Proc. Natl. Acad. Sci. USA Vol. 76, No. 7, pp. 3107-31101, July 1979 Biochemistry

Resolution of the ATP-dependent proteolytic system from reticulocytes: A component that interacts with ATP ( degradation/energy/cell-free system) AVRAM HERSHKO*, AHARON CIECHANOVER*, AND IRWIN A. ROSEt *Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel; and tThe Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111 Communicated by Alton Meister, March 26, 1979

ABSTRACT The ATP-dependent proteolytic cell-free system MATERIALS AND METHODS from reticulocytes has been resolved into three components, each of which is absolutely required for acid solubilization of Fractionation of Reticulocyte Extracts. Soluble lysates were 125I-labeled bofine serum albumin radioactivity. In addition prepared from ATP-depleted rabbit reticulocytes and separated to the previously reported heat-stable polypeptide [Ciechanover, on DEAE-cellulose into fractions I and II as described (7), ex- A., Hod, Y. & Hershko, A. (1978) Biochem. Biophys. Res. Com- cept that the column was washed with a solution containing 20 mun. 81, 1100-1105], we now describe a protein of high mo- mM lecular weight (nt450,000) that is labile at 420C. The extremely KC1, 1 mM dithiothreitol, and 3 mM potassium phosphate heat-labile factor is remarkably stabilized by ATP. GTP and (pH 7.0) before the elution of fraction II. APF-1 was purified CTI, which do not stimulate , do not stabilize this frim fraction I by heat treatment, ammonium sulfate precip- factor. Adenylate such as ADP or the nonhydroly- itation, and gel filtration on a column of Sephadex G-75, as zable #,'y imidb or methylene analogues of ATP cause stabili- described (7). zation although they do not activate proteolysis. A third protein Fraction II was separated by ammonium sulfate fractionation component of the rotease system, stable at 420C, has been separated from theheat-labile species by salt precipitation. All to fractions TIA (0-38% saturation) and IIB (42-75% saturation). three components are required with ATP for proteolytic activity, The precipitates were dissolved in a minimal volume of 20 mM but thus far only the heat-labile factor has been shown to in- Tris-HCl (pH 7.2)/1 mM dithiothreitol and passed through teract directly with ATP. Sephadex G-25 equilibrated with the same buffer. ATP (0.5 A universally observed aspect of intracellular protein degra- mM) was added to fraction IIA and both fractions were stable dation is the marked dependence on adequate levels of ATP when stored at -80°C. (1, 2) as shown by its almost total depression in the presence of Assay of ATP-Dependent Proteolysis. 125I-Labeled bovine a wide range of inhibitors of energy production. Only recently serum albumin (125I-albumin) (2 X 104-105 cpm/,ug) was have there been reports of ATP-dependent proteolysis in cell- prepared as described (8) and passed through a Sephadex G-25 free extracts. Goldberg and colleagues have found that ATP (fine) column (0.9 X 60 cm) to remove acid-soluble material. stimulates protein degradation in cell-free extracts of reticu- Unless otherwise stated, the reaction mixture contained in a locytes (3) and Escherichia coli (4), and Roberts et al. showed final volume of 100 ,ul: 100 mM Tris-HCl (pH 7.6), 2 mM di- that the proteolytic cleavage of bacteriophage X repressor in thiothreitol, 2 mM ATP, 5 mM NgCl2, 1 ,ug of 125I-albumin, vitro requires ATP (5). We have studied the role of ATP in the 3.6 ,g of purified APF-1, and other fractions as spec- degradation of abnormal globin chains in intact reticulocytes ified. After incubation at 37°C for 2 hr, the reaction was and in their soluble extracts and have found that in both cases stopped by chilling in ice, and 0.1 ml of carrier bovine serum ATP is required at or before the initial cleavage of the complete albumin (100 mg/ml) was added, followed by 0.8 ml of 5% polypeptide molecule (6). trichloroacetic acid. After centrifugation, radioactivity in the To gain insight into the mechanisms by which ATP partici- clear supernatant was determined. Acid-soluble radioactivity pates in protein breakdown, purification and characterization in zero-time samples (about 2-3% of the total) was subtracted of the responsible are required. We have reported (7) and the results were expressed as the percentage of '25I-albumin that