Figure 5.

ATP-independent HUB1–protein adduct formation. (A) Western blots of extracts from cells expressing HA-tagged HUB1. Cells were grown in YP medium lacking (−) or containing glucose (gluc). For ATP depletion, cells were grown either in NaF and NaN₃ (F/N3) or deoxyglucose and dinitrophenol (d-gluc/DNP) containing YP medium. Samples for the bottom panels were treated additionally with N-ethylmaleimide (NEM) for 30 min. The blots were probed either for HUB1 with anti-HA antibodies (left panels) or for SUMO/SMT3 using affinity-purified SUMO antibodies. Note that ATP depletion leads to a loss of SUMO conjugates. (B) In vitro adduct formation of purified recombinant HUB1 with proteins from yeast extracts (see Methods). The extracts were treated with the indicated reagents (25 U ml⁻¹ apyrase, 5 mM EDTA, 10 mM NEM) and incubated for 30 min at 30°C or for 5 min on ice. The bands labelled with asterisks are HUB1 dimers and trimers formed in vitro even without added extract (right lane). Free HUB1 runs at the bottom of the gel.