Figure 2 : USP30 is a Ub-specific deubiquitylase that prefers compact chain types.

From: USP30 and parkin homeostatically regulate atypical ubiquitin chains on mitochondria

Figure 2

(a) Lineweaver–Burk analysis of the catalytic domains of USP30, USP7 and USP2, obtained by measuring the initial rates at varying Ub-AMC concentrations. Error bars represent s.e.m. over n = 3 independent experiments. A standard Michaelis–Menten plot for each deubiquitylase is shown in Supplementary Fig. 3A for reference. (b) USP30 cleavage of AMC conjugated to Ub or Ubls over time. Representative data shown from one of three independent experiments. RFU, relative fluorescence units. (c) Dimeric Ub cleavage assay. USP30 cleavage of all eight Ub chain linkages over time was analysed using SDS–PAGE and SYPRO Ruby stain. Representative data shown from one of three independent experiments. (d) Tetrameric Ub cleavage assay. Cleavage of Lys 6, Lys 11, Lys 48, Lys 63 and linear tetrameric Ub chain types in the presence of USP30 was measured over time and analysed using SDS–PAGE and Coomassie staining. The star indicates Lys 63 chains that were formed as a side product during enzymatic formation of Lys 6 tetramers (Supplementary Fig. 3C–F). Owing to the extremely fast activity of USP30 on Lys 6 tetramers, early Lys 6 cleavage products appeared before the t = 0 reaction could be quenched (compare with gel of untreated tetramers, Supplementary Fig. 3C). Representative data shown from one of three independent experiments. (e) Quantification of the amount of dimer remaining from the data in c (Methods). Data were fitted using a one-phase exponential decay to guide the eye. Representative data shown from one of three independent experiments. (f) Quantification of the amount of tetramer remaining from the data in d (Methods). Data were fitted using a one-phase exponential decay to guide the eye. Representative data shown from one of three independent experiments. Uncropped images of blots/gels are shown in Supplementary Fig. 7.