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Recognition of Misfolding Proteins by PA700, the Regulatory Subcomplex of the 26 S Proteasome

Recognition of Misfolding Proteins by PA700, the Regulatory Subcomplex of the 26 S Proteasome THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 8, Issue of February 25, pp. 5565–5572, 2000 © 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Recognition of Misfolding Proteins by PA700, the Regulatory Subcomplex of the 26 S Proteasome* (Received for publication, September 7, 1999, and in revised form, November 8, 1999) Elizabeth Strickland‡, Kevin Hakala§, Philip J. Thomas‡§¶i, and George N. DeMartino§i From the ‡Program in Molecular Biophysics and the §Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235 The 26 S proteasome is a large protease complex that a polyubiquitin chain as a prerequisite for their proteolysis (1, 2). This requirement seems to have evolved, at least in part, catalyzes the degradation of both native and misfolded proteins. These proteins are known to interact with because of the unique structure of the 26 S proteasome. PA700, the regulatory subcomplex of the 26 S protea- The 26 S proteasome is composed of two 700,000-dalton some, via a covalently attached polyubiquitin chain. subcomplexes, the 20 S proteasome and PA700 (1, 2, 4). The 20 Here we provide evidence for an additional ubiquitin- S proteasome, the catalytic core of the complex, is a cylindrical independent mode of substrate recognition by PA700. particle consisting of four stacked heptameric rings that con- PA700 prevents the aggregation of three incompletely tain a hollow center in which all six of the catalytic sites are folded, nonubiquitinated substrates: the DF-508 mutant located (5, 6). This topology excludes both folded and aggre- form of cystic fibrosis transmembrane regulator, nucle- gated substrates from contact with, and hydrolysis by, the otide binding domain 1, insulin B chain, and citrate active sites. The role of initial substrate recognition and bind- synthase. This function does not require ATP hydroly- ing by the complex is relegated to PA700, a 20-subunit regula- sis. The stoichiometry required for this function, the tory complex (7–9). Because the 26 S proteasome typically effect of PA700 on the lag phase of aggregation, and the requires polyubiquitinated protein substrates for degradation, temporal specificity of PA700 in this process all indicate the polyubiquitin chain binding site on PA700 (10 –12) is an that PA700 interacts with a subpopulation of non-native important determinant of substrate specificity. In addition to conformations that is either particularly aggregation- binding protein substrates, PA700 also regulates the access of prone or nucleates misassociation reactions. The inhibi- substrates to the active sites of the proteasome. ATP-depend- tion of off-pathway self-association reactions is also re- ent binding of PA700 to the outer rings of the proteasome flected in the ability of PA700 to promote refolding of probably induces a conformational change in the proteasome citrate synthase. These results provide evidence that, in that creates narrow pores in the center of the outer rings, addition to binding polyubiquitin chains, PA700 con- thereby exposing the interior catalytic sites to the exterior (2, 3, tains a site(s) that recognizes and interacts with mis- folded or partially denatured polypeptides. This feature 13, 14). This effect explains, in part, the PA700-dependent supplies an additional level of substrate specificity to activation of the proteasome’s hydrolysis of short nonubiquiti- the 26 S proteasome and a means by which substrates nated peptides, which can freely cross the pores without need are maintained in a soluble state until refolding or deg- for interaction with or structural modification by PA700 (8). In radation is complete. contrast, the PA700-dependent opening of pores in the protea- some does not explain fully how PA700 promotes the degrada- tion of ubiquitinated proteins with native or partially folded The 26 S proteasome is a large proteolytic machine that structures because such proteins are too large to traverse the participates in nearly all of the multiple roles played by intra- pores (15). Therefore, it is commonly assumed that as an es- cellular protein degradation in cellular function (reviewed in sential feature of 26 S proteasome-catalyzed proteolysis, PA700 Refs. 1–3). For example, the proteasome catalyzes both the must unfold the bound, ubiquitinated proteins and processively constitutive turnover of the bulk of intracellular proteins and translocate them through the pores to the catalytic sites of the the conditional degradation of specific proteins that regulate proteasome. This model would explain the known ATP require- various cellular processes. The 26 S proteasome is also a com- ment for degradation of ubiquitinated proteins by the assem- ponent of the quality control machinery that selectively de- bled 26 S proteasome, because it predicts that substrate un- grades proteins with abnormal structures. Most known protein folding and/or translocation are coupled to ATP hydrolysis by substrates of the 26 S proteasome are covalently modified with one or more of the six AAA protein subunits of PA700 (7, 13, 14, 16). This attractive model, however, has little direct experi- mental support and, with the exception of ATPase activity, the * This work was supported by NIDDK, National Institutes of Health properties of PA700 required for such activities are poorly (NIH) Grant DK49835 (to P. J. T.), the Robert Welch Foundation I-1284 defined. (to P. J. T.), NIDDK, NIH DK46181 (G. N. D.), and the Muscular Dystrophy Association (G. N. D.). The costs of publication of this article To mediate unfolding and/or translocation, PA700 must con- were defrayed in part by the payment of page charges. This article must tain a binding site for the non-native structures characteristic therefore be hereby marked “advertisement” in accordance with 18 of both the products of the unfolding reaction and the sub- U.S.C. Section 1734 solely to indicate this fact. This work is dedicated to the late Dr. Paul Srere who was kind enough to have many helpful discussions with us. Established investigator of the American Heart Association The abbreviations used are: PA700, proteasome activator, 700 kDa; (9740033N). CFTR, cystic fibrosis transmembrane conductance regulator; NBD1, To whom correspondence may be addressed: 5323 Harry Hines Blvd., nucleotide binding domain 1; DF-NBD1, DF508 mutant N-terminal Dallas, TX 75235-9040. For G. N. D., Tel.: 214-648-3308; Fax: 214-648- nucleotide binding domain of CFTR; PA28, proteasome activator, 28 4771; E-mail: [email protected]. For P. J. T., Tel.: 214-648- kDa; CS, citrate synthase; ATPgS, adenosine 59-O-(thiotriphosphate); 8723; Fax: 214-648-9268; E-mail: [email protected]. GdnHCl, guanidinium hydrochloride; NEM, N- ethylmaleimide. This paper is available on line at http://www.jbc.org 5565 This is an Open Access article under the CC BY license. 5566 Recognition of Misfolding Proteins by PA700 strates for the translocation reaction. Precendent exists for this from studies of the bacterial Clp protease system, which, like the proteasome, has occluded active sites and capping ATPases ClpA and ClpX. These AAA ATPases are known to mediate molecular chaperone and unfoldase activities (17–20). To gain insight into the recognition of protein substrates by PA700 we have analyzed interactions of PA700 with several model pro- teins in native and non-native conformations. Our data dem- onstrate that PA700 can directly interact with non-native structures of nonubiquitinated proteins and suggest models for how such interactions can participate in the degradation or refolding of protein substrates. EXPERIMENTAL PROCEDURES Materials—PA700 (21), latent 20 S proteasome, PA28, and modula- tor were purified from bovine red blood cells as described previously (8, 22, 23). “Lid” and “base” subcomplexes of PA700 were identified during the purification of PA700 from bovine red blood cells and are essentially equivalent to their counterparts in yeast (24). Lid was purified as an isolated complex, whereas, the base was purified bound to 20 S protea- some. Details of the purification and characterization of these com- plexes will be published elsewhere. DF508 mutant CFTR-NBD1 from residues 404 –589 was expressed with a six-histidine tag and