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-ethyl- maleimide. 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-I conjugates are stable in sodium do- decyl sulfate/polyacrylamide gel electrophoresis and Sephadex G-75 isolation and are resistant to mild acid, alkali, heat dena- turation, and reduction; the conjugates are therefore cova- lent.

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 hand, Hough and Rechsteiner (14) reported that guanidinated proteins are degraded in reticulocyte lysates by an ATPdependent process. In the present study we have tried to use more selective methods, and we have found that a free aNH2 group has a special importance. MATERIALS AND METHODS Materials. Cytochrome c from horse heart (type III) or from yeast (Saccharomyces cerevisiae) (type VIII), enolase from baker's yeast (S. cerevisiae), and lysozyme from hen egg were purchased from Sigma, and enolase from rabbit muscle was obtained from Calbiochem, All the above proteins were found to be essentially homogeneous by PAGE. The sources of other proteins used in this study were as follows: bovine serum albumin, from Miles; aldolase (rabbit muscle), carbonic anhydrase (bovine erythrocyte), and glyceraldehyde-3-P dehydrogenase (rabbit muscle), from Boehringer Mannheim; actin (bovine muscle), lactate dehydrogenase (rabbit heart, H4 isoenzyme) and ribonuclease S-protein (bovine pancreas), from Sigma; and ovalbumin (hen egg), from Worthington. Ubiquitin was purified from human erythrocytes as described earlier (4, 15). All proteins were radioiodinated by the chloramine-T procedure (5). Protein Modification. Guanidination of proteins was performed according to Cupo et al. (16), except that the concentration of O-methylisourea was 1 M, and the reaction was carried out in 6 M urea at 4°C for 1 week' Acetylation was performed as described by Tanaka et al. (13). Reductive methylation (17) was carried out in 0.1 M Hepes-NaOH, pH 7.4/6 M urea, at 25°C for 1 hr; concentrations of formaldehyde and cyanoborohydride were varied as described in legends to figures. Carbamoylation of 125I-labeled lysozyme (1 mg/ml) was carried out in 0.2 M potassium phosphate, pH 6.0/6 M urea/50 mM potassium cyanate (Aldrich). After incubation at 37°C for the time periods indicated in Fig. 1, the reaction was stopped with 150 mM glycylglycine. The pH was adjusted to 8.1 with 0.5 M K2HPO4 and KOH, and the samples were incubated at 37°C for 1 hr. This latter treatment releases carbamoyl groups bound to nonamine residues in protein (18, 19). For polyalanylation, N-carboxy-L-alanine anhydride was prepared according to ref. 20. The anhydride (100 mM) was added to the protein solution (0.3 mg/ml in water) and the samples were incubated at 25°C for 2 hr. After all modification reactions, reagents were removed by dialysis at 4°C for 48 hr, with at least six changes of water. The extent to which NH2 groups were blocked was determined with fluorescamine (21). To prepare N`-carbamoylated [3H]globin, rabbit reticulocytes were labeled with [3H]leucine (500 ,Ci/ml, 2 hr, 1 Ci = Abbreviations: Ub, ubiquitin; Gdn, guanidine. *Present address: Biochemistry and Food Technology Division, Bhabha Atomic Research Center, Trombay 400 085, India. 7021 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 7022 Biochemistry: Hershko etaLP 37 GBq) and were lysed in water, as described (22). To remove most nonhemoglobin proteins, the lysate (5 ml) was passed through a 5-ml column of DEAE-Sephacel (Pharmacia) in 10 mM phosphate buffer (pH 7.0). The hemoglobin solution (36 mg/ml) was subjected to carbamoylation according to the procedure described by Manning (23), except that K CNO (New England Nuclear, 50 ,Ci/ml) was added and the final concentration of KCNO was 20 mM. The amount of carbamoyl groups bound to protein was estimated by the incorporation of the 14C label into material precipitated with 5% trichloroacetic acid. Both carbamoylated and unmodified hemoglobin preparations were extensively dialyzed against water, and globin was precipitated with acetone/HCI (24). The globin preparations were diluted to 2 mg/ml, brought to 0.01 M NaOH, and denatured by heating at 60'C for 1 hr (22). The extent of carbamoylation of a-NH2 and e-NH2 residues in globin was estimated by the method of Manning et al. (25). In this procedure, globin is treated with hot acid to release valine hydantoin from carbamoylated aNH2 residues. The hydantoin is extracted into ethyl acetate, while carbamoylated lysine (homocitrulline) remains in the aqueous phase. Since the specific radioactivity of [14C]carbamoyl groups is known, the amount of 14C in the organic and aqueous phases is a measure of carbamoyl groups bound to a-NH2 and E-NH2 residues, respectively. Assay of Protein Breakdown. Unless otherwise stated, the complete reaction mixture contained, in a final volume of 50 Al: 50 mM Tris HCI at pH 7.6, 5 mM MgCl2, 3 mM dithiothreitol, 1 mM ATP, 10 mM phosphocreatine, 2.5 ug of creatine kinase, 3 ,g of Ub, approximately 200 ,ug of protein of fraction II from reticulocytes, and 0.05-5 pg of labeled protein substrate. After incubation at 37°C for 1 hr, the release of radioactive material soluble in 5% trichloroacetic acid was determined as described (8). Fraction II (a Ub-free crude enzyme fraction) was prepared from lysates of rabbit reticulocytes as described previously (8). Preparations of fraction II used in this study contained ATP (0.5 mM), for stabilization in prolonged storage. Therefore, for incubations without ATP, hexokinase (3 ,ug, P-L Biochemicals, grade 300) was added together with 10 mM 2-deoxyglucose.