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Kidney Week

Abstract: PO1209

Interactions Between TULP3 and ARL13B in Lipidated Protein Transport to Cilia and Regulation of Renal Cystogenesis

Session Information

Category: Genetic Diseases of the Kidneys

  • 1001 Genetic Diseases of the Kidneys: Cystic

Authors

  • Mukhopadhyay, Saikat, The University of Texas Southwestern Medical Center, Dallas, Texas, United States
  • Palicharla, Vivek Reddy, The University of Texas Southwestern Medical Center, Dallas, Texas, United States
  • Hwang, Sun-hee, The University of Texas Southwestern Medical Center, Dallas, Texas, United States
Background

Signaling outputs from cilia maintain renal tubular homeostasis preventing cystogenesis; however, the ciliary proteins in this process are not well understood. The tubby family protein–TULP3 coordinates with the intraflagellar transport complex-A (IFT-A) in trafficking transmembrane proteins to cilia. Lack of Tulp3 in mouse promotes renal cystogenesis during embryogenesis but suppresses adult-onset polycystic kidney disease. Discovering cargoes of TULP3 and deciphering mechanisms underlying ciliary compartmentalization will help understand how signaling at and by cilia contributes to cystogenesis.

Methods

We used direct in vitro binding, proximity biotinylation and mass spectrometry to map TULP3-cargo interactions. We generated knockouts of Tulp3 and cargoes in kidney epithelial cells stably co-expressing different variants of TULP3 or cargoes, respectively. We generated conditional knockout of Tulp3 in mouse kidney nephrons using Cre-drivers and performed immunofluorescence for lipidated proteins with respect to kidney cystogenesis.

Results

The transmembrane cargoes have short motifs that are necessary and sufficient for TULP3-mediated trafficking. We now show that TULP3 is required for transport of the atypical GTPase ARL13B into cilia, and for ciliary enrichment of ARL13B-dependent farnesylated and myristoylated proteins. ARL13B transport requires TULP3 binding to IFT-A core but not to phosphoinositides, unlike transmembrane cargo transport that requires binding to both by TULP3. A conserved lysine in TULP3’s tubby domain mediates direct ARL13B binding and trafficking of lipidated and transmembrane cargoes. An N-terminal amphipathic helix in ARL13B flanking the palmitoylation site mediates binding to TULP3 and directs trafficking to cilia. Tulp3 trafficked lipidated proteins are depleted with distinctive temporal kinetics from kidney epithelial cilia during Tulp3 deletion-induced cystogenesis.

Conclusion

We conclude that TULP3 transports transmembrane proteins and ARL13B into cilia by capture of short sequences through a shared tubby domain site. Drugging this interaction domain could provide therapeutics in polycystic kidney disease. The depletion of lipidated cargoes with distinct kinetics from kidney epithelial cilia following Tulp3 deletion suggests their differential roles in cilia in regulating renal cystogenesis.

Funding

  • Other NIH Support