ATP-dependent reversible association of proteasomes with multiple protein components to form 26S complexes that degrade ubiquitinated proteins in human HL-60 cells

The role of proteasomes in ubiquitin (Ub)-dependent protein degradation was studied by analyzing lysates of human promyelocytic leukemia HL-60 cells by glycerol density gradient centrifugation. High succinyl-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide hydrolyzing activity was found in the 26S fraction, whereas the 20S fraction containing proteaomes had no activity. Addition of 0.05% sodium dodecylsulfate to the latter fraction, however, induced marked activity. The 26S, but not the 20S fraction catalyzed ATP-dependent degradation of [125I]lysozyme-Ub conjugate. Depletion from the lysate of ATP caused complete shift of the active 26S complex to the latent 20S form, whereas in the lysate prepared from ATP-depleted cells, ATP converted 20S proteasomes to 26S complexes. The immunoprecipitated 26S complexes were found to consist of proteasomes and 13–15 other proteins ranging in size from 35 to 110 kDa. We conclude that in the lysate, latent proteasomes undergo reversible, ATP-dependent association with multiple protein components to form 26S complexes that catalyze ATP-dependent degradation of Ub-protein conjugates.