Developmental Cell
Volume 35, Issue 4, 23 November 2015, Pages 497-512
Journal home page for Developmental Cell

Resource
Proteomics of Primary Cilia by Proximity Labeling

https://doi.org/10.1016/j.devcel.2015.10.015Get rights and content
Under an Elsevier user license
open archive

Highlights

  • APEX labeling enables proteomic analyses of a non-membrane-enclosed compartment

  • Cilia-APEX identifies the kinases PKA, AMPK, and LKB1 in primary cilia

  • PKA functions inside cilia to phosphorylate GLI3 and regulate Hedgehog signaling

  • Proteomic profiling of Ift27/Bbs19 cilia detects ciliary accumulation of BBSome

Summary

While cilia are recognized as important signaling organelles, the extent of ciliary functions remains unknown because of difficulties in cataloguing proteins from mammalian primary cilia. We present a method that readily captures rapid snapshots of the ciliary proteome by selectively biotinylating ciliary proteins using a cilia-targeted proximity labeling enzyme (cilia-APEX). Besides identifying known ciliary proteins, cilia-APEX uncovered several ciliary signaling molecules. The kinases PKA, AMPK, and LKB1 were validated as bona fide ciliary proteins and PKA was found to regulate Hedgehog signaling in primary cilia. Furthermore, proteomics profiling of Ift27/Bbs19 mutant cilia correctly detected BBSome accumulation inside Ift27/ cilia and revealed that β-arrestin 2 and the viral receptor CAR are candidate cargoes of the BBSome. This work demonstrates that proximity labeling can be applied to proteomics of non-membrane-enclosed organelles and suggests that proteomics profiling of cilia will enable a rapid and powerful characterization of ciliopathies.

Cited by (0)