Figure 7. UBE2G1 functions redundantly with UBE2R1/2 in cells and in SCF enzyme reactions in vitro.

(a) Comparison of the steady state stabilities of HIF1α, CYCLIN E, and p27 proteins in HEK 293T control or UBE2R1/2 double knockout cells treated with siRNA targeting UBE2G1 expression. (b) Quantitative comparison of the steady state levels for HIF1α (open or closed black circles), CYCLIN E (open or closed gray squares), and p27 protein (open or closed orange diamonds). P-values were calculated using an unpaired t-test (* and ** denote values of less than 0.05 or 0.01, respectively). P-values for all relevant combinations are provided in Figure 7—source data 1. Three biological replicates each for control and UBE2R1/2 double knockout cells were used to generate the figure with duplicate technical replicates. (c) Representative autoradiogram of a Cyclin E peptide ubiquitylation reaction with either ARIH1, UBE2D3, or UBE2G1 alone or in combination. (d) Graphical representation of the levels of unmodified substrate and ubiquitylated products from the reactions shown in panel c. Duplicate technical replicates were performed to generate the figure. Source data have been provided in Figure 7—source data 2 for panel d. (e) Representative autoradiogram of a Cyclin E ubiquitylation reaction with UBE2G1 levels (12.5 μM) sufficient to saturate the SCF complex. Figure 7—figure supplement 1d shows the fit of the data to the kinetic model. The enzyme concentrations have been provided in Supplementary file 2.

Figure 7—figure supplement 1. UBE2G1 displays robust activity in the presence of mono-ubiquitylated peptide substrates.

(a) Single encounter ubiquitylation reactions containing either saturating UBE2R2 or UBE2G1 and mono-ubiquitylated β-Catenin peptide substrate and SCF^βTRCP or mono-ubiquitylated Cyclin E peptide substrate and SCF^FBW7. Reactions were quenched at 10 s (see Materials and methods). (b) Multi-turnover ubiquitylation reactions were assembled in the presence of constant amounts of SCF^FBW7 and ³²P-labeled Ub-Cyclin E peptide and increasing amounts of UBE2G1 protein. Each lane represents a single ubiquitylation reaction used to calculate the reaction velocity (see Materials and methods). Representative data from two technical replicates are shown for panels (a) and (b). (c) The velocities from the reactions in (b) were plotted as a function of the UBE2G1 protein concentration and the data were fit to the Michaelis-Menten equation, $velocity=\frac{k_{cat}\left[S\right]}{\left(\right[K_m+\left[S\right])}$, using nonlinear curve fitting. Data points are shown from duplicate technical replicates. (d) Data points and fit to the kinetic model of the reaction in Figure 7d for mono-ubiquitylated substrate. Data points are shown from duplicate technical replicates. All relevant enzyme concentrations have been provided in Supplementary file 2.