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.
Orino
E
E
Third Department of Internal Medicine, School of Medicine, University of Tokushima, Japan.
Tanaka
K
K
Tamura
T
T
Sone
S
S
Ogura
T
T
Ichihara
A
A
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D002499:Centrifugation, Density Gradient; D003374:Coumarins / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007163:Immunosorbent Techniques; D015473:Leukemia, Promyelocytic, Acute; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism*; D009113:Muramidase / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D011506:Proteins / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014452:Ubiquitins / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D003374:Coumarins; D009097:Multienzyme Complexes; D009842:Oligopeptides; D011506:Proteins; D014452:Ubiquitins; C059197:succinyl-leucyl-leucyl-valyl-tyrosyl-4-trifluoromethylcoumarin-7-amide; D000255:Adenosine Triphosphate; D009113:Muramidase; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex