Proteasome Activation by REG Molecules Lacking Homolog-specific Inserts*

Zhiguo Zhang, Claudio Realini, Andrew Clawson, Scott Endicott, and Martin Rechsteiner

From the Department of Biochemistry, University of Utah, Salt Lake City, Utah 84132

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The peptidase activities of eukaryotic proteasomes are markedly activated by the 11 S REG or PA28. The three identified REG subunits, designated alpha , beta , and gamma , differ significantly in sequence over a short span of 15-30 amino acids that we call homolog-specific inserts. These inserts were deleted from each REG to produce the mutant proteins REGalpha Delta i, REGbeta Delta i, and REGgamma Delta i. The purified recombinant proteins were then tested for their ability to oligomerize and activate the proteasome. Both REGalpha Delta i and REGgamma Delta i formed apparent heptamers and activated human red cell proteasomes to the same extent as their full-length counterparts. By contrast, REGbeta Delta i exhibited, at low protein concentrations, reduced proteasome activation when compared with the wild-type REGbeta protein. REGbeta Delta i was able to form hetero-oligomers with a single site, monomeric REGalpha mutant and with REGalpha Delta i. At low concentrations, the REGalpha Delta i/REGbeta Delta i hetero-oligomers stimulated the proteasome less than REGalpha /REGbeta oligomers formed from wild-type subunits, and the reduced activation by REGalpha Delta i/REGbeta Delta i was due to removal of the REGbeta insert, not the REGalpha insert. These studies demonstrate that the REGalpha and REGgamma inserts play virtually no role in oligomerization or in proteasome activation. By contrast, removal of REGbeta insert reduces binding of this subunit and REGalpha /REGbeta oligomers to proteasomes. On the whole, however, our findings show that REG inserts are not required for binding and activating the proteasome. We speculate that they serve to localize REG-proteasome complexes within cells, possibly by binding components in endoplasmic reticulum membranes.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The proteasome is a major proteolytic organelle in the cytosol and nucleus of eukaryotic cells (1-3). The enzyme is a cylindrical structure containing 28 subunits arranged as four rings of seven subunits each. The two end rings are composed of catalytically inactive subunits belonging to the alpha  subunit family based on homology to proteasome subunits from the archebacterium, Thermoplasma. The two central rings are composed of members of the beta  subunit family (4-6), some of which are proteolytically active (7). Crystal structures of Thermoplasma and yeast proteasomes reveal that the beta  subunits form a central proteolytic chamber far from the particle's surface and that entry to this central chamber is greatly restricted (8, 9).

By itself, the proteasome does not degrade intact proteins. Association with a 19 S regulatory complex converts the proteasome to the 26 S protease, an energy-dependent enzyme capable of degrading intact proteins (10-13), including those marked by ubiquitin (14). The proteasome also binds an 11 S protein complex, which we have termed REG and others have named PA28 (15-17). REG binding to the proteasome can stimulate peptide hydrolysis as much as 100-fold. Human red blood cell REG is composed of two ~30 KDa subunits, REGalpha and REGbeta . These two proteins are closely related to each other and to a third protein, REGgamma (18). The three proteins show extensive sequence similarities, except for a region of 15-32 amino acids where they diverge significantly (18, Fig. 1A). We call the divergent regions "homolog-specific inserts" or, for sake of discussion, simply inserts.

Using random mutagenesis, we recently isolated 45 single-site REGalpha mutants with impaired ability to activate the proteasome; none of the 45 amino acid changes was found in the REGalpha insert (19). It should be noted, however, the REGalpha insert is a KEKE motif, which consists largely of "alternating" glutamate and lysine residues and which has been hypothesized to mediate protein-protein associations (20). Hence, the apparent absence of single-site mutations in the REGalpha insert region could simply reflect redundant information within the insert (see Fig. 1A). The crystal structure of REGalpha reveals that it is a heptameric ring with a conical pore measuring 20 Å in diameter on one face and 30 Å in diameter on the other (Ref. 21 and Fig. 1B). The 39 amino acids that encompass the insert region are disordered, and the seven inserts present in the assembled heptamer are located on the surface opposite the presumed proteasome binding face (21). One can calculate that even if these 39 amino acids were maximally condensed (i.e. globular), they could interact with each other (see Fig. 1C). Furthermore, four proteasome alpha  subunits possess long C-terminal extensions that are capable of interacting with REG inserts (see Fig. 1D and Ref. 22 for a description of the C-terminal extensions). Therefore, even though the REGalpha inserts appear to be some distance from the presumed proteasome binding surface, they could interact with each other and/or with proteasome alpha  subunits. These interactions could, in turn, affect REG oligomerization or the ability of REG homologs to bind and/or activate the proteasome. To test these possibilities, we have deleted the inserts from each REG homolog and have characterized the three mutant proteins. At low concentrations, REGbeta lacking its insert stimulates the proteasome less than wild-type REGbeta and produces REGalpha /beta hetero-oligomers that bind and activate the proteasome less efficiently than hetero-oligomers formed from wild-type REGalpha and REGbeta subunits. However, the biochemical properties of REGalpha and REGgamma insert deletion mutants demonstrate that the inserts are not required for oligomerization or activation of the proteasome by these two REG homologs.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- The fluorogenic peptides t-butyloxycarbonyl (Boc)1-Phe-Ser-Arg-aminomethylcoumarin (FSR-MCA), Boc-Val-Leu-Lys-MCA (VLK-MCA), Suc-Leu-Leu-Val-Tyr-MCA (LLVY-MCA), Suc-Ala-Ala-Phe-MCA (AAF-MCA), Pro-Phe-Arg-MCA (PFR-MCA), Suc-Leu-Tyr-MCA (LY-MCA), and Cbz-Leu-Leu-Glu-beta NA (LLE-beta NA) were purchased from Sigma. Boc-Leu-Leu-Arg-MCA (LRR-MCA) was obtained from Peptide International. Nitrocellulose membranes were from Schleicher and Schuell. Peroxidase-conjugated goat anti-rabbit IgG was from Cappel. Western blot chemiluminescence reagents were from DuPont NEN. Site-directed mutagenesis kits were from Bio-Rad. Isopropyl-beta -thiogalactopyranoside was from Boehringer Mannheim. BL-21(DE3) competent cells were from Novagen.

