Proposed role of ATP in protein breakdown: conjugation of protein with multiple chains of the polypeptide of ATP-dependent proteolysis.

Abstract

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...

Formation of conjugates by 125I‐labelled ubiquitin microinjected into cultured hepatoma cells

“…It is more difficult to interpret the significance of the bands found predominantly in the cytoplasm. Conjugates of ubiquitin with cellular proteins have been formed in extracts of reticulocytes [6,7], by the ATP-dependent linkage of the carboxyl terminal of ubiquitin to the lysine residues of other proteins via an isopeptide bond [7,16]. These conjugates were rapidly broken down in the absence of ATP [7].…”

“…Conjugates of ubiquitin with cellular proteins have been formed in extracts of reticulocytes [6,7], by the ATP-dependent linkage of the carboxyl terminal of ubiquitin to the lysine residues of other proteins via an isopeptide bond [7,16]. These conjugates were rapidly broken down in the absence of ATP [7]. In our experiments ATP-depletion decreased the amount of band X and bands in the W region ( fig.4), which is consistent with the interpretation that these conjugates consist of ubiquitin linked by its carboxyl-terminal to lysine residues of other proteins.…”

“…It is widely distributed in animals, plants, yeasts and bacteria [1,2]. In animal cells ubiquitin has been found both in free form [1,4] or covalently bound by its carboxyl terminal to other proteins [5][6][7][8]. In the chromatin of interphase cells, ~10% of histone 2A and -,-1% of histone 2B are covalently linked to ubiquitin [8].…”

“…Amounts of ubiquitin linked to H2A decrease in nucleoli undergoing hypertrophy [ I 1 ], in 'active' chromatin [ 12] and during chicken erythrocyte maturation [ 13]. Ubiquitin may be involved in intracellular protein degradation in reticulocytes [7,[14][15][16]. Conjugates of ubiquitin with other proteins could act as substrates of intracellular proteolytic systems [6,7].…”

“…Ubiquitin may be involved in intracellular protein degradation in reticulocytes [7,[14][15][16]. Conjugates of ubiquitin with other proteins could act as substrates of intracellular proteolytic systems [6,7].…”