Figure 5

Migration pattern of proteasome from WT and rpn11 mutants by non-denaturing PAGE. Cell extracts were prepared from Logarithmically growing WT or the rpn11 MPN+ mutants that were brought to an identical OD600 level. Extracts were clarified by centrifugation, and identical amounts of total protein were separated on native gel. The gel was then incubated for 10 minutes with the fluorescent peptide LLVY-AMC at 30°C, and photographed under UV light (380/440 nm). Fluorescent bands indicate migration of Doubly capped and Singly capped 26S proteasome forms [14]. There is no noticeable change in migration pattern or overall levels of proteasomes from the different rpn11 mutants, indicating that mutated Rpn11 is properly incorporated into the lid and no gross structural changes in proteasome composition or amounts are due to the mutations.