ATP-dependent protease acting on labeled globin can be converted to acid-soluble material. fractionated into a heat-stable protein that is not retained on Materials. For protein iodination, carrier-free Na'25I was DEAE-cellulose (fraction 1) and a crude fraction eluted with obtained from Amersham and crystalline bovine serum albu- 0.5 M KC1. The active principle of fraction 1, now designated min from Sigma. All nucleotides were purchased from Sigma, APF-1, is a heat-stable and relatively small (Mr ; 9000) poly- except for adenosine 5'-[f3,y-imido]triphosphate (Ado- peptide that has no proteolytic activity but restores ATP-de- PP[NH]P), which was from Nutritional Biochemicals. Yeast pendent proteolysis when combined with fraction 11 (7). We hexokinase (140 units/mg) was obtained from Boehringer, and report here the further resolution of fraction II into two com- creatine kinase (115 units/mg) from Sigma. plementary activities, and identify one of the components as capable of interacting directly with ATP. Abbreviations: APF-1, active principle of fraction 1 [previously des- ignated Fraction I (7)1; APF-2 is defined as the 0-38% saturated am- The publication costs of this article were defrayed in part by page monium sulfate precipitate from Fraction II (7); AdoPP[CH2JP, charge payment. This article must therefore be hereby marked "ad- adenosine 5'-[3,ly-methyleneltriphosphate; AdoPP[NH]P, adenosine vertisement" in accordance with 18 U. S. C. §1734 solely to indicate 5'-[,,y-imido]triphosphate; l25I-albumin, l25I-labeled bovine serum this fact. albumin. 3107 Downloaded by guest on October 3, 2021 3108 Biochemistry: Hershko et al. Proc. Natl. Acad. Sci. USA 76 (1979) Table 1. Separation of fraction II into complementing activities of fractions IIA (0-38% cut) and IIB (42-75% cut), when in- Degradation of cubated without the other, had very little proteolytic activity Fraction added 1251-albumin, % in either the presence or the absence of ATP, but ATP-depen- IIA, gl IIB, tdl APF-1 -ATP +ATP dent proteolysis was reconstituted upon the combination of these two fractions. Activity was proportional with increasing 10 + 0.3 0.7 amounts of either fraction IIA or IIB, until it became limited 10 + 1.3 0.7 by the complementing fraction. As in the case of crude fraction 2 10 + 11.7 II, ATP-dependent proteolysis in the reconstituted IIA + TIB 10 5 + 13.0 system was almost completely dependent upon the presence 10 10 + 0.2 21.1 of APF-1 (Table 1). 10 10 - 1.4 Stabilization of the Activity of Fraction ILA by ATP. It was Fractions HA (32.4 mg of protein per ml) and IIB (30.0 mg/ml) were observed previously that fraction II activity is extremely heat added at the amounts specified. Where indicated, the mixtures were labile (7). We have now found that this is due to the lability of supplemented with ATP (2 mM) or purified APF-1 (3.6 jsg). fraction 11A, whereas the activity of fraction IIB is relatively stable at 420C (Fig. 1). The half-time of inactivation of fraction RESULTS IIA at 420C varied between 10 and 30 min in different prepa- Resolution of Fraction II into Complementing Activities. rations. We furthermore found that ATP effectively protects Initial attempts to separate or purify the active components in fraction 11A activity against heat inactivation (Fig. 1). fraction II were hindered by its extreme lability and high and By utilizing ATP to stabilize fractior IIA activity during gel variable content of ATP-independent protease activity (7). The filtration chromatography, the molecular size of the factor could lability problem was solved, at least in part, by the finding that be determined (Fig. 2): it is large, eluting around the region of ATP stabilizes the labile component (see below), while the high ferritin on a Sepharose 4B column (i.e., Mr 450,000). We call protease background could be minimized by using a suitable this ATP-stabilized high-molecular-weight component in substrate that is'not hydrolyzed significantly by these enzymes. fraction IIA factor 2 of the ATP-dependent proteolytic system We found that, in contrast to other polypeptide substrates such (APF-2). as globin (7), 125I-albumin is attacked only slightly by the It might be asked whether the apparent stimulation of protein non-ATP-dependent proteases, whereas it is effectively pro- degradation by ATP is