Construction of REGalpha , REGbeta , and REGgamma Insert Deletion Mutants-- Site-directed mutagenesis (23, 24) was used to produce REGalpha , REGbeta , and REGgamma insert deletion mutants. The three specific DNA primers used for REGalpha , REGbeta , and REGgamma were 5'-GTTCACTGGGCCACAGGGAGGACCGACTGGATCAGGCACTGG-3', 5'-GGAGAAATCCACACTTAGGGACTGGAGGGTCTGGGATGGG-3', and 5'-GCATCCCATTGGGCATCACAATGGGGTCAGGGACTGG-3', respectively. To prepare REGalpha , REGbeta , and REGgamma single-strand DNAs containing uracil residues, each REG homolog cDNA was subcloned into the phagemid pET26b and used to transform CJ236 cells, which lack dUTPase and uracil N-glycosylase and thus allow incorporation of deoxyuridine into DNA. A helper phage strain VCS-M13 (Stratagene) was used to infect the CJ236 cells containing REGalpha , REGbeta , or REGgamma plasmids, and single-strand DNAs were isolated according to a standard protocol (23, 24). DNAs from selected clones were purified and sequenced.

Expression and Purification of Recombinant REGalpha Delta i, REGbeta Delta i, and REGgamma Delta i and Their Wild-type Counterparts-- Escherichia coli BL-21 (DE3) were grown at 30 °C overnight and induced for 2 h with 0.4 mM isopropyl-beta -thiogalactopyranoside when the A600 was between 0.2 and 0.3 or with 0.6 mM isopropyl-beta -thiogalactopyranoside when the A600 was between 0.3 and 0.5. Lysates were prepared by resuspending the bacteria pellets in TSD (10 mM Tris, 25 mM KCl, 10 mM NaCl, 1.1 mM MgCl2, 0.1 mM EDTA, 1 mM dithiothreitol), pH 8.8, with 1 µg/ml pepstatin A and 20 µg/ml phenylmethylsulfonyl fluoride and passing the suspension through a French press. The lysates were then cleared by centrifugation and applied to an ion exchange column packed with diethylaminoethyl fast flow resin (Pharmacia Biotech Inc.). All the recombinant proteins used in this study were purified to homogeneity using a combination of diethylaminoethyl chromatography and Superdex 200 gel filtration as described (18, 19). Fractions from the sizing column were pooled, concentrated with a Centriprep 10, and dialyzed against 0.5 × TSD for at least 20 h prior to biochemical characterization.

Generation of (His)10-REGalpha Delta i and (His)6-REGgamma Delta i Mutants-- REGalpha Delta i cDNA in pET26b was digested with NdeI and BamHI and ligated into pET16b to produce (His)10-REGalpha Delta i constructs. Site-directed mutagenesis was used to insert Gly-Gly-(His)6 after the first N-terminal Met of REGgamma Delta i.

Electrophoresis-- SDS-polyacrylamide gels consisted of a 4% stacking gel and a 15% resolving gel. Minigels prepared using a Mini-Protean apparatus were used to separate (His)10-REGalpha Delta i from REGbeta Delta i and REGalpha (N50Y), an inactive monomeric REGalpha mutant, from REGbeta Delta i and to check protein purity. After gel electrophoresis, proteins were visualized using Coomassie Brilliant Blue staining or silver staining.

Hetero-oligomerization between REGalpha Delta i and REGbeta Delta i-- To determine whether REGalpha and REGbeta inserts affected their hetero-oligomerization, (His)10-REGalpha Delta i instead of REGalpha Delta i was used as REGalpha Delta i could not be separated from REGbeta Delta i by SDS-PAGE or HPLC. Bacteria expressing (His)10-REGalpha Delta i and those expressing REGbeta Delta i were combined together and resuspended in TBS (20 mM Tris, pH 7.9, 150 mM NaCl) plus protease inhibitors (1 µg/ml pepstatin A, 20 µg/ml phenylmethylsulfonyl fluoride). A French press was used to lyse the bacteria, and the lysates were cleared by centrifugation at 40,000 × g for 1 h. The lysates were then incubated for 1 h with Ni-NTA beads equilibrated with TBS. After discarding the supernatants, the beads were washed three times with TBS plus protease inhibitors and three times with TBS and 25 mM imidazole plus protease inhibitors. The bound proteins were eluted with TBS, 0.5 M imidazole and applied to a Superdex 200 gel filtration column (see Fig. 4). To determine whether REGbeta Delta i formed hetero-oligomers with REGgamma Delta i, we used (His)6-REGgamma Delta i and followed the same procedures described above; no interactions were observed between REGbeta Delta i and REGgamma Delta i under these conditions.

Enzymatic Assay of the Proteasome Using Fluorogenic Substrates-- Human red blood cell 20 S proteasomes (typically 170 ng) were incubated with various amounts of wild-type or insert-deleted REG at 37 °C for 10 min in 50 µl of buffer (10 mM Tris, pH 7.5). Enzymatic reactions were started by adding 50 µl of a 200 µM solution of fluorogenic peptide substrate. After further incubation as indicated in the figure legends, the reactions were quenched with 200 µl of ice-cold ethanol, and fluorescence was measured as described (18).

Hydrolysis of Natural Synthetic Peptides-- The experiment was performed by incubating peptide P21 (SADPELALALRVSME-EQRQRQ) or BBC1 (MKKEKARVITEEEKNFKAFASLRMARANARLFGIRAKRAKEAAEQDGSG) with REG homologs or deletion mutants and human red blood cell proteasomes as described (25). The digested products were then separated on a C18 HPLC column using a gradient of 0-45% acetonitrile containing 0.1% trifluoroacetic acid.

