1. The trimeric coiled‐coil HSBP1 protein promotes WASH complex assembly at centrosomes
MDA‐MB‐231 clones stably transfected with plasmids expressing control or HSBP1 targeting shRNAs were obtained. HSBP1‐depleted cells (shHSBP1) display significant downregulation of the CCDC53 and WASH subunits compared to control cells (shCtrl). Mean ± s.e.m. of densitometric signals; three independent experiments; ANOVA, *P < 0.05, **P < 0.01. Cytosolic extracts from control or HSBP1‐depleted cells were fractionated by ultracentrifugation in sucrose gradients, and fractions were analyzed by Western blot with the indicated antibodies. Three color‐coded pools containing CCDC53 were detected in control cells, whereas only the higher molecular weight pool was detected in HSBP1‐depleted cells. CCDC53 immunoprecipitations of the three pools from control cells were analyzed by Western blot using the indicated antibodies. IgG refers to the control immunoprecipitation performed with non immune IgG. Structural model of WASH complex assembly. HSBP1 promotes WASH complex assembly by dissociating the CCDC53 homotrimeric precursor and delivering a single CCDC53 molecule to the ternary complex composed of CCDC53, WASH, and FAM21. Based on the analogous structure of the WAVE complex, a new heterotrimeric coiled coil within WASH complexes, where CCDC53, WASH, and FAM21 contribute one strand each is proposed. Source data are available online for this figure.
IF THIS IMAGE HAS BEEN PROVIDED BY OR IS OWNED BY A THIRD PARTY, AS INDICATED IN THE CAPTION LINE, THEN FURTHER PERMISSION MAY BE NEEDED BEFORE ANY FURTHER USE. PLEASE CONTACT WILEY'S PERMISSIONS DEPARTMENT ON OR USE THE RIGHTSLINK SERVICE BY CLICKING ON THE 'REQUEST PERMISSIONS' LINK ACCOMPANYING THIS ARTICLE. WILEY OR AUTHOR OWNED IMAGES MAY BE USED FOR NON-COMMERCIAL PURPOSES, SUBJECT TO PROPER CITATION OF THE ARTICLE, AUTHOR, AND PUBLISHER.
EMBO J, Volume: 37, Issue: 13, First published: 29 May 2018, DOI: ( /embj )