merely due to the stabilization of APF-2 teolyzed by the ATP-dependent system. Control experiments activity during assay. It may also be that ATP stabilizes this showed that: (i) 125I-albumin is not cleaved significantly even factor by its . Comparison of the action of to large (acid-insoluble) fragments in the absence of ATP, ac- various nucleotides on the stimulation of protein breakdown cording to autoradiograms of 15% polyacrylamide/0.1% so- dium dodecyl sulfate slab gels in which fragments below 20,000 daltons would have been detected; (ii) more than 90% of the acid-soluble radiqactive material produced in the presence of ATP is iodotyrosine, as analyzed by paper chromatography with 1-butanol/glacial acetic acid/water, 12:3:5 vol/vol (9). By utilizing the assay system indicated earlier, crude fraction II could be readily resolved into two mutually dependent ac- tivities by ammonium sulfate fractionation (see Table 1). Each -0a) 0 IIA IIB TC 0i Cu 100 E U 0c ±ATP T-_<_+A+TP 8-6 OC C 50j - 0\ .C ._o E

CD

20[ Fraction -ATP FIG. 2. Gel filtration analysis of fraction 11A on Sepharose 4B. A column of Sepharose 4B (0.9 x 48 cm) was equilibrated with a buffer I I consisting of 20 mM Tris-HCl (pH 7.6), 1 mM dithiothreitol, and 0.5 20 40 60 20 40 60 mM ATP. Fraction IIA (15 mg) was applied and eluted with the above Time at 420C, min buffer. Fractions (0.95 ml) were collected and activity was assayed FIG. 1. Heat inactivation of fraction IIA and its protection by in 50-,ul aliquots with fraction IIB (140 jug) and partially purified ATP. Fraction IIA was diluted to a concentration of 6.5 mg of protein APF-1 (14.5 ,ug). Assay conditions were as described under Materials per ml in 20 mM Tris.HCl buffer (pH 7.6) containing 5 mM MgCl2 and Methods, except that creatine phosphate (10 mM) and creatine and 1 mM dithiothreitol and heated at 420C in the presence (0) or kinase (100 ,ug/ml) were added with ATP (0.5 mM). The absorbance absence (0) of ATP (2 mM). At different times, 10-pl aliquots were of ATP at 280 nm was subtracted from the total absorbance. Markers withdrawn and assayed with 2 mM ATP, 150 pig of fraction 1IB, and (arrows): BD, blue dextran (Mr > 2 X 106); TG, thyroglobulin (Mr 3.6 ,g of APF-1. Fraction hIB was diluted and heat treated similarly, 6.7 X 105); F, ferritin (Mr % 4.6 X 105); BSA, bovine serum albumin and assayed with 165 pig of untreated fraction IIA. (Mr 69,000). Downloaded by guest on October 3, 2021 Biochemistry: Hershko et al. Proc. Ndtl. Acad. Sci. USA 76 (1979) 3109 and the stabilization of APF-2 rules out the above possibilities. Stimulation of protein degradation is specific for ATP. ADP is not effective unless an ATP-generating system such as cre- atine phosphate and creatine kinase is added; unlike a recently discovered protease from liver cytosol (unpublished data), the reticulocyte system is neither activated nor stabilized by cre- atine phosphate. Other nucleoside triphosphates, such as GTP, CTP, or UTP, as well as the nonhydrolyzable 3,7y imido or methylene analogues of ATP, cannot replace ATP in the acti- vation of proteolysis. In fact, AdoPP[NH]P inhibits ATP- stimulated protein degradation (not shown). The stabilization of APF-2 is also specific for adenine nucleotides (GTP, CTP, or UTP do not stabilize). However, ADP (but not AMP) and the ATP analogues also partially protect APF-2 against heat inac- tivation. Furthermore, while Mg2+ is absolutely required for .0- ATP-dependent protein degradation, ATP can effectively 10 Br APF-2 stabilize APF-2 also in the absence of Mg2+ ions (Table 2). It is evident, therefore, that APF-2 is not stabilized by phos- phorylation; neither is the stimulation of proteolysis by ATP 5- solely due to the stabilization of APF-2, otherwise the other stabilizing nucleotides would also stimulate protein degradation. It rather seems that the interaction of ATP, its analogues, or ADP with adenine site(s) on APF-2 converts this 15- factor to a more stable conformation. C Continued Requirement for ATP in the Action of All Three APF-1 Components. The question arises as to whether one of the components is an ATP-stimulated protease that produces initial 10 cleavage(s) in the polypeptide substrate, whereas the other factors are ordinary (non-ATP-dependent) proteases or pep- tidases that convert the cleavage fragments