The ubiquitin-mediated degradation of mitotic cyclins is required for cells to exit from mitosis. Previous work with cell-free systems has revealed four components required for cyclin-ubiquitin ligation and proteolysis: a nonspecific ubiquitin-activating enzyme E1, a soluble fraction containing a ubiquitin carrier protein activity called E2-C, a crude particulate fraction containing a ubiquitin ligase (E3) activity that is activated during M-phase, and a constitutively active 26S proteasome that degrades ubiquitinated proteins. Here, we identify a novel approximately 1500-kDa complex, termed the cyclosome, which contains a cyclin-selective ubiquitin ligase activity, E3-C. E3-C is present but inactive during interphase; it can be activated in vitro by the addition of cdc2, enabling the transfer of ubiquitin from E2-C to cyclin. The kinetics of E3-C activation suggest the existence of one or more intermediates between cdc2 and E3-C. Cyclosome-associated E3-C acts on both cyclin A and B, and requires the presence of wild-type N-terminal destruction box motifs in each cyclin. Ubiquitinated cyclins are then rapidly recognized and degraded by the proteasome. These results identify the cyclosome-associated E3-C as the component of the cyclin destruction machinery whose activity is ultimately regulated by cdc2 and, as such, the element directly responsible for setting mitotic cyclin levels during early embryonic cell cycles.

The heat-stable polypeptide ATP-dependent proteolysis factor 1 (APF-1) of the reticulocyte proteolytic system forms covalent compounds with proteins in an ATP-requiring reaction. APF-1 and lysozyme, a good substrate for ATP-dependent proteolysis, form multiple conjugates, as was shown by comigration of label from each upon gel electrophoresis.Multiple bands were also seen with other substrates of the ATP-dependent proteolytic system, such as globin or a-lactalbumin. Analysis of the ratio of APF-1 to lysozyme radioactivities and of the molecular weights of the bands indicated that they consist of increasing numbers of the APF-1 polypeptide bound to one molecule of lysozyme. The covalent linkage is probably of an isopeptide nature, because it is stable to hydroxylamine and alkali, and polylysine is able to give conjugates of APF-1. Removal of ATP after formation of the 125I-labeled APF-1 conjugates with endogenous proteins caused the regeneration of APF-1, indicating presence of an amidase. This reaction is thought to compete with proteases that may act on APF-1-protein conjugates, especially those containing several APF-1 ligands. A sequence of reactions in which the linkage of APF-1 to the substrate is followed by the proteolytic breakdown of the substrate is proposed to explain the role of ATP.Since the discovery of the energy requirement of protein breakdown by Simpson in 1953 (1), this feature has been found to be universal in a variety of biological systems (for review, see ref.2). Only recently have cell-free systems in which protein breakdown is stimulated by ATP been delineated (3-6).We have resolved the ATP-dependent proteolytic system from reticulocytes into several components (7,8). In the preceding paper (9) it was found that the heat-stable polypeptide of this system, ATP-dependent proteolysis factor 1 (APF-1), is bound to reticulocyte proteins in an ATP-specific reaction. We now show that this is due to the formation of a covalent, probably amide, linkage between the protein substrate and one or more APF-1 molecules. The role of this reaction in protein breakdown is considered. METHODS Preparation of Radiolabeled Proteins and EnzymeFractions. The purification and radiolabeling of APF-1 from rabbit reticulocytes were described in the previous article (9). Crystalline hen egg white lysozyme (Worthington) and purified bovine a-lactalbumin (a generous gift of W. Klee) were labeled with Na'25I by a method similar to that described for the radioiodination of APF-1 (9). When used in unlabeled form these proteins were treated in the same way without isotope. Globin labeled with [3H]leucine was prepared as described (7). Fraction 11 (0.5 M KCI eluate from DEAE-cellulose) was prepared from ATP-depleted rabbit reticulocytes (7). Poly(L-lysine) was obtained from Miles-Yeda (Mr 1500-8000). (vol/vol) glycerol, 0.5% NaDodSO4, and 0.5% 2-mercaptoethanol. The samples were electrophoresed on NaDodSO4/polyacrylamide slab gels (13 X 13 X 0.12 cm) with the system of Laemmli (10).Unless otherwise stated, 10-14...

The heat-stable polypeptide (APF-1) required for ATP-dependent proteolysis in reticulocytes enters into high molecular weight conjugates upon incubation with the fraction of reticulocytes that is retained by DEAE-cellulose. Conjugate formation requires ATP and Mg2+ and is inhibited by N-ethylmaleimide. UTP and GTP are inactive. These properties are identical to those of ATP-dependent protein breakdown in the same system, suggesting that the conjugates are intermediates in this process. The APF-1 conjugates are stable in sodium dodecyl sulfate/polyacrylamide gel electrophoresis and Sephadex C-75 isolation and are resistant to mild acid, alkali, heat denaturation, and reduction; the conjugates are therefore covalent.Although most cellular proteins turn over rapidly, the enzymic reactions of protein degradation have not yet been identified. A basic feature of intracellular protein breakdown is its absolute requirement for cellular energy. Inhibitors of ATP production inhibit the degradation of liver proteins (1, 2), of tyrosine aminotransferase in hepatoma cells (3), of normal proteins in bacteria (4, 5) and cultured cells (6, 7), and of abnormal proteins in Escherichia coli (8, 9) and reticulocytes (10, 11). Recently several reports have shown an ATP requirement for protein breakdown in cell-free systems. ATP stimulates the degradation of abnormal proteins in crude soluble extracts of reticulocytes (10, 11) and E. coli (12), and a requirement for ATP has been found in the cleavage of bacteriophage X repressor in vitro (13,14).We have shown that the ATP-dependent proteolytic system from rabbit reticulocytes is composed of several required components. A heat-stable polypeptide of a relatively small size, (Mr -8000) designated ATP-dependent proteolysis factor 1 (APF-1), has been resolved (15). APF-1 has no proteolytic activity itself but stimulates ATP-dependent protein breakdown by a crude protein fraction eluted from DEAE-cellulose, fraction 11 (15). Fraction II has been resolved into two subfractions, both required for protein breakdown by the ATP-APF-1 system (16).To gain a better insight into the roles of the different factors and of ATP in protein breakdown, we have now purified APF-1, radiolabeled it, and observed its association with other reticulocyte components. ATP is required for binding of APF-1 to reticulocyte proteins and the binding seems to be covalent in nature. METHODS Preparation of Enzyme Fractions and Purification of APF-1. Lysates from ATP-depleted rabbit reticulocytes were prepared and separated on DEAE-cellulose into fraction I (unadsorbed material) and fraction 11 (0.5 M KCI eluate), as described earlier (15, 16). Fraction I was subjected to heat treatment (90'C, 20 min) and gel filtration on a column (1.5 X 90 cm) of Sephadex G-75, as described (15). Then 10 mg of this material was adsorbed onto a column (1.5 X 30 cm) of CM-Sephadex equilibrated with 10 mM potassium phosphate and was eluted with a 0-150 mM linear gradient of KCl. The active peak, designated APF-1, eluted at about 6...

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