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 (protein 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 proteolysis, do not stabilize this frim fraction I by heat treatment, ammonium sulfate precip- factor. Adenylate nucleotides 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 enzyme 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 enzymes 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 phosphorylation. 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).