to acid-soluble APF-25- orfractionIPFI2Bo(C) Pricuae witou AP- GInren products. In fact, most of the reticulocyte aminopeptidase and *APF-1 + Hk +Gc trypsin-like protease activities are present in fraction IIB (un- published results). To examine this possibility, the reaction 30 60 90 120 mixture was preincubated without one of the components and Time, min then ATP was trapped with hexokinase and glucose and the FIG. 3. Continued requirement for ATP for the action of the missing factor was added. If one of the components is a terminal various components. The complete system cohtained(sbg of protein): protease, it should under these conditions act on the primary APF-1, 3.6; APF-2, 81; fraction IIB, 150. At 60 min, where indicated, cleavage products of 125I-albumin (which should accumulate hexokinase (Hk, 1 pg) and glucose1.(Glc, mM) were added. (A) during preincubation), even after the removal~of ATP. In the Complete system. (h)Preincubated at 37C for 60 mn without either complete reaction mixture, the addition of hexokinase with APF-2 or fraction IIB. (C) Preincubated without APF-1. In prein- glucose immediately arrested ATP-dependent proteolysis, ei- cubation, all components of the reaction mixture (except for the one omitted) were incubated with 1251-albumin and ATP, and then the ther at the onset of the incubation or later (Fig. 3A). Control missing component was added without (open symbols) or with (closed hexokinase and Table 2. Nucleotide specificity of the stabilization of APF-2 symbols) glucose. Relative activity Stimulation experiments showed that hexokinase or glucose, each without of protein Stabilization the other, did not significantly inhibit protein degradation. Addition breakdown of APF-2 When the reaction mixture was preincubated without APF-2, ATP 1.00 1.00 fraction TIB (Fig. 3B) or APF-1 (Fig. 3C) and the missing factor ATP, -Mg2+ 0 0.98 then added, the restoration of protein degradation was com- ADP 0 0.56 pletely inhibited by the ATP trap in all cases. This shows that ADP + creatine-P + kinase 1.57 - the action of all three components requires the continued AMP 0 0 presence of ATP, and no single component is an ATP-inde- AdoPP[NH]P 0 0.53 pendent terminal protease or peptidase. AdoPP[CH2IP 0 0.54 Examination of the time course of protein degradation in the GTP 0 0 above experiments shows that, in the complete reaction mixture, CTP 0 0 there is a time lag in the degradation of 125I-albumin' during the Creatine-P 0 0 initial 30 min of incubation', following which the reaction All nucleotides were added to give 2 mM, creatine phosphate to 15 proceeds linearly (Fig. 3A). Preincubation without APF-2 or mM, and creatine kinase to 100 ,ug/ml. Stimulation of protein fraction t eB(followed by their respective addition) did not breakdown is expressed relative to the activity obtained with ATP, abolish the lag significantly (Fig. 3B). However, when the which was 17.4% of 125I-albumin solubilized. Stabilization of APF-2 mixture was preincubated without APF-1 (i.e., 1251-albumin was tested by treating at 42°C for 25 min. % 1251-albumin degraded incubated in the 'of fraction and was: untreated, 14.3; heated without additions, 2.5; heated with ATP, presence A.PF-2, TIB, ATP), and then APF-1 was the reaction 10.0. In the case of AdoPP[NHIP, the results were corrected for the added, immediately pro- inhibition (38%) caused by a similar final concentration of the ana- ceeded at a high linear rate (Fig. 3C). These observations may logue in the assay mixture. indicate that the rate of formation of the active ATP-dependent Downloaded by guest on October 3, 2021 3110 Biochemistry: Hershko et al. Proc. Natl. Acad. Sci. USA 76 (1979) protease complex may be limited by interaction of APF-2 and that serve as substrates and compete with 125I-albumin; fraction IIB (see Discussion). these denatured proteins might be rapidly broken down during preincubation by other non-ATP-dependent proteases present I5ISCUSSION in these fractions. ATP-dependent protein degradation in reticulocytes is ap- The multi-component reticulocyte system appears to differ parently carried out by a complex sy'stem consisting of at leZ'st from the ATP-dependent proteolytic cleavage of the phage X three components of widely differing properties. Among the repressor (5), In the latter, the reaction