Proteasome Binding Assay-- Proteasome binding assays were performed as described (18). Briefly, various amounts of REG homologs or hetero-oligomers were incubated with human red cell proteasomes tethered to enzyme-linked immunosorbent assay plates by the monoclonal antibody MCP20. After washing away unbound REGs, the bound proteins were eluted with 20 mM Tris, 0.5 M NaCl, pH 7.5, and dot-blotted onto a nitrocellulose membrane. The membrane was blocked with 5% nonfat dry milk in TBS, pH 7.5, 0.1% Tween 20, and bound REG proteins were detected with rabbit anti-REG followed by peroxidase-conjugated goat anti-rabbit and chemiluminescence.

Analysis of Subunit Ratio in REGalpha /REGbeta Hetero-oligomers Using HPLC-- After REGalpha (N50Y)/REGbeta and REGalpha (N50Y)/REGbeta Delta i hetero-oligomers were isolated from the Superdex 200 gel filtration column as described above, three fractions across each peak were applied to a C18 column separately, and a gradient of 30-70% acetonitrile containing 0.1% trifluoroacetic acid was used to separate REGalpha (N50Y) from REGbeta or REGbeta Delta i. The ratio of REGbeta or REGbeta Delta i to REGalpha (N50Y) was determined by the HPLC peak area computed by an HP ChemStation (version A. 04.01, Hewlett Packard) based on the absorbance at 214 nm. The area of each peak was divided by the molecular weight of the corresponding protein to produce a subunit molar ratio.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Purification and Oligomerization of REGalpha , REGbeta , and REGgamma Lacking Insert Regions-- The crystal structure of REGalpha reveals that the insert regions are disordered and located on the upper structure of a heptameric REGalpha ring, whereas the lower surface of the ring is the presumed proteasome binding face (Ref. 21, Fig. 1, B and C). To address the functions of the inserts, defined by the bold letters in Fig. 1A, we used recombinant DNA techniques to delete them from each homolog (see "Experimental Procedures"). The mutant proteins and their wild-type counterparts were expressed in E. coli and purified to near homogeneity (Fig. 1D). It was clear from the gel filtration step used during purification that the REGalpha insert deletion mutant (REGalpha Delta i) and REGgamma insert deletion mutant (REGgamma Delta i) formed oligomers. Nonetheless, after purification, we directly compared the oligomerization state of each deletion mutant with its full-length counterpart. REGalpha Delta i eluted from a Superdex 200 column at 183 ml, whereas wild-type REGalpha elutes at ~175 ml (data not shown). REGgamma Delta i proteins also eluted later than wild-type REGgamma (180 ml versus 172 ml); REGbeta Delta i eluted at 230 ml compared with 226 ml for wild-type REGbeta . We attribute the slight reduction in the apparent hydrodynamic radius of REGalpha Delta i and REGgamma Delta i to removal of the 28 or 32 amino acids present in the inserts rather than to changes in the oligomerization state from a heptamer to, say, a hexamer. However, we cannot strictly exclude the latter possibility.


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Fig. 1.   REG homologs with inserts deleted. A, sequence alignment of REGalpha , REGbeta , and REGgamma . Amino acids deleted from each homolog are highlighted. B, a schematic representation of a section of the REGalpha heptamer as viewed from the side. The dimensions are taken from the crystal structure (21). Note that only two of the seven inserts are shown, and their shapes are arbitrarily drawn because they are disordered in the crystal structure (21). C, schematic representation of the REGalpha heptamer viewed from the top. The central circle (p) is the pore leading down to the proteasome, and it is about 20 Å in diameter. Each of the seven peripheral circles (i) represents the sphere that would be formed if the 39 amino acids (Pro-64 to Gly-102) disordered in the REGalpha crystal structure adopted a globular conformation. The radius of each "insert" was calculated by the formula V in Å3 = 1.27 × the molecular weight in Daltons (31). The distance between each insert is about 3 Å. As this is the tightest packing possible, it is clear that interactions are possible between the seven inserts in the REGalpha heptamer. D, potential interactions between REGalpha inserts and C-terminal tails of proteasome alpha subunits. The proteasome is represented by four rectangles, and the REGalpha heptamer is shown in the same perspective as in panel B. If the insert and the tail adopt beta  conformations, they could overlap significantly as shown at the left. If the insert and the tail adopt helical conformations (right), the insert could still touch the tail of the proteasome alpha subunit C2. Proteasome alpha  subunit tails are discussed in Ref. 22. Only two REGalpha inserts and two proteasome alpha tails are shown. Their positions in the diagram are hypothetical. The lengths of beta  strands and alpha  helices are calculated using normal secondary structure geometries (31). E, SDS-PAGE analysis of insert deletion mutants REGalpha Delta i, REGbeta Delta i, REGgamma Delta i, and their wild-type counterparts. Purified recombinant proteins were separated on an SDS-PAGE gel (15% acrylamide) and visualized with Coomassie Brilliant Blue. The numbers at the left are molecular weight standards in kDa.

Activation of Fluorogenic Peptide Hydrolysis by REGs Lacking Inserts-- Activation of the proteasome by each REGDelta i was compared with the activation properties of its full-length counterpart. REGalpha Delta i and REGgamma Delta i stimulated the proteasome to the same extent as their full-length counterparts at all protein concentrations tested (Fig. 2, A and B). By contrast, REGbeta Delta i activated the proteasome less than wild-type REGbeta at low protein concentrations, although the degree of stimulation by each REGbeta molecule was equivalent at high concentrations (Fig. 2C, Table I). Because decreased activation by REGbeta Delta i was observed using REGbeta and REGbeta Delta i proteins obtained in three independent purifications, we do not attribute the differences to denaturation or variabilities in a given purification (see also Fig. 5 for similar results with REGalpha Delta i/REGbeta Delta i hetero-oligomers at low protein concentrations).


