MultiDsk: a ubiquitin-specific affinity resin

Abstract

Ubiquitylation is a highly diverse and complex post-translational modification for the regulation of protein function and stability. Studies of ubiquitylation have, however, been hampered by its rapid reversal in cell extracts, for example through the action of de-ubiquitylating enzymes (DUBs). Here we describe a novel ubiquitin-binding protein reagent, MultiDsk, composed of an array of five UBA domains from the yeast ubiquitin-binding protein Dsk2, fused to GST. MultiDsk binds ubiquitylated substrates with unprecedented avidity, and can be used as both an affinity resin to study protein ubiquitylation, and to effectively protect ubiquitylated proteins from the action of DUBs and the proteasome in crude cell extracts. We use the resin to show that the Def1 protein becomes ubiquitylated in response to DNA damage, and to isolate ubiquitylated forms of RNA polymerase II.

Figure 1. MultiDsk binds efficiently to ubiquitylated proteins. A

. Schematic representation of the MultiDsk protein. B. 2 mg of yeast whole cell lysate from strain SUB592 expressing Myc-His-tagged ubiquitin was incubated with affinity beads. Differing amounts of GST protein, alone or as a mixture with GST-MultiDsk protein, were mixed with agarose beads so that equal amounts of total protein and bead bed volumes were used in each experiment. The flow-through not bound to the beads was retained and loaded at equivalent levels to the Input (Lane 1). After Western transfer, the membrane was stained with Ponceau S to reveal total protein in samples (lower panel), and Western blot was performed using anti-Myc antibodies to detect ubiquitylated species (upper panel). C. As in B, except performed on a human cell whole cell extract. 100 µg of protein was incubated with the indicated amounts of GST, GST-Dsk2, or MultiDsk protein and purified via agarose beads. D. As in B, but using purified ubiquitin chains. GST alone, full length GST-Dsk2 protein, or MultiDsk, bound to beads, were incubated with 100 µg synthetic K48- or K63-linked ubiquitin chains for 2 hours. After Western transfer, the membrane was stained with Ponceau S to reveal total protein in samples (lower panel), and Western blot was performed using anti-ubiquitin antibodies to detect ubiquitylated species (upper panel).

Figure 2. Protection of poly-ubiquitin chains in extract.

Extract from strain SUB592 was incubated with equivalent amounts of GST, GST-Dsk2, commercially available TUBE-1, and MultiDsk and incubated at 30°C for the indicated time. Total protein extracts were subject to Western blot and probed using anti-myc antibody.

Figure 3. MultiDsks can be used to characterise the kinetics of ubiquitylation of a specific protein species. A.

Exponentially growing yeast cells were either treated with 10 µg/ml of 4-NQO for one hour, or left untreated, as indicated. Equal amounts of extracts were incubated with MultiDsk resin. Dilute Input extract (1%) and washes from the beads were also loaded. Proteins were analysed by Western blotting using anti-Rpb1 antibody, 4H8. B. Exponentially growing yeast cells were either treated with 10 µg/ml of 4-NQO for one hour, or left untreated, as indicated. Equal amounts of the extracts were incubated with agarose beads loaded with GST alone, GST-Dsk2 protein, commercial TUBE1, or MultiDsk. Proteins were eluted via boiling in sample buffer and subjected to Western blot analysis using the anti-Rpb1 antibody, 4H8 (upper panel). Ponceau S staining (lower panel) shows relative amounts of affinity proteins used. C. Yeast cells were harvested at the indicated time after treatment with 4-NQO (10 µg/ml), incubated with MultiDsk resin, and isolated proteins were analysed by Western blotting using either 4H8, or an anti-Def1 antibody, as indicated.

Figure 4. MultiDsks can be used to purify a specific ubiquitylated protein. A

A schematic of purification of ubiquitylated RNAPII from 4-NQO (10 µg/ml) treated Rpb3-FLAG tagged cells. Cells were grown to logarithmic phase and treated with 4-NQO for one hour before extract was created as described earlier. B Western blot of two-step purification, probed with anti-Rpb1 antibody (4H8) and anti-ubiquitin antibody (P4D1). Lanes correspond to; 1- Input extract, 2- Flow through from MultiDsk resin, 3-MultiDsk resin and bound ubiquitin proteins prior to elution, 4- Gluathione beads post elution, 5- Eluted MultiDsk-ubiquitin conjugates, 6- FLAG Immunoprecipitate and 7- Flow through form the FLAG immunoprecipitate. C Silver stain comparing FLAG immunoprecipitate post MultiDsk enrichment (lane 6 from B) to pure Rpb3-FLAG tagged RNAPII. Different concentrations of pure RNAPII are loaded for comparison. Band at corresponding heights are labelled Rpb1 mono-ubiquitin and Rpb1 poly-ubiquitin.