Role of C-terminal Extensions of Subunits (cid:1) 2 and (cid:1) 7 in Assembly and Activity of Eukaryotic Proteasomes*

A close inspection of the crystal structure of the yeast 20 S proteasome revealed that a prominent connection between the two (cid:1) -rings is mediated by the subunit (cid:1) 7/ Pre4. Its C-terminal extension intercalates between the (cid:1) 1/Pre3 and (cid:1) 2/Pup1 subunits on the opposite ring. We show that the interactions promoted by the (cid:1) 7/Pre4 tail are important to facilitate the formation of 20 S particles from two half-proteasome precursor complexes and/or to stabilize mature 20 S proteasomes. The deletion of 19 residues from the (cid:1) 7/Pre4 C terminus leads to an accumulation of half-proteasome precursor complexes containing the maturation factor Ump1. The C-terminal extension of (cid:1) 7/Pre4, which forms several hydrogen bonds with (cid:1) 1/Pre3, is in addition required for the post-acidic activity mediated by the latter subunit. Deletion of the C-terminal tail of (cid:1) 7/Pre4 results in an inhibition of (cid:1) 1/Pre3 propeptide processing and abrogation of post-acidic activity. Our data obtained with yeast strains that expressed

Ubiquitin-mediated proteolysis is the main pathway for ATPdependent non-lysosomal degradation of intracellular proteins in eukaryotes (1,2). Polyubiquitylated substrates are recognized and degraded to small peptides by a multisubunit protease termed 26 S proteasome. This large proteolytic complex is composed of a 20 S barrel-shaped complex (the 20 S proteasome) that encloses the proteolytic sites, to which at both ends 19 S regulatory complexes are attached. The latter harbor isopeptidase and ATPase activities as well as binding sites for polyubiquitin chains (3)(4)(5). 20 S proteasomes are present in all eukaryotes, as well as in some archaea and eubacteria. In 1995, the structure of the 20 S proteasome of the archaea Thermoplasma acidophilum solved by x-ray crystallography was reported. This proteasome is composed of two distinct types of subunits, ␣ and ␤, that are organized in four heptameric stacked rings with the arrangement ␣ 7 ␤ 7 ␤ 7 ␣ 7 . The ␤-rings form a central cavity that contains 14 active sites located on the inner surface (5). Two years later the crystal structure of the eukaryotic Saccharomyces cerevisiae 20 S proteasome was determined revealing a similar organization. However, the two identical halves contain seven different ␣-type subunits and seven distinct ␤-type subunits occupying unique locations within the structure. Only three of the seven different ␤-subunits are active (6). Based on mutational and structural studies it was found that subunits ␤1/ Pre3, ␤2/Pup1, and ␤5/Pre2 harbor the peptidase sites, which mediate activities classified as post-acidic, tryptic, and chymotryptic, respectively (6 -9). In mammalian proteasomes, the constitutive active subunits ␤1, ␤2, and ␤5, upon immune stress, can be replaced by the interferon-␥-induced subunits ␤1i, ␤2i, and ␤5i, respectively, resulting in the formation of so-called "immunoproteasomes" (10). More recently, the crystal structure of a constitutive mammalian 20 S proteasome was determined showing that the overall structure of eukaryotic proteasomes is highly conserved (11).
In all proteasomes studied, ␣-type and ␤-type subunits share a common "␤-sandwich" fold, in which two antiparallel ␤-sheets are sandwiched in between two layers of ␣-helices (3). Major structural differences exhibited by the individual subunits, such as insertions or C-and N-terminal extensions, are important features that determine the specific subunit interactions within the ␣or ␤-rings (cis contacts) and between the rings (␣-trans-␤ and ␤-trans-␤ contacts). These interactions target each of the 14 subunits to unique locations within the structure of the eukaryotic 20 S proteasome (6,11).
In Thermoplasma, the ␤-subunits are synthesized as precursors that require posttranslational processing for activation. Similarly, all active eukaryotic ␤-type subunits are synthesized as inactive precursors with N-terminal propeptides. These propeptides are cleaved off by intrasubunit autolysis yielding the mature forms with N-terminal threonine residues that act as nucleophiles attacking peptide bonds of substrates (7,(12)(13)(14). This property places proteasomes into the family of "Ntn hydrolases" that is characterized by an N-terminal nucleophile (15,16).
Another structural aspect of several ␤-subunits is their Cterminal extensions that are involved in interactions within or across the rings. Subunit ␤2 has the longest C-terminal extension (37 residues) with which it embraces ␤3 within the same ring. The C-terminal extension of ␤7 resembles a clamp that projects from one half of the proteasome to reach out to the opposing half entering into its ␤-ring between ␤1 and ␤2. In the present study, we have investigated the role of the C-terminal extensions of ␤2 and ␤7. We present evidence showing that, in yeast, the ␤7/Pre4 C-terminal domain has a role in mediating efficient generation of 20 S proteasome from its precursors. In addition, we show that specific interactions between this domain and ␤1/Pre3 are essential for post-acidic peptidase activity. The C-terminal extension of ␤2/Pup1 is essential for yeast cell viability indicating that functional proteasomes cannot form in its absence.
