secondary structural motifs from diverse cargoes.

Human IFT-A complex structures provide molecular insights into ciliary transport

“…The molecular docking also revealed the membrane orientation of IFT-A complexes. As expected, the last resolved residue of TULP3, which is linked to the cargo-interacting tubby domain, 18,22 points toward the membrane (Fig. 5d).…”

“…To test how IFT-A–TULP3 interactions affect cargo transportation, we mutated the TULP3 interface residues at both the helical and loop regions and examined cargo trafficking in the mutants. By stably expressing wild-type (WT) and helical/loop mutants of TULP3 in a Tulp3 KO IMCD3 cell line, 22 we monitored the ciliary levels of ARL13B (a palmitoylated cargo) and GPR161, whose ciliary transportation is dependent on TULP3. 18 , 22 , 23 , 54 , 55 We determined that mutations in the TULP3 helical region (K41A/K42A/R43A), loop region (F47D/V49D) or a combination of both (K41A/K42A/V49D) could significantly disrupt TULP3–IFT-A interaction and reduce ARL13B and GPR161 trafficking to cilia (Fig.…”

“…By stably expressing wild-type (WT) and helical/loop mutants of TULP3 in a Tulp3 KO IMCD3 cell line, 22 we monitored the ciliary levels of ARL13B (a palmitoylated cargo) and GPR161, whose ciliary transportation is dependent on TULP3. 18 , 22 , 23 , 54 , 55 We determined that mutations in the TULP3 helical region (K41A/K42A/R43A), loop region (F47D/V49D) or a combination of both (K41A/K42A/V49D) could significantly disrupt TULP3–IFT-A interaction and reduce ARL13B and GPR161 trafficking to cilia (Fig. 4b, c ; Supplementary information, Figs.…”

“…21 The tubby family protein, TULP3, functions as an adapter linking IFT-A to cargos in mammalian cells. 16,18,22 IFT-A and TULP3 mediate ciliary transport of the orphan GPCR, GPR161, which has been implicated in the negative regulation of vertebrate hedgehog signaling. [23][24][25][26] In Drosophila, the altered localization of TRPV channels in the specialized mechanosensory chordotonal cilia, 27 caused by IFT-A mutations, phenocopies the deficiency of the tubby homolog, dTULP.…”

Ciliary ARL13B inhibits developmental kidney cystogenesis in mouse

“…ARL13B localization in renal cilia requires TULP3, which is not the case in other cell types (Ferent et al, 2019;Hwang et al, 2019;Legue and Liem, 2019;Palicharla et al, 2023). Previous work on TULP3 proposed that it regulates a cilia-specific pathway that normally inhibits cystogenesis, termed the cilia localized cyst inhibitory (CLCI) pathway (Walker et al, 2022).…”