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Fig. 2.   Proteasome activation by REGalpha Delta i, REGbeta Delta i, and REGgamma Delta i and their wild-type counterparts. A, proteasome stimulation by REGalpha Delta i (open circles) and REGalpha (closed circles). Human red cell proteasomes (170 ng) were mixed with increasing amounts of purified REGalpha Delta i or REGalpha in a final volume of 50 µl of 10 mM Tris, pH 7.5, and incubated at 37 °C for 10 min. The reaction was started by adding 50 µl of 200 µM Suc-LLVY-MCA in 10 mM Tris, pH 7.5. After an additional 15-min incubation, the reaction was stopped with 200 µl of cold 100% ethanol, and the enzymatically released MCA was measured. B, proteasome stimulation by REGgamma Delta i (open squares) and REGgamma (closed squares). The experiment was performed essentially as described in Fig. 2A except that Boc-LRR-MCA was used as the substrate. C, proteasome stimulation by REGbeta Delta i (open diamonds) and REGbeta (closed diamonds). The experiment was performed as described in A. Each data point in this figure represents the mean of three measurements from a single experiment. Equivalent results were observed in at least two experiments using recombinant REG proteins from different purifications.

                              
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Table I
Substrate-specific stimulation of the proteasome by wild-type and insert-deleted REGs
Proteasomes (170 ng) were mixed with 50 µl of 10 mM Tris, pH 7.5, alone or with 100 nmol of REGalpha or 100 nmol of REGalpha Delta i in 10 mM Tris, pH 7.5. After 10 min at 37 °C, reactions were initiated by adding 50 µl of 200 µM fluorogenic peptide in 10 mM Tris, pH 7.5. After an additional 10 min, the reaction was quenched by addition of 200 µl of ice-cold ethanol. Fluorescence was measured, and the results are expressed as fold-stimulation, e.g., the ratios of fluorescence generated by proteasomes plus REGalpha or REGalpha Delta i divided by fluorescence produced by proteasomes alone. Identical experimental protocols were used for REGgamma and REGgamma Delta i, whereas 440 nmol of REGbeta and REGbeta Delta i were used to obtain the data shown. At lower concentrations of REGbeta , the wild-type molecule activated proteolysis much better than REGbeta Delta i (data not shown).

REGgamma preferentially activates hydrolysis of fluorogenic peptides with positive residues adjacent to the fluorescent MCA leaving group (18). REGalpha and REGbeta stimulate the proteasome to cleave after basic, acidic, or hydrophobic residues at the P1 position. Because the greatest sequence divergence between REGgamma and REGalpha or REGbeta is in the insert region, differential activation of specific peptide hydrolysis might result from interactions between REG inserts and the proteasome. This possibility was tested by measuring cleavage of various fluorogenic peptides in the presence of wild-type and insert-deleted REGs. There were no significant changes in peptide cleavage patterns activated by REGalpha Delta i, REGbeta Delta i, and REGgamma Delta i and their corresponding wild-type counterparts (Table I). Therefore, the homolog-specific inserts do not account for the distinct patterns of activated peptide hydrolysis observed with REGgamma , on one hand, and REGalpha or REGbeta on the other.

Enhanced Cleavage of Long Synthetic Peptides by REGalpha Lacking Its Insert-- The longest fluorogenic peptide surveyed in Table I consists of four amino acids. Whether proteasome specificities revealed by short fluorogenic peptides are relevant to the enzyme's preference for natural peptide substrates has generated some debate (26-28). As mentioned, the crystal structure of REGalpha reveals that the insert lies on a surface opposite to the presumed proteasome binding face (21). Its position suggests that REG inserts might bind natural peptide substrates and either promote or retard their entry into the proteasome's central chamber. If so, REG molecules lacking their inserts should exhibit altered ability to promote cleavage of long, natural peptides by the proteasome. To explore this possibility, we used a 21-residue peptide and a 49-residue peptide as proteasome substrates and analyzed the cleavage products in the presence of REGalpha Delta i or wild-type REGalpha . It is evident from the HPLC profiles in Fig. 3 that there was no significant difference between REGalpha Delta i and REGalpha in their ability to stimulate cleavage of two long peptides by the proteasome. However, we cannot exclude the possibility that removal of the REGalpha insert affects the rate at which the proteasome degrades the two substrates, P21 and BBC1. Nor can we exclude the possibility that removal of the REGalpha insert affects Km values for peptide hydrolysis by REG-proteasome complexes.


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Fig. 3.   Activated cleavage of long natural peptides by REGalpha Delta i and REGalpha . Left panels, HPLC analysis of proteasome-generated cleavage products from peptide P21 (SADPELALALRVSMEEQRQRQ). The upper panel shows the pattern of cleavage products formed in the absence of REGs; the middle and lower panels are the patterns in the presence of REGalpha and in the presence of REGalpha Delta i, respectively. Human red blood cell proteasomes (600 ng) were incubated with 0.1 mg/ml P21 alone or in the presence of REGalpha or REGalpha Delta i for 12 h, and the cleavage products were analyzed as described under "Experimental Procedures." Right panels, HPLC analysis of products from peptide BBC1 (MKKEKARVITEEEKNFKAFASLRMARANARLFGIRAKRAKEAAEQDGSG) generated by the proteasome alone (upper panel), the proteasome in the presence of REGalpha (middle panel), and the proteasome in the presence of REGalpha Delta i (lower panel).

