The proteasome (multicatalytic protease) is a component of the 1500-kDa proteolytic complex which degrades ubiquitin-conjugated proteins

• Published in: The Journal of biological chemistry 1990-03, Vol.265 (9), p.4789-4792
• Main Authors: DRISCOLL, J, GOLDBERG, A. L
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - pharmacology; Adenylyl Imidodiphosphate - pharmacology; Ammonium Sulfate; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Cysteine Endopeptidases - blood; Cysteine Endopeptidases - isolation & purification; Endopeptidases - blood; Endopeptidases - isolation & purification; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Hydrolases; Kinetics; Magnesium - pharmacology; Molecular Weight; Multienzyme Complexes - blood; Multienzyme Complexes - isolation & purification; Proteasome Endopeptidase Complex; Rabbits; Reticulocytes - enzymology; Substrate Specificity
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(19)34041-4

Mammalian cells contain two large proteolytic complexes, the 650-kDa proteasome (or multicatalytic protease) and the 1500-kDa (26 S) Ubiquitin-conjugate-degrading enzyme. Since the proteasome is also required for the ATP-dependent degradation of ubiquitinated proteins, we tested whether it may be a component of the larger complex. The proteasome normally is soluble in 38% ammonium sulfate. However, after preincubation of reticulocyte extracts with ATP, several proteasome activities appeared in the 38% ammonium sulfate pellet, including the ability to degrade hydrophobic peptides and 14C-casein. Also, following preincubation with ATP, the precipitable fraction could degrade 125I-lysozyme-ubiquitin (Ub) conjugates. The activities were not present after incubation without ATP or with a nonmetabolizable ATP analog. Nondenaturing gel electrophoresis indicated the ATP-dependent appearance of a new band which degraded proteasome substrates, and reacted with an anti-proteasome monoclonal antibody on Western blot. This new band appeared larger than the proteasome and migrated similarly to the larger Ub-conjugate-degrading complex. The formation of the larger complex required factor(s) present in the 38% ammonium sulfate pellet and either the 40-80% fraction or the purified proteasome from reticulocytes or muscle. After complex formation, hydrolysis of Ub-protein conjugates and also the non-ubiquitinated substrate, casein, was stimulated severalfold by ATP, but non-metabolizable ATP analogs had little or no effect. Thus, the proteasome corresponds to component CF-3 of Ganoth et al. (Ganoth, D., Leshinisky, E., Eytan, E., and Hershkov, A. (1989) J. Biol. Chem. 263 12412-12419) and undergoes an energy-dependent association with other factors to form the 1500-kDa, ATP-requiring proteolytic complex.