title: Proteomics of Primary Cilia by Proximity Labeling.
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.
authors.affiliation: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305-5345, USA.
authors.forename: David U
authors.initials: DU
authors.lastname: Mick
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Rachel B
authors.initials: RB
authors.lastname: Rodrigues
authors.affiliation: Stanford University Mass Spectrometry, Stanford University, Stanford, CA 94305, USA.
authors.forename: Ryan D
authors.initials: RD
authors.lastname: Leib
authors.affiliation: Stanford University Mass Spectrometry, Stanford University, Stanford, CA 94305, USA.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Adams
authors.affiliation: Stanford University Mass Spectrometry, Stanford University, Stanford, CA 94305, USA.
authors.forename: Allis S
authors.initials: AS
authors.lastname: Chien
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Steven P
authors.initials: SP
authors.lastname: Gygi
authors.affiliation: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305-5345, USA. Electronic address: nachury@stanford.edu.
authors.forename: Maxence V
authors.initials: MV
authors.lastname: Nachury
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D019390:Arrestins / Q000378:metabolism; D060387:Ascorbate Peroxidases / Q000737:chemistry / Q000378:metabolism*; D001692:Biological Transport; D002478:Cells, Cultured; D002923:Cilia / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D053823:Hedgehog Proteins / Q000378:metabolism; D007091:Image Processing, Computer-Assisted; D051379:Mice; D018345:Mice, Knockout; D008853:Microscopy; D008869:Microtubule-Associated Proteins / Q000502:physiology; D008969:Molecular Sequence Data; D015388:Organelles / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D020691:rab GTP-Binding Proteins / Q000502:physiology
chemical_list: C000605258:Arrb2 protein, mouse; D019390:Arrestins; C506280:Bbs1 protein, mouse; D053823:Hedgehog Proteins; D008869:Microtubule-Associated Proteins; D020543:Proteome; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; C000595889:intraflagellar transport 27 protein, mouse; D060387:Ascorbate Peroxidases; D017346:Protein Serine-Threonine Kinases; C491654:Stk11 protein, mouse; D017868:Cyclic AMP-Dependent Protein Kinases; D055372:AMP-Activated Protein Kinases; D020691:rab GTP-Binding Proteins