Tandem affinity purification of the BBSome, a critical regulator of Rab8 in ciliogenesis.
Bardet-Biedl syndrome (BBS) is a hereditary disorder whose symptoms include obesity, retinal degeneration, and kidney cysts. Intriguingly, the cellular culprit of BBS seems to lie in the primary cilium, a "cellular antenna" used by a number of signaling pathways. Yet, despite the identification of 12 BBS genes, a consistent molecular pathway for BBS had so far remained elusive. The recent discovery of a stable complex of seven BBS proteins (the BBSome) considerably simplifies the apparent molecular complexity of BBS and provides a clear insight into the molecular basis of BBS. Most tellingly, the BBSome associates with Rabin8, the guanine nucleotide exchange factor for the small GTPase Rab8, and Rab8-GTP enters the primary cilium to promote extension of the ciliary membrane. Thus, BBS is likely caused by defects in vesicular transport to the primary cilium. This chapter describes methods used to purify the BBSome using a tandem affinity purification method and presents a variation of this technique to demonstrate the existence of a stable complex of BBS proteins by sucrose gradient fractionation. When combined with state-of-the art mass spectrometry, these methods can provide a nearly complete BBSome interactome containing factors such as Rabin8.
Nachury
Maxence V
MV
Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California, USA.
D020788:Bardet-Biedl Syndrome / Q000503:physiopathology*; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity / Q000379:methods; D002923:Cilia / Q000502:physiology*; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008869:Microtubule-Associated Proteins; D010857:Pigment Epithelium of Eye / Q000166:cytology; D011506:Proteins / Q000235:genetics
C429655:BBS4 protein, human; D008869:Microtubule-Associated Proteins; D011506:Proteins