purified as described previously (25). Porcine heart citrate synthase was purchased from Sigma; it was dialyzed into TE buffer (50 mM Tris-HCl, pH 8.0, 2 mM EDTA), concentrated to 4.65 mg/ml, and stored at 220 °C in ali- quots. Porcine insulin was from Calbiochem. Aggregation of DF-NBD1—Aggregation of DF-NBD1 was performed as described previously (26) at 2 mM final protein concentration in Buffer R (100 mM Tris-HCl, pH 7.4, 0.385 ML-arginine hydrochloride, 2 mM EDTA, 10 mM dithiothreitol, and 25 mM guanidine hydrochloride (GdnHCl)). Aggregation was initiated by rapid dilution from 6 M FIG.1. Components of the proteasomal machinery inhibit GdnHCl, while vortexing into a solution of 37 °C Buffer R, and was CFTR DF-NBD1 aggregation. GdnHCl-denatured DF-NBD1 was di- monitored by the increase in turbidity at 400 nm. 26 S proteasome, luted into Buffer R under conditions (2 mM DF-NBD1, 37 °C) where PA700, or 20 S proteasome were present at 100 nM prior to the addition folding to the native state is disfavored and DF-NBD1 aggregates. A, of DF-NBD1 unless otherwise noted. aggregation in the absence or presence of 100 nM 26 S proteasome (126 Assembly of 26 S proteasome—The 26 S proteasome was assembled S). B: Aggregation of DF-NBD1, DF-NBD1 with 100 nM 20 S proteasome in an ATP-dependent reaction from purified 20 S proteasome and (120 S), or DF-NBD1 with 100 nM PA700 (1PA700). PA700 components, as described previously (8). Assembly conditions contained 2-fold molar excess of 20 S proteasome with respect to PA700. done at single time points were assayed after a 45-min reactivation. The Less than 10% of the PA700 was unbound to the proteasome after the slope of the initial rate during the linear portion of the progress curve assembly reaction. was plotted relative to the activity of mock-treated native CS. During Reductive Aggregation of Insulin—A2mM stock solution of porcine the time course of the activity assay, which is short relative to the time insulin was prepared in 0.1 M potassium phosphate buffer, pH 7.6. course of reactivation, no further reactivation occurred. To ensure uni- Aggregation assays were performed by a modification of previously formity in initiating the reactivation, denatured CS was added to the reported methods (27, 28). Reactions were performed at 37 °C in a final reactivation mixture while vortexing. The fraction of citrate synthase volume of 250 ml containing 45 mM Tris-HCl, pH 7.8, 4 mM reduced that could be reactivated was reproducible within an experiment but glutathione, 0.4 mM oxidized glutathione, 160 mM insulin, and concen- varied between 25 and 75% among different citrate synthase prepara- trations of various proteins as indicated for specific experiments. Ag- tions. Each experiment was repeated multiple times with consistent gregation was monitored continuously as an increase in turbidity at 650 results obtained with multiple preparations. nm. In independent experiments, the length of the lag phase prior to initial detection of turbidity with insulin alone varied typically from RESULTS about 300 s to about 1100 s. However, within a given experiment, PA700, the Regulatory Component of the 26 S Proteasome, repetitions varied by less than 10%. All reported results were obtained Inhibits Aggregation of Misfolding DF-NBD1—To understand in at least three to four independent experiments. Thermal Aggregation of Citrate Synthase—Thermal aggregation of better the cellular and molecular mechanisms involved in the citrate synthase (CS) was carried out by adding 150 nM (monomer) CS recognition and processing of misfolded proteins, we studied to a solution of 40 mM HEPES-KOH, pH 7.5, heated to 43 °C (29). the interaction of the 26 S proteasome with DF-NBD1. DF- Aggregation was followed by light scattering at 500 nm in a PTI fluo- NBD1 is the nucleotide binding domain of CFTR that contains rometer at right angles with 2 nm excitation and 4 nm emission slit the common cystic fibrosis disease-causing mutation responsi- widths. PA700 was present before the addition of CS unless stated ble for impaired folding of the full-length CFTR protein (25, otherwise. Reactivation of Citrate Synthase—The reactivation of citrate syn- 32–34). In refolding experiments, less DF-NBD1 reaches the thase was monitored in a multi-step assay as described previously (30). native state than does wild-type NBD1. A certain fraction, In the first step, native CS was denatured in 6 M GdnHCl, 50 mM instead, forms large aggregates of misfolded, mutant protein Tris-HCl, pH 7.9, 2 mM dithiothreitol for1hat room temperature. (25, 26, 34). In the cell this misfolding protein is recognized by Reactivation of denatured CS was then initiated by dilution into a molecular chaperones (35, 36) and degraded by the ubiquitin- reactivation buffer containing 100 mM Tris-HCl, pH 7.9, 2 mM dithio- proteasome pathway (37, 38). Therefore, DF-NBD1 aggregation threitol, 10 mM KCl and allowed to continue at room temperature. Experiments containing ATP (2 mM) also included 5 mM MgCl . The provides a useful and physiologically relevant in vitro model activity of the reactivated CS was then assayed spectrophotometrically system for studying interactions of unfolded or misfolded pro- according to the method of Srere and Kosicki (31) at various time points teins with components of the cellular quality control with freshly prepared oxaloacetic acid and acetyl-CoA. Experiments machinery. As shown in Fig. 1A, the 26 S proteasome completely inhib- K. Hakala and G. N. DeMartino, manuscript in preparation. ited aggregation of DF-NBD1. The lack of turbidity observed in Recognition of Misfolding Proteins by PA700 5567 the presence of the 26 S proteasome was not due to the degra- dation of DF-NBD1 into small, soluble peptides (data not shown). To determine whether either subcomplex of the 26 S proteasome was sufficient to inhibit aggregation, 20 S protea- some and PA700 were assayed individually for their ability to inhibit the aggregation of DF-NBD1. The 20 S proteasome did not affect aggregation, whereas PA700 inhibited aggregation as effectively as the intact 26 S proteasome (Fig. 1B). These data demonstrate that the inhibitory activity of the 26 S pro- teasome can be attributed to PA700. PA700 Inhibits the Aggregation of Other Misfolding Pro- teins—To determine whether the ability of PA700 to interact with the non-native conformation of DF-NBD1 is a general property, analogous experiments were conducted with two ad- ditional proteins whose misfolding can be initiated under dif- ferent conditions. Insulin is composed of two polypeptide chains, A and B, linked by two disulfide bonds. Upon reduction of the disulfides, the chains separate, and the B chain aggre- gates (27, 28). As shown in Fig. 2, PA700 inhibited this aggre- gation as effectively as the 26 S proteasome, whereas the 20 S proteasome had no inhibitory effect. To extend further the generality of this effect, we tested the ability of PA700 to inhibit the aggregation of thermally dena- tured citrate synthase, a protein commonly utilized for study- ing chaperone activity (29, 30, 39). Native citrate synthase is a homodimer; when heated to 43 °C, the population of partially folded conformations of citrate synthase increases, leading to aggregation. As with the other tested proteins, citrate synthase aggregation was inhibited by PA700 at low molar ratios (Fig. 3). PA700 increased the lag period of citrate synthase aggrega- tion in a manner similar to its effect on insulin. Together, these data demonstrate that PA700 interacts with non-native forms of a variety of proteins. The ability of PA700 to inhibit aggregation is specific to PA700 and is not a property of either of two related multipro- tein proteasome regulator complexes. First, a protein complex, termed “modulator,” consisting of two of the ATPase subunits found in PA700 and a third novel protein not found in PA700, enhances PA700-dependent activation of the proteasome (21) but was not effective at inhibiting the aggregation of insulin (Fig. 2B). Second, the proteasome activator PA28 (23), which is known to associate with the 20 S proteasome, did not inhibit the aggregation of insulin (Fig. 2B). In addition, PA700 that has been heat-treated for 15 min at 75 °C is no longer effective in retarding the aggregation of insulin (data not shown). These FIG.2. PA700 inhibits the aggregation of insulin. Aggregation data provide further evidence for specificity of the PA700-de- was