Formation of Hetero-oligomers from REGalpha and REGbeta Subunits Lacking Inserts-- REGalpha and REGbeta preferentially form hetero-oligomers. By contrast, REGgamma only forms homo-oligomers (18). The REGalpha and REGbeta inserts are similar, being composed largely of alternating lysine and glutamic acid residues, i.e. KEKE motifs. The REGgamma insert, on the other hand, contains blocks of positive and negative amino acids sandwiched between two stretches of hydrophobic residues (see Fig. 1A). Conceivably, the similar chemical nature of the REGalpha and REGbeta inserts promotes preferential association of REGalpha with REGbeta . If this were an important function of the REGalpha and REGbeta inserts, hetero-oligomerization of insert-deleted subunits should be impaired. This possibility was tested by mixing (His)10-REGalpha Delta i or (His)6-REGgamma Delta i with REGbeta Delta i and assaying hetero-oligomer formation by gel filtration. Bacteria that expressed REGbeta Delta i were mixed with bacteria that expressed either (His)10-REGalpha Delta i or (His)6-REGgamma Delta i prior to lysis. After clearing the lysate, the His-tagged proteins were captured on Ni-NTA beads, eluted with 0.5 M imidazole, and applied to a Superdex 200 filtration column (Fig. 4, A and B). (His)10-REGalpha Delta i formed active, stable hetero-oligomers with REGbeta Delta i, whereas (His)6-REGgamma Delta i did not (data not shown). To rule out the possibility that association of (His)10-REGalpha Delta i with REGbeta Delta i was due to the interactions between REGbeta Delta i and the polyhistidine extension on REGalpha Delta i, we used a REGalpha inactive monomeric mutant, REGalpha (N50Y), that forms hetero-oligomers with wild-type REGbeta .2 REGalpha (N50Y) was mixed with REGbeta Delta i, and gel filtration revealed that the two proteins formed stable active hetero-oligomers (Fig. 4, C and D). From these experiments, we conclude that the homolog-specific inserts do not determine the preferential association of REGalpha and REGbeta .


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Fig. 4.   Hetero-oligomer formation between insert-deleted REGalpha and REGbeta . A, (His)10-REGalpha Delta i and REGbeta Delta i were expressed in E. coli separately. The bacteria were mixed prior to lysis by high pressure, and the lysates were then incubated with Ni-NTA beads. After extensive washing, the bound proteins were eluted with 0.5 M imidazole and loaded onto a Superdex 200 sizing column. The peak fractions corresponding to oligomers were pooled, concentrated, and rechromatographed producing the profile (His)10-REGalpha Delta i/REGbeta Delta i (thick solid line) shown in A. Selected fractions were assayed for proteasome activation (dashed line with squares). As a control, purified REGbeta Delta i proteins were also subjected to gel filtration analysis (thin solid line), and selected fractions were used to perform proteasome activation assay (thin dashed line with triangles). Note that REGbeta Delta i is shown to be inactive in this assay because of low protein concentration. B, SDS-PAGE analysis of selected fractions of (His)10-REGalpha Delta i/REGbeta Delta i visualized using Coomassie staining. The numbers at the left are the molecular weight standards in kDa. The numbers at the top identify fractions from the sizing column. C, gel filtration profile of REGalpha (N50Y)/REGbeta Delta i. An inactive monomeric mutant REGalpha (N50Y) was mixed with REGbeta Delta i and incubated overnight at 4 °C prior to gel filtration analysis (thick solid line). Selected fractions were assayed for proteasome activation (dash line with solid circles) and subjected to SDS-PAGE analysis (panel D). Arrow head in panel C shows where monomeric REGalpha (N50Y) elutes from the sizing column.

Activity of REGalpha /beta Hetero-oligomers Formed from Subunits Lacking Inserts-- The formation of stable hetero-oligomers between REGalpha Delta i and REGbeta Delta i allowed us to measure the ability of this complex to activate the proteasome and compare it with activation by wild-type REGalpha /REGbeta hetero-oligomers. As shown in Fig. 5A, higher concentrations of REGalpha Delta i/REGbeta Delta i were needed to activate the proteasome to the same extent as wild-type REGalpha /REGbeta oligomers. Reduced proteasome activation at low concentrations of REGalpha Delta i/REGbeta Delta i might result from the lack of the REGalpha insert, the REGbeta insert, or both. To distinguish among these possibilities, we measured proteasome activation by hetero-oligomers of REGalpha (N50Y)/REGbeta or REGalpha (N50Y)/REGbeta Delta i and found that REGalpha (N50Y)/REGbeta Delta i produced less activation than REGalpha (N50Y)/REGbeta at low concentrations (Fig. 5B). By contrast, REGalpha Delta i/REGbeta activated the proteasome in a manner indistinguishable from REGalpha /REGbeta (Fig. 5C). Therefore, the reduced stimulation by REGalpha Delta i/REGbeta Delta i can be attributed to the absence of the REGbeta insert, not the REGalpha insert. This result is consistent with the impaired ability of REGbeta Delta i to stimulate the proteasome (Fig. 2C). Apparently, the REGbeta insert affects proteasome activation even in the REGalpha /REGbeta hetero-oligomer.


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Fig. 5.   Proteasome activation by alpha /beta hetero-oligomers formed from subunits lacking inserts. A, proteasome stimulation by REGalpha Delta i/REGbeta Delta i (open triangles) and REGalpha /REGbeta (closed triangles). REGalpha Delta i/REGbeta Delta i hetero-oligomers were formed by mixing REGalpha Delta i with an equal amount of REGbeta Delta i, and the mixture was incubated overnight at 4 °C. REGalpha /REGbeta was formed in the same way. The combinations were then used to assay for proteasome activation as described in Fig. 2. B, proteasome stimulation by REGalpha (N50Y)/REGbeta Delta i (open diamonds) and REGalpha (N50Y)/REGbeta (closed diamonds). REGalpha (N50Y), a monomeric REGalpha mutant, was mixed with either REGbeta Delta i or REGbeta and incubated at 4 °C overnight. The resulting hetero-oligomers were purified using a Superdex 200 sizing column and assayed for proteasome activation as described in Fig. 2 except that reactions were incubated for 10 min after substrate addition. C, proteasome stimulation by REGalpha /REGbeta (closed squares) and REGalpha Delta i/REGbeta (open squares). This experiment was performed as described in B. D, proteasome binding properties of the REGalpha (N50Y)/REGbeta Delta i and REGalpha (N50Y)/REGbeta hetero-oligomers. The concentrations of REGalpha (N50Y)/REGbeta Delta i or REGalpha (N50Y)/REGbeta hetero-oligomers indicated on the left were incubated with proteasomes tethered by antibodies to an enzyme-linked immunosorbent assay plate. After washing, the bound proteins were eluted with 0. 5 M NaCl and transferred to a nitrocellulose membrane; rabbit anti-REGalpha was used to probe the membrane. Chemiluminescence reagents from NEN were used to visualize bound horseradish peroxidase-goat versus rabbit antibodies. The box labeled C shows the signal obtained when 1 µM REGalpha was incubated in the wells coated with antibodies but in the absence of proteasomes. Each entry in panels A, B, and C represents the mean of three measurements from a single experiment. Similar results were obtained in at least two independent experiments using recombinant REG proteins from different preparations.

