Fig. 4
From: USP32 regulates late endosomal transport and recycling through deubiquitylation of Rab7
Ubiquitome analysis reveals small GTPase Rab7 as a substrate of USP32. a Schematic representation of stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative mass spectrometry (LC-MS/MS) workflow used to compare ubiquitylated proteomes of b control MelJuSo cells (siCtrl, green) vs. those where USP32 was knocked down (KD, siUSP32_2, magenta) and c HeLa cells overexpressing (OE) USP32-HA (magenta) vs. vector control (green). Cell growth media types: K0R0, light; K4R6, medium; K8R10, heavy. IP: immunoprecipitation, m/z: mass to charge ratio. b, c Volcano plots comparing abundance of detected peptides carrying a GlyGly (GG) modification expressed as Log 2 ratios of b USP32 knockdown (siUSP32_2, KD) vs. control (siCtrl, CTRL), n = 1 SILAC sample set independently validated using label-free quantitation (LFQ) with n = 2 biologically independent samples or c USP32-HA overexpression vs. vector control (Ctrl), n = 1 SILAC sample set. Small GTPases implicated in vesicular traffic whose modified peptides were detected are labeled according to their respective Log 2 ratios: magenta >1; green <−1; blue between −1 and 1, not significant. Analysis was performed using MaxQuant and Perseus software tools as described in the Methods under ubiquitome analysis. All mass spectrometry (MS) data can be accessed via the PRIDE repository (PXD011899). d Ubiquitylation status of GFP-Rab7 vs. GFP-Rab5 as a function of USP32 catalytic activity. GFP-Rabs, immunoprecipitated (IP) from HEK293T cells coexpressing HA-Ub and either USP32, C743A, or neither, was assessed by immunoblot against HA; WCL: whole cell lysate. See also Supplementary Fig. 5