initiated by dilution of native insulin (160 mM) into reducing buffer. pendent inhibition of aggregation. A, aggregation of insulin alone, insulin with 300 nM 20 S proteasome (120 S), insulin with 300 nM 20 S, 160 nM PA700-assembled 26 S The extent of inhibition of insulin aggregation was directly proteasome (126 S), or insulin with 160 nM PA700 (1PA700). B, ag- proportional to the concentration of PA700; but surprisingly, gregation of insulin alone, insulin with 300 nM modulator (1Modula- inhibition occurred at PA700 concentrations 1000-fold less tor), or insulin with 300 nM PA28 (1PA28). C, aggregation of insulin than those of the substrate insulin. Furthermore, PA700 in- with no PA700, 50 nM PA700 (3200:1), 100 nM PA700 (1600:1), 200 nM PA700 (800:1), or 400 nM PA700 (400:1). creased the lag time of insulin aggregation in a concentration- dependent manner (Fig. 2C). In contrast, simple reduction of the insulin concentration in the absence of PA700 reduced the by rapid dilution into a nondenaturing buffer. The restoration rate of aggregation but did not have a significant effect on the of native structure was monitored by the recovery of enzymatic lag phase in this concentration range (data not shown). These activity. Under these conditions, denatured citrate synthase results suggest that PA700 interacts with a subpopulation or refolds poorly in the absence of chaperones (Fig. 4 and Refs. 29, with a transient intermediate of misfolding insulin which is 30, and 39). However, in the presence of PA700, enzymatic particularly prone to aggregation (see “Discussion”). activity was recovered in both a time-dependent and PA700 PA700 Promotes Refolding of Misfolding Citrate Syn- concentration-dependent fashion (Fig. 4). These effects were thase—In addition to inhibiting aggregation of misfolding pro- achieved at the same low molar ratios required for inhibition of teins, many molecular chaperones promote refolding of these aggregation. Thus, by inhibiting aggregation of misfolded cit- proteins to native structures. Therefore, we tested the ability of rate synthase, PA700 promotes its refolding. PA700 to promote reactivation of chemically denatured citrate PA700 Does Not Depend on ATP to Inhibit Aggregation or synthase. Native citrate synthase was first denatured with 6 M Promote Refolding of Misfolded Proteins—Many well charac- guanidinium hydrochloride, and then refolding was initiated terized molecular chaperones utilize ATP to modulate the rate 5568 Recognition of Misfolding Proteins by PA700 with N-ethylmaleimide (NEM), which abolishes its ATPase activity (16). NEM-treated PA700 effectively inhibited the ag- gregation of insulin (Fig. 5D) and promoted the reactivation of citrate synthase (Fig. 5E). These data indicate that the inter- action of PA700 with non-native protein substrates does not depend upon ATP. PA700 Recognizes a Subset of Non-native Substrate Confor- mations—The substoichiometric PA700:substrate ratios re- quired for inhibition of aggregation and promotion of refolding and the progressively increased delay in onset of aggregation in the presence of PA700 suggest that PA700 interacts with a subpopulation of non-native conformations. To test this possi- bility further, PA700 was added to either the citrate synthase aggregation assay or reactivation assay after each process had begun. As shown in Fig. 6, PA700 remained effective at inhib- iting the thermal aggregation of initially folded citrate syn- FIG.3. PA700 inhibits the thermal aggregation of citrate syn- thase when added 20 s after the initiation of misfolding. How- thase. Native citrate synthase (150 nM) was heated to 43 °C either ever, PA700 was progressively less effective at inhibiting alone or in the presence of 0.9 nM PA700 (160:1), 1.9 nM PA700 (80:1), aggregation when addition was delayed for longer times. These or5nM PA700 (30:1). a.u., arbitrary units. results cannot be explained by simple stoichiometric binding of PA700 because of the large excess of unfolded monomer and, thus, imply that PA700 may act on a species critical for initi- ation of aggregation. Furthermore, once citrate synthase ag- gregates were formed, PA700 could not disassemble and solu- bilize them (data not shown). Likewise, PA700 promoted the reactivation of citrate synthase when present at the start of reactivation, but not when added 2 min later. In sum, these results suggest that PA700 acts on a non-native conformation not yet fully committed to off-pathway aggregation. The Base Subcomplex of PA700, Containing ATPase Sub- units, Inhibits Aggregation of Misfolded Proteins—PA700 is composed of 20 distinct subunits, including six AAA-ATPases (16, 21, 47, 48), at least one subunit that binds ubiquitin chains (10 –12), and an isopeptidase (49). The functions of the remain- ing subunits are unknown. To determine which PA700 sub- units are responsible for the effects on inhibition of protein aggregation we repeated some of the experiments described above using subcomplexes of PA700 essentially equivalent to the base and lid complexes described for yeast PA700 (24). The bovine base (in a complex with the 20 S proteasome) inhibited aggregation as effectively, if not more effectively, as intact PA700 when compared on a molar basis (Fig. 7A). The base contains all the ATPases of PA700 and two other subunits (data not shown). In contrast, lid, which contains the polyubiq- uitin chain binding subunit, inhibited aggregation of insulin very poorly (Fig. 7B). Similar effects of base and lid were observed with inhibition of citrate synthase aggregation (data not shown). These results suggest that the various functions of PA700 described here are localized to the base component of PA700. FIG.4. PA700 reactivates citrate synthase. GdnHCl-denatured DISCUSSION citrate synthase was diluted into refolding conditions, and the forma- tion of active, native protein was monitored. A, time course of CS This work demonstrates that PA700, the regulatory complex reactivation in the absence (open squares) or presence (closed circles)of of the 26 S proteasome, recognizes non-native conformations of 300 nM PA700. B, CS reactivation at 45 min with increasing concentra- proteins. PA700 inhibited the aggregation of three misfolding tions of PA700. proteins and promoted the refolding of a denatured protein to the native state. These newly discovered functions of PA700 of substrate turnover as they inhibit aggregation and/or pro- are defining features of molecular chaperones (50 –53) and, in mote refolding (40 – 46). To determine whether PA700, a known combination with the previously established function of PA700 ATPase (16, 47), requires ATP for these activities, we repeated in regulating the degradation of ubiquitinated proteins, place the described assays in the presence and absence of Mg or PA700 in a unique and central position for determining the fate of protein substrates as part of the quality control machinery of ATP. Mg or ATP had no significant effect on the ability of PA700 to inhibit the aggregation of DF-NBD1, insulin, or cit- the cell. Like known chaperones, PA700 could bind to non- rate synthase (Fig. 5, A–C). In addition, neither ADP nor the native proteins, thereby inhibiting their aggregation and per- nonhydrolyzable analog ATPgS influenced the results. ATP mitting refolding subsequent to their release (Fig. 8). Alterna- also did not affect the ability of PA700 to reactivate citrate tively, by inhibiting the competing aggregation reaction, PA700 synthase (Fig. 5E). To confirm this result, PA700 was treated could promote entry of non-native proteins into the degradation Recognition of Misfolding Proteins by PA700 5569 FIG.5. ATP is not required for PA700 inhibition of aggregation or the reactivation of CS. A, DF-NBD1 (2 mM) was aggregated at 37 °C as in Fig. 1. Aggregation was either with no PA700, 100 nM PA700 (1PA700), or 100 nM PA700, 20 mM MgCl ,2mM ATP (1PA700 (1 ATP)). B, insulin (160 mM) was aggregated under reducing conditions as in Fig. 2A. Reactions contained insulin, insulin 1 160 nM PA700 (1PA700), or insulin 1 3.6 mM MgCl , 800 mM ATP, 160 nM PA700 (1 PA700 (1ATP)). C, CS (150 nM) thermal aggregation was carried out with CS, CS 1 7.5 nM PA700 (1 PA700), CS 1 200 mM ATP, 7.5 nM PA700 (1 PA700 (1ATP)), or CS 1 200 mM ADP, 7.5 nM PA700 (1 PA700 (1ADP)). MgCl (1 mM) was present in all reactions. D, insulin (160 mM) aggregation was initiated with no additions, with 215 nM PA700 (1 PA700) or with 