An explanation for the reduced activation by REGalpha (N50Y)/REGbeta Delta i is a lower affinity of the complex for the proteasome. This was tested using the direct proteasome binding assay described earlier (18). The titration presented in Fig. 5D demonstrates that REGalpha (N50Y)/REGbeta Delta i does not bind the proteasome as well as REGalpha (N50Y)/REGbeta . We conclude that decreased proteasome activation by REGalpha (N50Y)/REGbeta Delta i reflects its impaired ability to bind the proteasome and that this lowered affinity is due to the absence of the REGbeta insert.

The reduced affinity of REGalpha (N50Y)/REGbeta Delta i for the proteasome could be due to changes in the subunit composition of the hetero-oligomers. Therefore, after isolation of REGalpha (N50Y)/REGbeta and REGalpha (N50Y)/REGbeta Delta i hetero-oligomers from a Superdex 200 gel filtration column, the apparent ratio of REGbeta to REGalpha was determined from the absorbance at 214 nm of each subunit peak after HPLC (see "Experimental Procedures"). Samples were taken from three fractions across the corresponding hetero-oligomer peaks, and the apparent molar ratios of REGbeta to REGalpha (N50Y) ranged from 1.30 to 1.38, whereas the apparent molar ratios of REGbeta Delta i to REGalpha (N50Y) ranged from 1.13 to 1.17. A ratio of 1.33 would be fully consistent with the REGalpha /REGbeta hetero-oligomer being a heptamer composed of four REGbeta and three REGalpha subunits. An average ratio of 1.15 observed for REGalpha (N50Y)/REGbeta Delta i cannot be explained by any integral combination of REGalpha and REGbeta subunits in a defined hetero-oligomer. However, it is not certain that the absorbance at 214 nm depends only on the number of peptide bonds in each subunit. Therefore, we cannot exclude the possibility that the absorbance ratio of 1.15 arises from hetero-oligomers consisting of four REGbeta Delta i and three REGalpha (N50Y). However, neither can we exclude the possibility that removal of the REGbeta insert produces small changes in the relative abundance of REGalpha and REGbeta subunits in a mixture of hetero-oligomers.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

The experiments presented above address four hypotheses regarding the functions of REG homolog-specific inserts. We previously hypothesized that KEKE motifs promote protein-protein association (20). More recently, we proposed that the KEKE motifs in REGalpha and REGbeta might interact with KEKE motifs on the proteasome subunits C6 and C9 (18). The geometric considerations presented in Fig. 1D show that even though the KEKE inserts in REGalpha are located on the surface opposite to the presumed proteasome binding surface, they could still interact with C-terminal extensions on proteasome alpha subunits. Thus, it is reasonable to imagine that REG inserts might promote binding to the proteasome, and this constitutes the first hypothesis tested. Fig. 2, A and B, demonstrates that proteasome activation by REGalpha and REGgamma deletion mutants are virtually identical to their wild-type counterparts, indicating that binding of REGalpha or REGgamma to the proteasome is not impaired by removal of their corresponding inserts. Although REGalpha and REGgamma inserts do not promote proteasome binding, several experiments show that the REGbeta insert is important for binding to the proteasome. First, the results in Fig. 2C demonstrate that, at low concentrations, REGbeta Delta i stimulates the proteasome much less than wild-type REGbeta . Likewise, low concentrations of REGalpha Delta i/REGbeta Delta i hetero-oligomers activate the proteasome to a lesser extent than REGalpha /REGbeta hetero-oligomers (Fig. 5A). Impaired proteasome activation by REGalpha Delta i/REGbeta Delta i was due to removal of REGbeta inserts because REGalpha Delta i/REGbeta hetero-oligomers stimulate the proteasome equally as well as REGalpha /REGbeta do at all concentrations tested (Fig. 5C). REGalpha (N50Y)/REGbeta Delta i, on the other hand, stimulates the proteasome less than REGalpha (N50Y)/REGbeta at low concentrations (Fig. 5B). Reduced proteasome activation by REGbeta Delta i and REGalpha Delta i/REGbeta Delta i could reflect weaker binding to the proteasome; indeed, a direct proteasome binding assay revealed that REGalpha (N50Y)/REGbeta Delta i binds less tightly than REGalpha (N50Y)/REGbeta (Fig. 5D). It is possible that the reduced activity of REGbeta Delta i results from structural alterations in the subunit produced by removal of the insert. However, in view of the high degree of sequence homology surrounding the alpha , beta , and gamma  inserts (Fig. 1A), it seems unlikely that structural perturbations produced by deleting the inserts would be confined to the beta  subunit. For this reason, we favor the idea that the REGbeta insert actively contributes to proteasome binding by this subunit and by hetero-oligomers containing this subunit. Still, we must admit that the reduced activation by REGbeta Delta i and hetero-oligomers formed from REGbeta Delta i can also be explained by subtle structural alterations of the REGbeta subunit upon removal of its insert.

