Fig. 7. Encore is required for proteolysis. (A) Proteasome assays measuring the hydrolysis of the fluorogenic peptide Suc-LLVYMCA. The 20S-CP proteolysis activity is not significantly compromised in encore mutant germarium-enriched extracts at 29°C (yellow) or room temperature (light blue) compared with wild-type extracts at 29°C (pink). Control assays contain no substrate peptide (dark blue). (B) Western blot using anti p27 antibodies show that Ubiquitination reactions produce the same polyubiquitinated p27 forms (arrowheads) in wild-type and encore mutant extracts. The deubiquitination and degradation reaction is much slower when encore mutant extract was used as a source of the degradation machinery. (C) Model of Encore function. Cul1 (pink) is localized to the fusome (green) in an Encore (blue)-dependent manner (Encore may directly or indirectly modify Cul1 and thus influence its subcellular localization). Cul1 may serve as an anchor where the SCF E3 complex is assembled and a phosphorylated substrate (yellow) is recognized and ubiquitinated. The polyubiquitinated substrate is then recognized by the 19S-recognition particle. 19S-RP and presumably the 20S-core particle are recruited to the fusome where the substrate is de-ubiquitinated, unfolded and degraded by the 26S-subunit of the proteasome.