Figure 4.

A GFP reporter for polyubiquitination and proteasomal degradation. (A) Schematic representation of the Ub-GFP and Ub^G76V-GFP chimeras. The amino-acid sequences for ubiquitin are in yellow, whereas the sequences for GFP are in green. The Ub-GFP chimera is co-translationally cleaved, releasing a stable GFP protein. In contrast, the Ub^G76V-GFP chimera, which contains a mutation in the terminal residue of ubiquitin, cannot be cleaved. The resulting protein is a substrate for polyubiquitination (indicated by the circles labelled with ‘u’) and degradation. (B) Co-expression of Ub^G76V-GFP (green) and mRFP (red) in C. elegans epithelia using the col-19 promoter. Ub^G76V-GFP fluorescence is visible 24 h after L4 stage, but gone 48 h after L4. (C) Relative levels of Ub^G76V-GFP (green line) and mRFP (red line) fluorescence quantified (AU indicates arbitrary units) from micrographs at different time points after L4 stage. Black line indicates the relative ratio of Ub^G76V-GFP to actin from western blot analysis. Error bars indicate s.e.m. (D) GFP and actin detected by western blot at specific times (in hours) after L4. ‘Load’ indicates that equivalent amounts of protein were loaded into each lane, except for the ‘36 h’ and ‘48 h’ lanes, in which twice the amount was loaded, so as to maximize Ub^G76V-GFP detection. For the anti-GFP blot, the multiple bands represent the Ub^G76V-GFP chimeric protein with different numbers of additional ubiquitins attached. (E) Co-expression of Ub^G76V-GFP (green, top panels) and mRFP (red, bottom panels) proteins in C. elegans at 48 h after L4 stage. ‘Control RNAi’ indicates wild-type animals raised on bacteria containing an empty RNAi vector. N2 animals raised on pas-5(RNAi) or pbs-3(RNAi) bacteria, both of which knockdown proteasome subunit genes, showed stable GFP. Mutants for rpn-10(ok1865), which lack the RPN-10 proteasome regulatory subunit, also contained stable GFP. Bar, 50 μm.