The second hypothesis asked whether the inserts are responsible for the fact that REGalpha and REGbeta activate cleavage of a variety of fluorogenic substrates, whereas REGgamma mostly activates cleavage after basic amino acids (18). The results presented in Table I show that the patterns of peptide cleavage are identical regardless of whether full-length REG homologs or their insert-deleted counterparts were used to activate the proteasome. It is clear, therefore, that the homolog-specific inserts do not determine the patterns of activated fluorogenic peptide cleavage by the proteasome. The unique pattern of activation by REGgamma , as opposed to REGalpha and REGbeta , may be defined by other region(s) in the molecules, e.g. C-terminal and/or N-terminal sequences. Future experiments in which these regions are exchanged between REGalpha and REGgamma should provide answers to this question.

The crystal structure of REGalpha reveals that the inserts surround a pore through the REGalpha heptamer (Fig. 1, B and C). Because of their location, the inserts could bind long peptides and promote entry of these substrates to the active sites of the proteasome. Alternatively, the inserts might prevent longer substrates from being degraded by blocking the pore of REGalpha or by directly binding longer peptides. These two possibilities constitute the third hypothesis tested. The HPLC profiles in Fig. 3 show that there are no significant differences in the ability of REGalpha and REGalpha Delta i to stimulate the digestion of two long peptide substrates by the proteasome. However, we consistently observe that REGalpha Delta i stimulates the proteasome slightly better than wild-type REGalpha using both Suc-LLVY-MCA and long peptides as substrates (Fig. 2A, Fig. 3, and data not shown). Because the differences are within the experimental error, they may simply result from experimental variation. In any event, removal of the REGalpha insert produces, at most, a small effect on substrate degradation.

The last hypothesis concerns the possibility that the inserts are responsible for the fact that REGalpha preferentially binds REGbeta , and neither of these subunits binds REGgamma (18). The results presented in Fig. 4 clearly rule out a requirement for REGalpha and REGbeta inserts in hetero-oligomer formation. While this manuscript was in review, a study from DeMartino's laboratory (29) reported that deletion of the insert from rat REGalpha does not affect its activity, a finding that agrees with results presented here. However, Song et al. (29) found reduced activation by REGalpha /REGbeta hetero-oligomers formed from rat REGalpha molecules lacking inserts, whereas removal of the human REGalpha insert did not affect proteasome activation by REGalpha /REGbeta (Fig. 5C). Song et al. (29) speculate that decreased activation resulted from impaired interactions between REGalpha and REGbeta upon removal of the REGalpha inserts. Although we cannot rule out the possibility that the inserts facilitate association of these two subunits, they are clearly not required for human REGalpha to oligomerize with REGbeta (see Fig. 4). Song et al. (29) also reported that recombinant rat REGbeta is inactive. The results presented here (Fig. 2C and Table I) show that recombinant human REGbeta stimulates the proteasome. Two other studies from our laboratory have shown that human REGbeta is active (18, 19). Moreover, a single-site mutation in the activation region of REGbeta results in loss of this activity (19). Therefore, we do not believe that the activity observed for human REGbeta is an experimental artifact. The discrepancy between our findings and those of Song et al. (29) could reflect differing properties of rat and human REGbeta molecules or differences in assay conditions. Regarding the latter possibility, we note that the assay of Song et al. uses REGbeta concentrations 10-fold lower than those employed by us.

In summary, the results presented above clearly demonstrate that the presence of REGbeta inserts increases the affinity of REGbeta and REGalpha /REGbeta hetero-oligomers for the proteasome. By contrast, REGalpha and REGgamma inserts are not required for proteasome binding or activation. We have also demonstrated that the REGalpha , REGbeta , and REGgamma inserts are not responsible for their differing abilities to activate cleavage of specific fluorogenic peptides, nor do the inserts define the specific patterns of homo-oligomerization and/or the hetero-oligomerization observed with these three homologs. In addition, removal of the REGalpha inserts did not have any pronounced effect on the extent of the degradation of long peptides. If the REGalpha and REGgamma inserts are not important for activating the proteasome, what do they do? Although it is possible that they have no biological functions, the high degree of conservation observed among each homolog-specific insert from several mammalian species would suggest otherwise. Possibly, the inserts have some regulatory function. For example, REGgamma has been reported to disappear upon interferon-gamma treatment (30), raising the possibility that the REGgamma insert harbors a degradation signal. As for REGalpha and REGbeta , we have observed that KEKE motifs are found in chaperonins such as hsp90 and hsp70 (20). Conceivably, the KEKE insert of REGalpha may bind heat shock proteins. Alternatively, we have proposed that the inserts serve to couple the proteasome to the calnexin-TAP-MHC I complexes in the endoplasmic reticulum, thereby promoting transfer of antigenic peptides to MHC I molecules (20). The availability of insert-deleted forms of each REG homolog should allow direct tests of the latter hypothesis.

    ACKNOWLEDGEMENTS

We thank Robert Schackmann for oligonucleotide synthesis. We are also grateful to Drs. Carlos Gorbea, Patrick Young, Vicenca Ustrell, David Mahaffey, Laura Hoffman, and Daniel Taillandier for comments on the manuscript and to other members of the Rechsteiner laboratory for technical advice.

    FOOTNOTES

* These studies were supported by National Institutes of Health Grant GM37009 and by grants from the Lucille P. Markey Charitable Trust and the American Cancer Society.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 The abbreviations used are: Boc, t-butyloxycarbonyl; beta NA, beta -naphthylamide; Cbz, benzyloxycarbonyl; HPLC, high performance liquid chromatography; MCA, aminomethylcoumarin; MHC, major histocompatibility complex; REG, 11 S regulator; Suc, succinyl; TAP, transporter associated with antigen processing; PAGE, polyacrylamide gel electrophoresis.

