Figure 6: Quantification of the Urm1-GFP fusion substrate processing by the core 20S proteasome, and unfolding by the PAN ATPase. | Nature Communications

Figure 6: Quantification of the Urm1-GFP fusion substrate processing by the core 20S proteasome, and unfolding by the PAN ATPase.

From: Involvement of a eukaryotic-like ubiquitin-related modifier in the proteasome pathway of the archaeon Sulfolobus acidocaldarius

Figure 6

(a) 20S core processing of the superfolder GFP protein and the 1XUrm1:GFP, 2XUrm1:GFP and 3XUrm1:GFP N-terminal fusion substrates in triplicate and visualized by SDS-PAGE and Coomassie staining. Equimolar quantities (6 μM) of the substrates were incubated with 10 μg Saci0613/Saci0662ΔN/Saci0909ΔN active 20S complex at 69 °C. Lanes 1–4 display the reactions before heating, while lanes 5–8 represent the reactions upon termination after 75 min at 69 °C. (b) Quantification of the GFP-sized intermediates (boxed region) after resolution by SDS-PAGE. Gels were scanned using the ‘Coomassie Brilliant Blue Digitization’ mode on a Typhoon Imaging system (GE Healthcare). The histograms display the mean of three independent repeats and the error bars one s.d. The data were quantified using the ImageQuant software (GE Healthcare) and plotted in Microsoft Excel. (c) Verification of the direct degradation of the Urm1-GFP fusion substrates by the 20S core proteasome (at 69 °C), demonstrated by the reduction in the GFP fluorescence. (d) ATP-dependent unfolding of a (4X) Urm1:GFP fusion protein by the PAN ATPase (at 60 °C) reflected by the decrease in the GFP fluorescence. Untagged GFP is not unfolded by PAN, even in the presence of ATP. In (c,d) the data points represent the mean of three independent repeats and the error bars one s.d. Data were plotted in Microsoft Excel. The GFP fluorescence was detected at 485 nm excitation and 520 nm emission wavelengths, respectively, using a PheraStar (BMG LABTECH) plate reader.

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