A spectrophotometric assay for conjugation of ubiquitin and ubiquitin-like proteins

Abstract

Ubiquitination is a widely studied regulatory modification involved in protein degradation, DNA damage repair, and the immune response. Ubiquitin is conjugated to a substrate lysine in an enzymatic cascade involving an E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme, and an E3 ubiquitin ligase. Assays for ubiquitin conjugation include electrophoretic mobility shift assays and detection of epitope-tagged or radiolabeled ubiquitin, which are difficult to quantitate accurately and are not amenable to high-throughput screening. We have developed a colorimetric assay that quantifies ubiquitin conjugation by monitoring pyrophosphate released in the first enzymatic step in ubiquitin transfer, the ATP-dependent charging of the E1 enzyme. The assay is rapid, does not rely on radioactive labeling, and requires only a spectrophotometer for detection of pyrophosphate formation. We show that pyrophosphate production by E1 is dependent on ubiquitin transfer and describe how to optimize assay conditions to measure E1, E2, and E3 activity. The kinetics of polyubiquitin chain formation by Ubc13-Mms2 measured by this assay are similar to those determined by gel-based assays, indicating that the data produced by this method are comparable to methods that measure ubiquitin transfer directly. This assay is adaptable to high-throughput screening of ubiquitin and ubiquitin-like conjugating enzymes.

Figure 1. Schematic of Molybdenum Blue Assay

The ubiquitination cascade (bottom) involves the sequential action of E1, E2 and E3 enzymes, leading to ubiquitination of the substrate lysine. In the first step, the ubiquitin activating enzyme, E1, adenylates ubiquitin, forming ubiquitin-AMP and releasing pyrophosphate. To assay ATP consumption (top), the pyrophosphate product is broken down by pyrophosphatase into two phosphate molecules. Addition of ascorbic acid and ammonium molybdate in hydrochloric acid halts the reaction progress and initiates phosphomolybdate formation. The solution is then developed by addition citric acid and sodium arsenite and the phosphomolybdate formed measured by visible spectroscopy.

Figure 2. Pyrophosphate production during diubiquitin synthesis

The plot shows pyrophosphate formation during diubiquitin synthesis by Ubc13-Mms2 in the absence (open circles) and presence (black circles) of the RING domain of the Rad5 E3. The control (black triangles) shows the background signal in the absence of Ubc13-Mms2. Reactions contained 0.6 μM Ubc13-Mms2, 1 μM Rad5 RING domain, 0.3 μM E1, 80 μM ubiquitin^K63R (the donor ubiquitin), and 800 μM ubiquitin^D77 (the acceptor ubiquitin. -Data points are an average of three independent measurements and the error bars are the standard deviation of the experiments. Lower panel shows a gel of diubquitin synthesis using fluorescently labeled ubiquitin.

Figure 3. Absorbance of phosphomolybdate

(A) Absorbance spectrum of phosphomolybdate from 100 μM (solid line), 200 μM (dotted line), and 500 μM (dashed line) sodium phosphate in 20 mM HEPES, pH 7.5 and 100 mM NaCl. Spectra were taken of 200 μL solutions in a 96-well plate using a POLARStar Omega plate reader (B) Linearity of phosphomolybdate absorbance at 600 nm (filled circles), 710 nm (open circles), and 850 nm (filled triangles). (C) Molybdenum blue assay of time course of Ubc13-Mms2 ubiquitin chain formation measured at 600 nm (filled circles), 710 nm (filled triangles), and 850 nm (open circles).

Figure 4. High throughput screen for the effect of ubiquitin mutations on polyubiquitin chain formation

The effect of ubiquitin mutations on polyubiquitin chain formation by Ubc13-Mms2. Wells in row 1 contain E1, Ubc13-Mms2 and ubiquitin, row 2 contains E1 and ubiquitin, and row 3 contains ubiquitin alone. Protein concentrations used were 2 μM Ubc13-Mms2, 0.5 μM E1, 500 μM ubiquitin (except for no-lysine ubiquitin which was at 100 μM). Reactions were incubated for 1 hour at 37 °C, quenched and the absorbance measured. Assay was performed in duplicate, with representative data shown.

Figure 5. Rate-limiting concentration range of Ubc13-Mms2

Rate of pyrophosphate formation in μM/sec at 0, 0.4, 0.8, 1.2, 2, and 4 μM Ubc13-Mms2 at 0.3 μM E1 (filled circles) and 0.6 μM E1 (open circles). Grey box indicates concentrations where ubiquitin transfer by Ubc13-Mms2 is directly correlated with pyrophosphate production by E1.

Figure 6. Ubiquitin^ΔG75,ΔG76 saturation curves for Ubc13-Mms2

Reactions contained 0.3 μM Ubc13-Mms2, 0.2 μM E1, 0 to 950 μM ubiquitin^ΔG75,ΔG76, and 80 μM ubiquitin K63R. For molybdenum blue assay reactions (black circles), 500 Units of pyrophosphatase was present and for the band shift assay (open circles), 10 μM ubiquitin labeled with fluorescein at position 63 and with 70 μM unlabeled ubiquitin^K63R. Each rate is an average of 3 to 5 separate experiments. Error bars are not shown for clarity.