2 Z. Zhang, A. Clawson, and M. Rechsteiner, manuscript in preparation.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Coux, O., Tanaka, K., and Goldberg, A. (1996) Annu. Rev. Biochem. 65, 801-847[CrossRef][Medline] [Order article via Infotrieve]
  2. Hoffman, L., and Rechsteiner, M. (1996) Curr. Top. Cell. Regul. 34, 1-32[Medline] [Order article via Infotrieve]
  3. Hochstrasser, M. (1995) Curr. Opin. Cell Biol. 7, 215-223[CrossRef][Medline] [Order article via Infotrieve]
  4. Grziwa, A., Baumeister, W., Dahlmann, B., and Kopp, F. (1991) FEBS Lett. 290, 186-190[CrossRef][Medline] [Order article via Infotrieve]
  5. Lupas, A., Koster, A. J., and Baumeister, W. (1993) Enzyme Protein 47, 252-273[Medline] [Order article via Infotrieve]
  6. Kopp, F., Kristensen, P., Hendil, K. B., Johnsen, A., Sobek, A., and Dahlmann, B. (1995) J. Mol. Biol. 248, 264-272[CrossRef][Medline] [Order article via Infotrieve]
  7. Seemuller, E., Lupas, A., Stock, D., Lowe, J., Huber, R., and Baumeister, W. (1995) Science 268, 579-582[Medline] [Order article via Infotrieve]
  8. Lowe, J., Stock, D., Jap, B., Zwickl, P., Baumeister, W., and Huber, R. (1995) Science 268, 533-539[Medline] [Order article via Infotrieve]
  9. Groll, M., Ditzel, L., Lowe, J., Stock, D., Bochtler, M., Bartunik, H., and Huber, R. (1997) Nature 386, 463-471[CrossRef][Medline] [Order article via Infotrieve]
  10. Hoffman, L., Pratt, G., and Rechsteiner, M. (1992) J. Biol. Chem. 267, 22362-22368[Abstract/Free Full Text]
  11. Udvardy, A. (1993) J. Biol. Chem. 268, 9055-9062[Abstract/Free Full Text]
  12. Peters, J. M., Franke, W. W., and Kleinschmidt, J. A. (1994) J. Biol. Chem. 269, 7709-7718[Abstract/Free Full Text]
  13. Ma, C.-P., Vu, J. H., Proske, R. J., Slaughter, C., A., and DeMartino, G. N. (1994) J. Biol. Chem. 269, 3539-3547[Abstract/Free Full Text]
  14. Hershko, A., and Ciechanover, A. (1992) Annu. Rev. Biochem. 61, 761-807[CrossRef][Medline] [Order article via Infotrieve]
  15. Ma, C.-P., Slaughter, C. A., and DeMartino, G. N. (1992) J. Biol. Chem. 267, 10515-10523[Abstract/Free Full Text]
  16. Dubiel, W., Pratt, G., Ferrell, K., and Rechsteiner, M. (1992) J. Biol. Chem. 267, 22369-22377[Abstract/Free Full Text]
  17. Realini, C., Dubiel, W., Pratt, G., Ferrell, K., and Rechsteiner, M. (1994) J. Biol. Chem. 269, 20727-20732[Abstract/Free Full Text]
  18. Realini, C., Jensen, C., Zhang, Z., Johnston, S., Knowlton, J. R., Hill, C. P., and Rechsteiner, M. (1997) J. Biol. Chem. 272, 25483-25492[Abstract/Free Full Text]
  19. Zhang, Z, Clawson, A., Realini, C., Jensen, C., Knowlton, J. R., Hill, C. P., and Rechsteiner, M. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 2807-2811[Abstract/Free Full Text]
  20. Realini, C., Rogers, S. W., and Rechsteiner, M. (1994) FEBS Lett. 348, 109-113[CrossRef][Medline] [Order article via Infotrieve]
  21. Knowlton, J. R., Johnston, S., Whitby, F., Realini, C., Zhang, Z., Rechsteiner, M., and Hill, C. P. (1997) Nature 390, 639-642[CrossRef][Medline] [Order article via Infotrieve]
  22. Rechsteiner, M., Hoffman, L., and Dubiel, W. (1993) J. Biol. Chem. 268, 6065-6068[Free Full Text]
  23. Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 488-492[Abstract]
  24. Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987) Methods Enzymol. 154, 367-382[Medline] [Order article via Infotrieve]
  25. Niedermann, G., Butz, S., Ihlenfeldt, H. G., Grimm, R., Lucchiari, M., Hoschutzky, H., Jung, G., Maier, B., and Eichmann, K. (1995) Immunity 2, 289-299[Medline] [Order article via Infotrieve]
  26. Ustrell, V., Realini, C., Pratt, G., and Rechsteiner, M. (1995) FEBS Lett. 376, 155-158[CrossRef][Medline] [Order article via Infotrieve]
  27. Groettrup, M., Soza, A., Kuckelhorn, U., and Kloetzel, P.-M. (1996) Immunol. Today 17, 429-435[CrossRef][Medline] [Order article via Infotrieve]
  28. Tanaka, K., Tanahashi, N., Tsurumi, C., Yokota, K., and Shimbara, N. (1997) Adv. Immunol. 64, 1-38[Medline] [Order article via Infotrieve]
  29. Song, X., von Kampen, J., Slaughter, C. A., and DeMartino, G. N. (1997) J. Biol. Chem. 272, 27994-28000[Abstract/Free Full Text]
  30. Tanahashi, N., Yokota, K., Ahn, J. Y., Chung, C., Fujiwara, T., Takahashi, E., Demartino, G. N., Slaughter, C. A., Toyonaga, T., Yamamura, K., Shimbara, N., and Tanaka, K. (1997) Genes Cells 2, 195-211[Abstract/Free Full Text]
  31. Creighton, T. E. (1993) Proteins: Structure and Molecular Properties, 2nd Ed., pp. 182-187, 229, W. H. Freeman & Co., New York


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