215 nM NEM-treated PA700 NEM (1 PA700 ). E, Gdn-HCl-denatured CS (150 nM) was reactivated either alone or with the addition of 2 mM ATP; 2 mM ATPgS; 300 nM PA700; 300 nM PA700, 2 mM ATP; 300 nM NEM-treated PA700, 2 mM ATP; or 300 nM PA700, 2 mM ATPgS. All reactions contained 5 mM MgCl . Bars represent the average of three experiments. pathway, as has been reported for a known molecular chaper- atypical of many well characterized molecular chaperones and one (54). The possible role of PA700 as a molecular chaperone provide insights regarding their possible physiological signifi- in the cell may be analogous to known chaperone functions cance. PA700 inhibited the aggregation of misfolded proteins at played by ClpA and ClpX, the regulatory subcomplexes of pro- very low PA700:protein substrate ratios, whereas other chap- karyotic ATP-regulated proteases (55). In addition, PA700 re- erones normally require near stoichiometric ratios for function. cently has been shown to play a nonproteolytic role in nucleo- For example, 30 –100 times more Hsc70 was required to tide excision repair in yeast, perhaps by exerting a chaperone- achieve the same degree of inhibition accomplished by PA700 like activity that disassembles or structurally rearranges the in the assays reported here (data not shown and Ref. 26). These repair complex (56). results indicate that PA700 interacts with a subpopulation of Several features of the effects of PA700 reported here are proteins in a conformation prone to aggregation. Thus, to in- 5570 Recognition of Misfolding Proteins by PA700 FIG.7. The base subcomplex of PA700, but not the lid subcom- FIG.6. PA700 acts on a partially folded subpopulation. A, cit- plex, inhibits aggregation. The reductive aggregation of insulin (160 mM) was performed as in Fig. 2. A, effect of base on insulin aggregation. rate synthase (150 nM) misfolding was initiated as in Fig. 3 in the presence of 7.5 nM PA700 (0 s) or with 7.5 nM PA700 added after the Insulin aggregation was initiated in the absence of other proteins (up- per line), with 300 nM base-proteasome complex (1 “base”), and with initiation of aggregation (20, 60, or 120 s). B, the reactivation of citrate synthase was assayed and reported as in Fig. 4 with either citrate 300 nM PA700 (1 PA700). B, effect of lid on insulin aggregation. Insulin aggregation was initiated in the absence of any other proteins (upper synthase alone, citrate synthase with PA700 present at the beginning of reactivation, or citrate synthase with PA700 added 2 min after the start line) and in the presence of either 200 nM lid (1 “lid”) or 130 nM PA700 (1PA700). of reactivation. hibit aggregation effectively, PA700 need not bind each sub- for those needed here. Second, ATP promoted a small stimula- strate molecule, but only those disposed to off-pathway associ- tion of inhibition in their experiments. Third, consistent with ations. Additionally, order-of-addition experiments suggest the low stoichiometry or possibly the dynamic nature of the that PA700 likely recognizes a conformation early in the mis- complex, we were unable to detect significant amounts of as- folding process. These features are consistent with the obser- sociation of either insulin or citrate synthase with PA700 dur- vation that PA700 lengthens the lag phase for aggregation of ing density gradient centrifugation (data not shown). The basis both insulin (Fig. 2C) and citrate synthase (Fig. 3) in a concen- for these differences is unclear but may have significant con- tration-dependent manner. The lag phase observed in each of sequences for interpretation of the mechanisms in which these these aggregation processes is likely due to the formation of a processes are involved (see below). specific conformation or nucleus prone to self-association that What are the physiological implications of the current re- PA700 may recognize and interact with. sults? Regardless of whether PA700, either alone or as part of Finally, PA700 does not require ATP either to inhibit aggre- the 26 S proteasome, functions independently as a molecular gation or to promote refolding. Many previously characterized chaperone in the cell, the novel activities described here are molecular chaperones utilize ATP hydrolysis as a timing mech- directly relevant to its established function in the degradation anism to coordinate the rate of substrate binding and release of ubiquitinated proteins. For example, PA700 must contain a with the rate of substrate folding, rather than utilizing the binding site for non-native protein structures as an essential hydrolytic energy to promote an otherwise unfavorable reac- feature for its role in proteolysis because non-native structures tion. Therefore, the current results may indicate that the are the products of unfolding reactions and the substrates for known ATP requirement for protein degradation by the 26 S translocation reactions that are part of the putative proteolytic proteasome supports energy-dependent unfolding or processive mechanism. The potential function of this site for both protein translocation of unfolded substrates to the proteasome active folding and protein degradation is illustrated in the model sites by PA700. proposed in Fig. 8. In this model, PA700 may bind to: (i) native While this manuscript was in preparation, Braun et al. (57) ubiquitinated proteins (via recognition of polyubiquitin reported that the 26 S proteasome inhibited the aggregation chains), (ii) non-native nonubiquitinated proteins (via recogni- and promoted the refolding of citrate synthase, one of the tion of non-native protein structure), or (iii) non-native ubiq- substrates used in the current study. Both of these activities uitinated proteins (via recognition of both polyubiquitin chains were localized to the base of PA700. Although the results of the and non-native protein structure). These multiple recognition two studies are in general agreement, they differ in several and binding modes would allow PA700 to serve a central edit- important ways. First, much higher protein:substrate ratios ing function that determines the final fate of substrates. For were required for the effects reported by Braun et al. (57) than example, the known isopeptidase activity of PA700 may pro- Recognition of Misfolding Proteins by PA700 5571 FIG.8. Model for the interactions of the 26 S proteasome with protein substrates. Established (black lines) and hypothetical (gray lines) actions of the 26 S proteasome with protein substrates are depicted. Pathways denoted with heavy arrows represent reactions directly related to the studies in the current report. The model distinguishes four different levels through which proteins are processed. At the first level, protein structure is converted between native and non-native forms, either of which may be modified further by ubiquitination. At the second level, the PA700 subcomplex of the 26 S proteasome can recognize and interact with each of the protein forms except native nonubiquitinated proteins, for which it has very low affinity. Otherwise, it can bind to the polyububiquitin chain on either the native or non-native protein or with lower affinity to the non-native, nonubiquitinated protein via a second site that recognizes features of non-native structure, as described in the text. Assuming the two sites are energetically coupled, the non-native ubiquitinated substrate would be the most likely to bind the PA700 subcomplex. At the third level, the substrate protein is edited by one or more of the activities of the PA700 subcomplex. The relative outcome of these activities is determined at the fourth level, substrate fate, and includes: recovery of the native substrate as a consequence of isopeptidase activity, degradation as a consequence of the action of ATP-dependent protease activity, or as described in this text, refolding of the non-native structure to a native structure. The model also depicts the formation of aggregates, which is the fate of non-native structures that escape recognition and editing by the 26 S proteasome. REFERENCES mote the removal of polyubiquitin chains from both native and 1. DeMartino, G. N., and Slaughter, C. A. (1999) J. Biol. Chem. 274, non-native proteins (49). 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Recognition of Misfolding Proteins by PA700, the Regulatory Subcomplex of the 26 S Proteasome

Strickland, Elizabeth; Hakala, Kevin; Thomas, Philip J.; DeMartino, George N.
