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 acid precipitate of the ubiquitin activating enzyme after reaction with ATP and ubiquitin contains one enzyme equivalent of ubiquitin adenylate in which the carboxyl-terminal glycine of ubiquitin and AMP are in an acyl-phosphate linkage. The recovered ubiquitin adenylate has the catalytic properties proposed for it as a reaction intermediate. Thus, upon reaction with fresh enzyme in the absence of Mg2+ or ATP, the product complex, E-ubiquitin . AMP-ubiquitin, is formed. This complex is capable of generating ubiquitin-protein isopeptide derivatives when added to a reticulocyte fraction that catalyzes protein conjugation. This reproduces the effect previously shown to require ubiquitin, ATP, and Mg2+. In the presence of activating enzyme, ubiquitin adenylate is converted to ATP and free ubiquitin in a step requiring PPi and Mg2+. On the basis of studies of [32P]PPi/nucleoside triphosphate exchange, the activating enzyme could be used to generate 2'-deoxy-AMP-, 2'-deoxy-IMP-, and 2'-deoxy-GMP-ubiquitin but not pyrimidine nucleotide-ubiquitin derivatives. The enzyme shows a modest preference for the pro-S diastereomers of adenosine 5'-O-(1-thiotriphosphate) and adenosine 5'-O-(2-thiotriphosphate). Inorganic phosphate, arsenate, methyl phosphate, and tripolyphosphate, but not nucleoside triphosphates, can serve as alternate substrates in place of PPi in the reverse of ubiquitin adenylate formation. Therefore, the enzyme catalyzes the unusual reaction ATP + Pi in equilibrium ADP + PPi in the presence of ubiquitin.

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Previous studies suggest that the conjugation of ubiquitin to NH2 groups of proteins is required for protein breakdown. We now show that the selective modification of NH2-terminal a-NH2 groups of globin and lysozyme prevents their degradation by the ubiquitin proteolytic system from reticulocytes. The conjugation by ubiquitin of e-NH2 groups of lysine residues, usually seen in multiples, was also inhibited in a-NH2-blocked proteins. Naturally occurring N-acetylated proteins are not degraded by the ubiquitin system at a significant rate, while their nonacetylated counterparts from other species are good substrates. This suggests that one function of N'-acetylation of cellular proteins is to prevent their degradation by the ubiquitin system. a-NH2-blocked proteins can have their activity as substrates for degradation increased by incorporation of a-NH2 groups through the introduction of polyalanine side chains. Proteins in which most e-NH2 groups are blocked but the a-NH2 group is free are degraded by the ubiquitin system, but at a reduced rate. It is therefore suggested *that the exposure of a free NH2 terminus of proteins is required for degradation and probably initiates the formation of ubiquitin conjugates committed for degradation.Studies on the mode of action of an ATP-dependent proteolytic system from reticulocytes revealed a pathway for the degradation of intracellular proteins (for reviews see refs. 1 and 2). That system requires for activity the 8500-dalton polypeptide ubiquitin (Ub) (3, 4). Ub is covalently conjugated to proteins (5) by a sequence of reactions in which the COOH-terminal residue of the polypeptide is first activated by a specific Ub-activating enzyme, E1 (6, 7), and activated Ub is transferred to protein by the action of two further enzymes, E2 and E3 (8). The structure of Ub-protein conjugates has not yet been characterized sufficiently, but at least some Ub molecules bind to E-NH2 groups of lysine residues by isopeptide linkages (5,9). Proteins conjugated to multiple molecules of Ub are degraded by an ATP-dependent enzyme system that does not degrade unconjugated proteins (10). The metabolic function of the Ub proteolytic system was strongly supported by a recent study in which a mammalian cell line found to have a temperature-sensitive Ub-activating enzyme was found to be defective in degrading most of its rapidly turning over protein (11).A central problem is what features of protein structure are recognized by the Ub conjugation system for commitment to proteolysis. Since most lysine residues are exposed at the surface of most proteins, the availability of any lysine does not seem to be sufficient for specific recognition. One approach is to study the influence of the modification of specific amino groups in proteins. Other investigators have used complete blocking of protein amino groups to distinguish between Ub-dependent and Ub-independent proteolytic systems (12, 13), and the requirement for free NH2 groups has been confirmed for the Ub-dependent system. On the other ha...

The generation and characterization of ubiquitin (Ub)-aldehyde, a potent inhibitor of Ub-C-terminal hydrolase, has previously been reported. We now examine the action of this compound on the Ub-mediated proteolytic pathway using the system derived from rabbit reticulocytes. Addition of Ub-aldehyde was found to strongly inhibit breakdown of added 12'I-labeled lysozyme, but inhibition was overcome by increasing concentrations of Ub. The following evidence shows the effect of Ub-aldehyde on protein breakdown to be indirectly caused by its interference with the recycling of Ub, leading to exhaustion of the supply of free Ub: (i) Ub-aldehyde markedly increased the accumulation of Ub-protein conjugates coincident with a much decreased rate of conjugate breakdown. (a) release of Ub from isolated Ub-protein conjugates in the absence of ATP (and therefore not coupled to protein degradation) is markedly inhibited by Ub-aldehyde. On the other hand, the ATP-dependent degradation of the protein moiety of Ub conjugates, which is an integral part of the proteolytic process, is not inhibited by this agent. (iii) Direct measurement of levels of free Ub showed a rapid disappearance caused by the inhibitor. The Ub is found to be distributed in derivatives of a wide range of molecular weight classes. It thus seems that Ub-aldehyde, previously demonstrated to inhibit the hydrolysis of Ub conjugates of small molecules, also inhibits the activity of a series of enzymes that regenerate free Ub from adducts with proteins and intermediates in protein breakdown.Current studies indicate that the polypeptide ubiquitin (Ub) has at least two types of cellular function. Ub plays a role in intracellular protein breakdown, in which conjugation with Ub commits proteins for degradation. The second role is modification of protein function, as seems to be the case in the attachment of Ub to certain histones. In both cases, Ub is linked to amino groups ofproteins by way of its C terminus (for reviews, see refs.