apparently requires only three components that haye been resolved, the high-molecu- one protein, recently identified as the product of the E. coli recA lar-weight factor described in this paper (APF-2) has been gene (11). Though the recA protein carries out a limited en- identified as interacting directly with ATP by, virtue of the doproteolytic cut in a specific polypeptide, while the reticu- stabilization at 420C resulting from ATP. Phosphorylation of locyte system breaks down various proteins to free amino acids, APF-2 cannot be part of the mechanism of stabilization because some similarities in the mechanisms of the involvement of ATP of the protective action of nonphosphorylating adenine nu- in cleavage might be expected. It should be noted cleotides as well (Table 2). However, APF-2 might be a protein that the possible contamination of purified recA protein by kinase in the proteolysis. reaction: the stabilization of an slight amounts of other factors(s) of high activity was not ruled erythrocyte protein kinase by ATP has been described (10), and out (11). It is also possible that the recA protein is a multifunc- preparations of APF-2 contain considerable protein kinase tional ehzyme in which different sites of the enzyme molecule activity with endogenous proteins as acceptors (unpublished catalyze different functions. These different functions may be observations). However, APF-2 cannot be identical to the above analogous to some of those carried out by the separate compo- erythrocyte enzyme,;because APF-2 Activity is lost during re- nents of the reticulocyte system. ticulocyte maturation (unpublished observations), as is protein degradation in intact cells (6). Some of this work was done during the stay of A.H. on sabbatical The elucidation of the nibde of action of APF-2, or the role leave at the Institute for Cancer Research, Philadelphia. The work was of ATP, depends upon further resolution, purification and supported by American Cancer Society Grant BC-24L. characterizatio. of the various components. ATP cannot act 1. Goldberg, A. L. & St. John, A. C. (1976) Annu. Rev. Biochem. simply by stabilization because nonphosphorylating ATP an- 45,747-803. alogues cannot replace ATP in the ptotease assay. ATP may act 2. Hershko, A. & Tomkins, G. M. (1971) J. Biol. Chem. 246, by the modification'(such As phosphorylation or adenylylation) 710-714. of an enzyme or a protein factor or by the modification of the 3. Etliniger, J. D. & Goldberg, A. L. (1977) Proc. Natl. Acad. Sci. polypeptide substrate, or it may be required for the interaction USA 74,54-58. 4. Murakami, K., Voellmy, R. & Goldberg, A. L. (1977) Fed. Proc. of these macromolecules. If a phosphorylation reaction occurs, Fed. Am. Soc. Exp. Biol. 36, 764 (abstr.). it will have to be concluded that is also very 5. Roberts, J. W., Roberts, C. W. & Mount, D. (1977) Proc. Natl. rapid in the partially purified system, because the addition of Acad. Sci. USA 74,2283-2287. an ATP trap stops protein breakdown immediately (Fig. 3 B 6. Hershko, A., Heller, H., Ganoth, D. & Ciechanover, A. (1978) and C). In the case of a phosphorylahbn mechanism, this might in Protein Turnover and Lysosonal Function, eds. Segal, H. L. mean that the latter factors are either continuously phos- & Doyle, D. J. (Academic, New York), pp. 149-169. phorylated (and dephosphorylated) themselves, or are closely 7. Ciechanover, A., Hod, Y. & Hershko, A. (1978) Biochem. Biophys. related to the phosphorylatipn process. Res. Commun. 81, 1100-1105. The observed lag in the time course of the reaction and its 8. Moore, A. T., Williams, K. E. & Lloyd, J. B. (1975) Biochem. J. 164,607-616. abolishment by the preincubation of all components except 9. Efron, M. L. (1968) Chromatographic and Electrophoretic APF-1 might mean that.this heat-stable polypeptide enters a Techniques, ed. Smith, I. (Heinman, New York), Vol. 2, pp. functional complex rapidly at a step subsequent to the initial 1r66182. interaction of APF-2 and fraction IIB. However, other expla- 10. Tao, M. (1971) Arch. Biochem. Biophys. 143, 151-157. nations cannot be eliminated at this stage of purification. For 11. Roberts, J. W., Roberts, C. W. & Craig, N. L. (1978) Proc. Natl. example, fractions APF-2 and IIB may contain denatured Acad. Sci. USA 75,4714-4718. Downloaded by guest on October 3, 2021