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Dev Cell. 2015 Nov 23;35(4):497-512. doi: 10.1016/j.devcel.2015.10.015. Epub 2015 Nov 12.

Proteomics of Primary Cilia by Proximity Labeling.

Author information

1
Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305-5345, USA.
2
Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
3
Stanford University Mass Spectrometry, Stanford University, Stanford, CA 94305, USA.
4
Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305-5345, USA. Electronic address: nachury@stanford.edu.

Abstract

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.

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PMID:
26585297
PMCID:
PMC4662609
DOI:
10.1016/j.devcel.2015.10.015
[Indexed for MEDLINE]
Free PMC Article

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