Journal of Biological Chemistry – Feb 1, 2000
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 8, Issue of February 25, pp. 5565–5572, 2000 © 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Recognition of Misfolding Proteins by PA700, the Regulatory Subcomplex of the 26 S Proteasome* (Received for publication, September 7, 1999, and in revised form, November 8, 1999) Elizabeth Strickland‡, Kevin Hakala§, Philip J. Thomas‡§¶i, and George N. DeMartino§i From the ‡Program in Molecular Biophysics and the §Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235 The 26 S proteasome is a large protease complex that a polyubiquitin chain as a prerequisite for their proteolysis (1, 2). This requirement seems to have evolved, at least in part, catalyzes the degradation of both native and misfolded proteins. These proteins are known to interact with because of the unique structure of the 26 S proteasome. PA700, the regulatory subcomplex of the 26 S protea- The 26 S proteasome is composed of two 700,000-dalton some, via a covalently attached polyubiquitin chain. subcomplexes, the 20 S proteasome and PA700 (1, 2, 4). The 20 Here we provide evidence for an additional ubiquitin- S proteasome, the catalytic core of the complex, is a cylindrical independent mode of substrate recognition by PA700. particle consisting of four stacked heptameric rings that con- PA700 prevents the aggregation of three incompletely tain a hollow center in which all six of the catalytic sites are folded, nonubiquitinated substrates: the DF-508 mutant located (5, 6). This topology excludes both folded and aggre- form of cystic fibrosis transmembrane regulator, nucle- gated substrates from contact with, and hydrolysis by, the otide binding domain 1, insulin B chain, and citrate active sites. The role of initial substrate recognition and bind- synthase. This function does not require ATP hydroly- ing by the complex is relegated to PA700, a 20-subunit regula- sis. The stoichiometry required for this function, the tory complex (7–9). Because the 26 S proteasome typically effect of PA700 on the lag phase of aggregation, and the requires polyubiquitinated protein substrates for degradation, temporal specificity of PA700 in this process all indicate the polyubiquitin chain binding site on PA700 (10 –12) is an that PA700 interacts with a subpopulation of non-native important determinant of substrate specificity. In addition to conformations that is either particularly aggregation- binding protein substrates, PA700 also regulates the access of prone or nucleates misassociation reactions. The inhibi- substrates to the active sites of the proteasome. ATP-depend- tion of off-pathway self-association reactions is also re- ent binding of PA700 to the outer rings of the proteasome flected in the ability of PA700 to promote refolding of probably induces a conformational change in the proteasome citrate synthase. These results provide evidence that, in that creates narrow pores in the center of the outer rings, addition to binding polyubiquitin chains, PA700 con- thereby exposing the interior catalytic sites to the exterior (2, 3, tains a site(s) that recognizes and interacts with mis- folded or partially denatured polypeptides. This feature 13, 14). This effect explains, in part, the PA700-dependent supplies an additional level of substrate specificity to activation of the proteasome’s hydrolysis of short nonubiquiti- the 26 S proteasome and a means by which substrates nated peptides, which can freely cross the pores without need are maintained in a soluble state until refolding or deg- for interaction with or structural modification by PA700 (8). In radation is complete. contrast, the PA700-dependent opening of pores in the protea- some does not explain fully how PA700 promotes the degrada- tion of ubiquitinated proteins with native or partially folded The 26 S proteasome is a large proteolytic machine that structures because such proteins are too large to traverse the participates in nearly all of the multiple roles played by intra- pores (15). Therefore, it is commonly assumed that as an es- cellular protein degradation in cellular function (reviewed in sential feature of 26 S proteasome-catalyzed proteolysis, PA700 Refs. 1–3). For example, the proteasome catalyzes both the must unfold the bound, ubiquitinated proteins and processively constitutive turnover of the bulk of intracellular proteins and translocate them through the pores to the catalytic sites of the the conditional degradation of specific proteins that regulate proteasome. This model would explain the known ATP require- various cellular processes. The 26 S proteasome is also a com- ment for degradation of ubiquitinated proteins by the assem- ponent of the quality control machinery that selectively de- bled 26 S proteasome, because it predicts that substrate un- grades proteins with abnormal structures. Most known protein folding and/or translocation are coupled to ATP hydrolysis by substrates of the 26 S proteasome are covalently modified with one or more of the six AAA protein subunits of PA700 (7, 13, 14, 16). This attractive model, however, has little direct experi- mental support and, with the exception of ATPase activity, the * This work was supported by NIDDK, National Institutes of Health properties of PA700 required for such activities are poorly (NIH) Grant DK49835 (to P. J. T.), the Robert Welch Foundation I-1284 defined. (to P. J. T.), NIDDK, NIH DK46181 (G. N. D.), and the Muscular Dystrophy Association (G. N. D.). The costs of publication of this article To mediate unfolding and/or translocation, PA700 must con- were defrayed in part by the payment of page charges. This article must tain a binding site for the non-native structures characteristic therefore be hereby marked “advertisement” in accordance with 18 of both the products of the unfolding reaction and the sub- U.S.C. Section 1734 solely to indicate this fact. This work is dedicated to the late Dr. Paul Srere who was kind enough to have many helpful discussions with us. Established investigator of the American Heart Association The abbreviations used are: PA700, proteasome activator, 700 kDa; (9740033N). CFTR, cystic fibrosis transmembrane conductance regulator; NBD1, To whom correspondence may be addressed: 5323 Harry Hines Blvd., nucleotide binding domain 1; DF-NBD1, DF508 mutant N-terminal Dallas, TX 75235-9040. For G. N. D., Tel.: 214-648-3308; Fax: 214-648- nucleotide binding domain of CFTR; PA28, proteasome activator, 28 4771; E-mail: [email protected]. For P. J. T., Tel.: 214-648- kDa; CS, citrate synthase; ATPgS, adenosine 59-O-(thiotriphosphate); 8723; Fax: 214-648-9268; E-mail: [email protected]. GdnHCl, guanidinium hydrochloride; NEM, N- ethylmaleimide. This paper is available on line at http://www.jbc.org 5565 This is an Open Access article under the CC BY license. 5566 Recognition of Misfolding Proteins by PA700 strates for the translocation reaction. Precendent exists for this from studies of the bacterial Clp protease system, which, like the proteasome, has occluded active sites and capping ATPases ClpA and ClpX. These AAA ATPases are known to mediate molecular chaperone and unfoldase activities (17–20). To gain insight into the recognition of protein substrates by PA700 we have analyzed interactions of PA700 with several model pro- teins in native and non-native conformations. Our data dem- onstrate that PA700 can directly interact with non-native structures of nonubiquitinated proteins and suggest models for how such interactions can participate in the degradation or refolding of protein substrates. EXPERIMENTAL PROCEDURES Materials—PA700 (21), latent 20 S proteasome, PA28, and modula- tor were purified from bovine red blood cells as described previously (8, 22, 23). “Lid” and “base” subcomplexes of PA700 were identified during the purification of PA700 from bovine red blood cells and are essentially equivalent to their counterparts in yeast (24). Lid was purified as an isolated complex, whereas, the base was purified bound to 20 S protea- some. Details of the purification and characterization of these com- plexes will be published elsewhere. DF508 mutant CFTR-NBD1 from residues 404 –589 was expressed with a six-histidine tag and purified as described previously (25). Porcine heart citrate synthase was purchased from Sigma; it was dialyzed into TE buffer (50 mM Tris-HCl, pH 8.0, 2 mM EDTA), concentrated to 4.65 mg/ml, and stored at 220 °C in ali- quots. Porcine insulin was from Calbiochem. Aggregation of DF-NBD1—Aggregation of DF-NBD1 was performed as described previously (26) at 2 mM final protein concentration in Buffer R (100 mM Tris-HCl, pH 7.4, 0.385 ML-arginine hydrochloride, 2 mM EDTA, 10 mM dithiothreitol, and 25 mM guanidine hydrochloride (GdnHCl)). Aggregation was initiated by rapid dilution from 6 M FIG.1. Components of the proteasomal machinery inhibit GdnHCl, while vortexing into a solution of 37 °C Buffer R, and was CFTR DF-NBD1 aggregation. GdnHCl-denatured DF-NBD1 was di- monitored by the increase in turbidity at 400 nm. 26 S proteasome, luted into Buffer R under conditions (2 mM DF-NBD1, 37 °C) where PA700, or 20 S proteasome were present at 100 nM prior to the addition folding to the native state is disfavored and DF-NBD1 aggregates. A, of DF-NBD1 unless otherwise noted. aggregation in the absence or presence of 100 nM 26 S proteasome (126 Assembly of 26 S proteasome—The 26 S proteasome was assembled S). B: Aggregation of DF-NBD1, DF-NBD1 with 100 nM 20 S proteasome in an ATP-dependent reaction from purified 20 S proteasome and (120 S), or DF-NBD1 with 100 nM PA700 (1PA700). PA700 components, as described previously (8). Assembly conditions contained 2-fold molar excess of 20 S proteasome with respect to PA700. done at single time points were assayed after a 45-min reactivation. The Less than 10% of the PA700 was unbound to the proteasome after the slope of the initial rate during the linear portion of the progress curve assembly reaction. was plotted relative to the activity of mock-treated native CS. During Reductive Aggregation of Insulin—A2mM stock solution of porcine the time course of the activity assay, which is short relative to the time insulin was prepared in 0.1 M potassium phosphate buffer, pH 7.6. course of reactivation, no further reactivation occurred. To ensure uni- Aggregation assays were performed by a modification of previously formity in initiating the reactivation, denatured CS was added to the reported methods (27, 28). Reactions were performed at 37 °C in a final reactivation mixture while vortexing. The fraction of citrate synthase volume of 250 ml containing 45 mM Tris-HCl, pH 7.8, 4 mM reduced that could be reactivated was reproducible within an experiment but glutathione, 0.4 mM oxidized glutathione, 160 mM insulin, and concen- varied between 25 and 75% among different citrate synthase prepara- trations of various proteins as indicated for specific experiments. Ag- tions. Each experiment was repeated multiple times with consistent gregation was monitored continuously as an increase in turbidity at 650 results obtained with multiple preparations. nm. In independent experiments, the length of the lag phase prior to initial detection of turbidity with insulin alone varied typically from RESULTS about 300 s to about 1100 s. However, within a given experiment, PA700, the Regulatory Component of the 26 S Proteasome, repetitions varied by less than 10%. All reported results were obtained Inhibits Aggregation of Misfolding DF-NBD1—To understand in at least three to four independent experiments. Thermal Aggregation of Citrate Synthase—Thermal aggregation of better the cellular and molecular mechanisms involved in the citrate synthase (CS) was carried out by adding 150 nM (monomer) CS recognition and processing of misfolded proteins, we studied to a solution of 40 mM HEPES-KOH, pH 7.5, heated to 43 °C (29). the interaction of the 26 S proteasome with DF-NBD1. DF- Aggregation was followed by light scattering at 500 nm in a PTI fluo- NBD1 is the nucleotide binding domain of CFTR that contains rometer at right angles with 2 nm excitation and 4 nm emission slit the common cystic fibrosis disease-causing mutation responsi- widths. PA700 was present before the addition of CS unless stated ble for impaired folding of the full-length CFTR protein (25, otherwise. Reactivation of Citrate Synthase—The reactivation of citrate syn- 32–34). In refolding experiments, less DF-NBD1 reaches the thase was monitored in a multi-step assay as described previously (30). native state than does wild-type NBD1. A certain fraction, In the first step, native CS was denatured in 6 M GdnHCl, 50 mM instead, forms large aggregates of misfolded, mutant protein Tris-HCl, pH 7.9, 2 mM dithiothreitol for1hat room temperature. (25, 26, 34). In the cell this misfolding protein is recognized by Reactivation of denatured CS was then initiated by dilution into a molecular chaperones (35, 36) and degraded by the ubiquitin- reactivation buffer containing 100 mM Tris-HCl, pH 7.9, 2 mM dithio- proteasome pathway (37, 38). Therefore, DF-NBD1 aggregation threitol, 10 mM KCl and allowed to continue at room temperature. Experiments containing ATP (2 mM) also included 5 mM MgCl . The provides a useful and physiologically relevant in vitro model activity of the reactivated CS was then assayed spectrophotometrically system for studying interactions of unfolded or misfolded pro- according to the method of Srere and Kosicki (31) at various time points teins with components of the cellular quality control with freshly prepared oxaloacetic acid and acetyl-CoA. Experiments machinery. As shown in Fig. 1A, the 26 S proteasome completely inhib- K. Hakala and G. N. DeMartino, manuscript in preparation. ited aggregation of DF-NBD1. The lack of turbidity observed in Recognition of Misfolding Proteins by PA700 5567 the presence of the 26 S proteasome was not due to the degra- dation of DF-NBD1 into small, soluble peptides (data not shown). To determine whether either subcomplex of the 26 S proteasome was sufficient to inhibit aggregation, 20 S protea- some and PA700 were assayed individually for their ability to inhibit the aggregation of DF-NBD1. The 20 S proteasome did not affect aggregation, whereas PA700 inhibited aggregation as effectively as the intact 26 S proteasome (Fig. 1B). These data demonstrate that the inhibitory activity of the 26 S pro- teasome can be attributed to PA700. PA700 Inhibits the Aggregation of Other Misfolding Pro- teins—To determine whether the ability of PA700 to interact with the non-native conformation of DF-NBD1 is a general property, analogous experiments were conducted with two ad- ditional proteins whose misfolding can be initiated under dif- ferent conditions. Insulin is composed of two polypeptide chains, A and B, linked by two disulfide bonds. Upon reduction of the disulfides, the chains separate, and the B chain aggre- gates (27, 28). As shown in Fig. 2, PA700 inhibited this aggre- gation as effectively as the 26 S proteasome, whereas the 20 S proteasome had no inhibitory effect. To extend further the generality of this effect, we tested the ability of PA700 to inhibit the aggregation of thermally dena- tured citrate synthase, a protein commonly utilized for study- ing chaperone activity (29, 30, 39). Native citrate synthase is a homodimer; when heated to 43 °C, the population of partially folded conformations of citrate synthase increases, leading to aggregation. As with the other tested proteins, citrate synthase aggregation was inhibited by PA700 at low molar ratios (Fig. 3). PA700 increased the lag period of citrate synthase aggrega- tion in a manner similar to its effect on insulin. Together, these data demonstrate that PA700 interacts with non-native forms of a variety of proteins. The ability of PA700 to inhibit aggregation is specific to PA700 and is not a property of either of two related multipro- tein proteasome regulator complexes. First, a protein complex, termed “modulator,” consisting of two of the ATPase subunits found in PA700 and a third novel protein not found in PA700, enhances PA700-dependent activation of the proteasome (21) but was not effective at inhibiting the aggregation of insulin (Fig. 2B). Second, the proteasome activator PA28 (23), which is known to associate with the 20 S proteasome, did not inhibit the aggregation of insulin (Fig. 2B). In addition, PA700 that has been heat-treated for 15 min at 75 °C is no longer effective in retarding the aggregation of insulin (data not shown). These FIG.2. PA700 inhibits the aggregation of insulin. Aggregation data provide further evidence for specificity of the PA700-de- was initiated by dilution of native insulin (160 mM) into reducing buffer. pendent inhibition of aggregation. A, aggregation of insulin alone, insulin with 300 nM 20 S proteasome (120 S), insulin with 300 nM 20 S, 160 nM PA700-assembled 26 S The extent of inhibition of insulin aggregation was directly proteasome (126 S), or insulin with 160 nM PA700 (1PA700). B, ag- proportional to the concentration of PA700; but surprisingly, gregation of insulin alone, insulin with 300 nM modulator (1Modula- inhibition occurred at PA700 concentrations 1000-fold less tor), or insulin with 300 nM PA28 (1PA28). C, aggregation of insulin than those of the substrate insulin. Furthermore, PA700 in- with no PA700, 50 nM PA700 (3200:1), 100 nM PA700 (1600:1), 200 nM PA700 (800:1), or 400 nM PA700 (400:1). creased the lag time of insulin aggregation in a concentration- dependent manner (Fig. 2C). In contrast, simple reduction of the insulin concentration in the absence of PA700 reduced the by rapid dilution into a nondenaturing buffer. The restoration rate of aggregation but did not have a significant effect on the of native structure was monitored by the recovery of enzymatic lag phase in this concentration range (data not shown). These activity. Under these conditions, denatured citrate synthase results suggest that PA700 interacts with a subpopulation or refolds poorly in the absence of chaperones (Fig. 4 and Refs. 29, with a transient intermediate of misfolding insulin which is 30, and 39). However, in the presence of PA700, enzymatic particularly prone to aggregation (see “Discussion”). activity was recovered in both a time-dependent and PA700 PA700 Promotes Refolding of Misfolding Citrate Syn- concentration-dependent fashion (Fig. 4). These effects were thase—In addition to inhibiting aggregation of misfolding pro- achieved at the same low molar ratios required for inhibition of teins, many molecular chaperones promote refolding of these aggregation. Thus, by inhibiting aggregation of misfolded cit- proteins to native structures. Therefore, we tested the ability of rate synthase, PA700 promotes its refolding. PA700 to promote reactivation of chemically denatured citrate PA700 Does Not Depend on ATP to Inhibit Aggregation or synthase. Native citrate synthase was first denatured with 6 M Promote Refolding of Misfolded Proteins—Many well charac- guanidinium hydrochloride, and then refolding was initiated terized molecular chaperones utilize ATP to modulate the rate 5568 Recognition of Misfolding Proteins by PA700 with N-ethylmaleimide (NEM), which abolishes its ATPase activity (16). NEM-treated PA700 effectively inhibited the ag- gregation of insulin (Fig. 5D) and promoted the reactivation of citrate synthase (Fig. 5E). These data indicate that the inter- action of PA700 with non-native protein substrates does not depend upon ATP. PA700 Recognizes a Subset of Non-native Substrate Confor- mations—The substoichiometric PA700:substrate ratios re- quired for inhibition of aggregation and promotion of refolding and the progressively increased delay in onset of aggregation in the presence of PA700 suggest that PA700 interacts with a subpopulation of non-native conformations. To test this possi- bility further, PA700 was added to either the citrate synthase aggregation assay or reactivation assay after each process had begun. As shown in Fig. 6, PA700 remained effective at inhib- iting the thermal aggregation of initially folded citrate syn- FIG.3. PA700 inhibits the thermal aggregation of citrate syn- thase when added 20 s after the initiation of misfolding. How- thase. Native citrate synthase (150 nM) was heated to 43 °C either ever, PA700 was progressively less effective at inhibiting alone or in the presence of 0.9 nM PA700 (160:1), 1.9 nM PA700 (80:1), aggregation when addition was delayed for longer times. These or5nM PA700 (30:1). a.u., arbitrary units. results cannot be explained by simple stoichiometric binding of PA700 because of the large excess of unfolded monomer and, thus, imply that PA700 may act on a species critical for initi- ation of aggregation. Furthermore, once citrate synthase ag- gregates were formed, PA700 could not disassemble and solu- bilize them (data not shown). Likewise, PA700 promoted the reactivation of citrate synthase when present at the start of reactivation, but not when added 2 min later. In sum, these results suggest that PA700 acts on a non-native conformation not yet fully committed to off-pathway aggregation. The Base Subcomplex of PA700, Containing ATPase Sub- units, Inhibits Aggregation of Misfolded Proteins—PA700 is composed of 20 distinct subunits, including six AAA-ATPases (16, 21, 47, 48), at least one subunit that binds ubiquitin chains (10 –12), and an isopeptidase (49). The functions of the remain- ing subunits are unknown. To determine which PA700 sub- units are responsible for the effects on inhibition of protein aggregation we repeated some of the experiments described above using subcomplexes of PA700 essentially equivalent to the base and lid complexes described for yeast PA700 (24). The bovine base (in a complex with the 20 S proteasome) inhibited aggregation as effectively, if not more effectively, as intact PA700 when compared on a molar basis (Fig. 7A). The base contains all the ATPases of PA700 and two other subunits (data not shown). In contrast, lid, which contains the polyubiq- uitin chain binding subunit, inhibited aggregation of insulin very poorly (Fig. 7B). Similar effects of base and lid were observed with inhibition of citrate synthase aggregation (data not shown). These results suggest that the various functions of PA700 described here are localized to the base component of PA700. FIG.4. PA700 reactivates citrate synthase. GdnHCl-denatured DISCUSSION citrate synthase was diluted into refolding conditions, and the forma- tion of active, native protein was monitored. A, time course of CS This work demonstrates that PA700, the regulatory complex reactivation in the absence (open squares) or presence (closed circles)of of the 26 S proteasome, recognizes non-native conformations of 300 nM PA700. B, CS reactivation at 45 min with increasing concentra- proteins. PA700 inhibited the aggregation of three misfolding tions of PA700. proteins and promoted the refolding of a denatured protein to the native state. These newly discovered functions of PA700 of substrate turnover as they inhibit aggregation and/or pro- are defining features of molecular chaperones (50 –53) and, in mote refolding (40 – 46). To determine whether PA700, a known combination with the previously established function of PA700 ATPase (16, 47), requires ATP for these activities, we repeated in regulating the degradation of ubiquitinated proteins, place the described assays in the presence and absence of Mg or PA700 in a unique and central position for determining the fate of protein substrates as part of the quality control machinery of ATP. Mg or ATP had no significant effect on the ability of PA700 to inhibit the aggregation of DF-NBD1, insulin, or cit- the cell. Like known chaperones, PA700 could bind to non- rate synthase (Fig. 5, A–C). In addition, neither ADP nor the native proteins, thereby inhibiting their aggregation and per- nonhydrolyzable analog ATPgS influenced the results. ATP mitting refolding subsequent to their release (Fig. 8). Alterna- also did not affect the ability of PA700 to reactivate citrate tively, by inhibiting the competing aggregation reaction, PA700 synthase (Fig. 5E). To confirm this result, PA700 was treated could promote entry of non-native proteins into the degradation Recognition of Misfolding Proteins by PA700 5569 FIG.5. ATP is not required for PA700 inhibition of aggregation or the reactivation of CS. A, DF-NBD1 (2 mM) was aggregated at 37 °C as in Fig. 1. Aggregation was either with no PA700, 100 nM PA700 (1PA700), or 100 nM PA700, 20 mM MgCl ,2mM ATP (1PA700 (1 ATP)). B, insulin (160 mM) was aggregated under reducing conditions as in Fig. 2A. Reactions contained insulin, insulin 1 160 nM PA700 (1PA700), or insulin 1 3.6 mM MgCl , 800 mM ATP, 160 nM PA700 (1 PA700 (1ATP)). C, CS (150 nM) thermal aggregation was carried out with CS, CS 1 7.5 nM PA700 (1 PA700), CS 1 200 mM ATP, 7.5 nM PA700 (1 PA700 (1ATP)), or CS 1 200 mM ADP, 7.5 nM PA700 (1 PA700 (1ADP)). MgCl (1 mM) was present in all reactions. D, insulin (160 mM) aggregation was initiated with no additions, with 215 nM PA700 (1 PA700) or with 215 nM NEM-treated PA700 NEM (1 PA700 ). E, Gdn-HCl-denatured CS (150 nM) was reactivated either alone or with the addition of 2 mM ATP; 2 mM ATPgS; 300 nM PA700; 300 nM PA700, 2 mM ATP; 300 nM NEM-treated PA700, 2 mM ATP; or 300 nM PA700, 2 mM ATPgS. All reactions contained 5 mM MgCl . Bars represent the average of three experiments. pathway, as has been reported for a known molecular chaper- atypical of many well characterized molecular chaperones and one (54). The possible role of PA700 as a molecular chaperone provide insights regarding their possible physiological signifi- in the cell may be analogous to known chaperone functions cance. PA700 inhibited the aggregation of misfolded proteins at played by ClpA and ClpX, the regulatory subcomplexes of pro- very low PA700:protein substrate ratios, whereas other chap- karyotic ATP-regulated proteases (55). In addition, PA700 re- erones normally require near stoichiometric ratios for function. cently has been shown to play a nonproteolytic role in nucleo- For example, 30 –100 times more Hsc70 was required to tide excision repair in yeast, perhaps by exerting a chaperone- achieve the same degree of inhibition accomplished by PA700 like activity that disassembles or structurally rearranges the in the assays reported here (data not shown and Ref. 26). These repair complex (56). results indicate that PA700 interacts with a subpopulation of Several features of the effects of PA700 reported here are proteins in a conformation prone to aggregation. Thus, to in- 5570 Recognition of Misfolding Proteins by PA700 FIG.7. The base subcomplex of PA700, but not the lid subcom- FIG.6. PA700 acts on a partially folded subpopulation. A, cit- plex, inhibits aggregation. The reductive aggregation of insulin (160 mM) was performed as in Fig. 2. A, effect of base on insulin aggregation. rate synthase (150 nM) misfolding was initiated as in Fig. 3 in the presence of 7.5 nM PA700 (0 s) or with 7.5 nM PA700 added after the Insulin aggregation was initiated in the absence of other proteins (up- per line), with 300 nM base-proteasome complex (1 “base”), and with initiation of aggregation (20, 60, or 120 s). B, the reactivation of citrate synthase was assayed and reported as in Fig. 4 with either citrate 300 nM PA700 (1 PA700). B, effect of lid on insulin aggregation. Insulin aggregation was initiated in the absence of any other proteins (upper synthase alone, citrate synthase with PA700 present at the beginning of reactivation, or citrate synthase with PA700 added 2 min after the start line) and in the presence of either 200 nM lid (1 “lid”) or 130 nM PA700 (1PA700). of reactivation. hibit aggregation effectively, PA700 need not bind each sub- for those needed here. Second, ATP promoted a small stimula- strate molecule, but only those disposed to off-pathway associ- tion of inhibition in their experiments. Third, consistent with ations. Additionally, order-of-addition experiments suggest the low stoichiometry or possibly the dynamic nature of the that PA700 likely recognizes a conformation early in the mis- complex, we were unable to detect significant amounts of as- folding process. These features are consistent with the obser- sociation of either insulin or citrate synthase with PA700 dur- vation that PA700 lengthens the lag phase for aggregation of ing density gradient centrifugation (data not shown). The basis both insulin (Fig. 2C) and citrate synthase (Fig. 3) in a concen- for these differences is unclear but may have significant con- tration-dependent manner. The lag phase observed in each of sequences for interpretation of the mechanisms in which these these aggregation processes is likely due to the formation of a processes are involved (see below). specific conformation or nucleus prone to self-association that What are the physiological implications of the current re- PA700 may recognize and interact with. sults? Regardless of whether PA700, either alone or as part of Finally, PA700 does not require ATP either to inhibit aggre- the 26 S proteasome, functions independently as a molecular gation or to promote refolding. Many previously characterized chaperone in the cell, the novel activities described here are molecular chaperones utilize ATP hydrolysis as a timing mech- directly relevant to its established function in the degradation anism to coordinate the rate of substrate binding and release of ubiquitinated proteins. For example, PA700 must contain a with the rate of substrate folding, rather than utilizing the binding site for non-native protein structures as an essential hydrolytic energy to promote an otherwise unfavorable reac- feature for its role in proteolysis because non-native structures tion. Therefore, the current results may indicate that the are the products of unfolding reactions and the substrates for known ATP requirement for protein degradation by the 26 S translocation reactions that are part of the putative proteolytic proteasome supports energy-dependent unfolding or processive mechanism. The potential function of this site for both protein translocation of unfolded substrates to the proteasome active folding and protein degradation is illustrated in the model sites by PA700. proposed in Fig. 8. In this model, PA700 may bind to: (i) native While this manuscript was in preparation, Braun et al. (57) ubiquitinated proteins (via recognition of polyubiquitin reported that the 26 S proteasome inhibited the aggregation chains), (ii) non-native nonubiquitinated proteins (via recogni- and promoted the refolding of citrate synthase, one of the tion of non-native protein structure), or (iii) non-native ubiq- substrates used in the current study. Both of these activities uitinated proteins (via recognition of both polyubiquitin chains were localized to the base of PA700. Although the results of the and non-native protein structure). These multiple recognition two studies are in general agreement, they differ in several and binding modes would allow PA700 to serve a central edit- important ways. First, much higher protein:substrate ratios ing function that determines the final fate of substrates. For were required for the effects reported by Braun et al. (57) than example, the known isopeptidase activity of PA700 may pro- Recognition of Misfolding Proteins by PA700 5571 FIG.8. Model for the interactions of the 26 S proteasome with protein substrates. Established (black lines) and hypothetical (gray lines) actions of the 26 S proteasome with protein substrates are depicted. Pathways denoted with heavy arrows represent reactions directly related to the studies in the current report. The model distinguishes four different levels through which proteins are processed. At the first level, protein structure is converted between native and non-native forms, either of which may be modified further by ubiquitination. At the second level, the PA700 subcomplex of the 26 S proteasome can recognize and interact with each of the protein forms except native nonubiquitinated proteins, for which it has very low affinity. Otherwise, it can bind to the polyububiquitin chain on either the native or non-native protein or with lower affinity to the non-native, nonubiquitinated protein via a second site that recognizes features of non-native structure, as described in the text. 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Published: Feb 1, 2000

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