Construction of Yeast Strains and Plasmids-The yeast strains used in this study are listed in Table I. Construction of chromosomal open reading frames that expressed C-terminally truncated versions of ␤7/ Pre4 was performed as follows. Different 3Ј-portions of PRE4 were generated by PCR using primers containing flanking EcoRI and XbaI sites. The 3Ј-primers were designed such that the codons for Ile 225 , Gly 219 , or Glu 207 (residue numbers refer to the processed subunits according to Ref. 6) were replaced by the stop codon TAA to produce ␤7/Pre4 mutant proteins lacking one (pre4-⌬C1), seven (pre4-⌬C7), or 19 (pre4-⌬C19) amino acid residues at the C terminus, respectively. The PCR fragments were cloned into the integrative plasmid YIplac204 (TRP1-marked) (18). All plasmids were verified by DNA sequencing. The resulting plasmids were linearized with BglII within the coding sequence for targeted integration into the S. cerevisiae genome. The generation of a chromosomal allele expressing a truncated version of ␤2/Pup1 lacking 30 residues at the C terminus (pup1-⌬C30) followed a similar strategy except that EcoNI was used for linearization. To follow the truncated Pup1, another genomic allele was generated that expressed a fusion with two ha epitopes (Pup1-⌬C30-ha) by an analogous strategy. The integrative plasmids that were used to generate in-frame fusions of full-length PUP1 and PRE3 with a sequence encoding two ha epitopes were described earlier (19). The same strategy was used to produce a genomically tagged strain expressing ␣7/Pre10-ha.
To express a ␤1/Pre3 protein that was identical to the mature processed form, the propeptide-encoding region of PRE3 gene was replaced in the genome, by a two-step gene transplacement protocol, with a sequence encoding ubiquitin (Ub) 1 (details available upon request). The Ub-Pre3 protein synthesized by the resulting strain is processed cotranslationally or nearly so by Ub-processing proteases (20). To exclude posttranslational acetylation of the N-terminal Thr residue of Pre3 generated from a Ub-Pre3 fusion, yeast strains with chromosomal deletions of NAT1 (nat1-⌬::KanMX4) were generated (21). NAT1 encodes a subunit of N-terminal acetylase responsible for this modification (20).
Fractionation of Whole-cell Extracts by Gel Filtration, Electrophoresis, and Immunoblotting-S. cerevisiae cells were grown at 30°C in SD medium to A 600 of 1.2 Ϯ 0.2 and treated as described by Ramos et al. (19). Protein extraction and fractionation on a Superose 6 column coupled to an Ä KTA FPLC (Amersham Biosciences) as well as SDS-PAGE and immunoblots were performed as described previously (19). Native PAGE was carried out according to published protocols (22). Polyclonal rabbit antibodies raised against ␤7/Pre4 were a kind gift from the laboratory of Dieter Wolf. Polyclonal anti-␣7/Pre10 antibodies were raised against Pre10-His 6 expressed in Escherichia coli.
Proteasomal Peptidase Activity Assays-Determination of proteasomal activities in crude extracts was performed as follows. Yeast cells FIG. 1. Structural aspects of the ␤7/Pre4 C terminus tail. A, structure of S. cerevisiae 20 S proteasome emphasizing the ␤7/Pre4 ␤-trans-␤ interactions with ␤1/Pre3 and ␤2/Pup1. This figure was prepared with RasMol 2.6. ␤7/Pre4 is shown in the spacefill representation; all other subunits are displayed as wireframe. B, detail of the contact sites between the ␤7/Pre4 C-terminal extension (orange and red) and ␤1/Pre3 (various shades of blue). The prediction of hydrogen bonds (shown as dotted lines) and the graphic representation were done using SETOR (25). Side chains of relevant residues are highlighted as balls and sticks. C, amino acid sequence alignments of the C-terminal regions of ␤7 subunits of the indicated species. Residues identical between ␤7/Pre4 from S. cerevisiae and other species are boxed. D, synthetic growth inhibition by ump1-⌬ and pre4-⌬C19. Spore clones (a-d) derived from JD281 (Table I) with the indicated genotypes were spotted in serial dilutions onto YPD medium and incubated at 30°C for 2 days. wt, wild type. E, analysis of the level of two proteasomal subunits, ␤7/Pre4 (top) and ␣7/Pre10 (bottom), in the same strains (a-d) as in D with anti-Pre4 and anti-Pre10 antibodies, respectively. Strain JD183 expressing a slower migrating Pre10-ha (wt*) was included as a control to demonstrate specificity of the anti-Pre10 antibodies. 30 g of crude extract proteins were loaded/lane. The propeptidecontaining precursor forms of Pre4 and Pre4-⌬C19 detected in the ump1-⌬ background are indicated by arrowheads. For the wild-type UMP1 strains, these forms were detectable only upon long exposure of the immunoblot (data not shown). from exponentially growing cultures (A 600 ϭ 0.8 -1.2) were harvested at 3000 ϫ g, washed with cold water, frozen in liquid nitrogen, and stored at Ϫ80°C. The cells were lysed with acid-washed glass beads (л 0.4 -0.5 mm, Sigma) in extraction buffer (50 mM Tris-HCl, pH 7.5, 2 mM ATP, 5 mM MgCl 2 , 1 mM dithiothreitol, 15% glycerol) by vortexing. Cell debris was removed by centrifugation at 15,800 ϫ g at 4°C. The protein content in the supernatant was determined using the Bio-Rad Bradford protein assay. For assaying chymotrypsin-like activity, reactions were set up with 5 g of extract protein and 5 g of succinyl-Leu-Leu-Val-Tyr 7-amido-4-methylcoumarin (Bachem) as substrate. The trypsin-like activity and the post-acidic activity were measured using 20 g of protein and 5 g of t-butoxycarbonyl-Leu-Arg-Arg 7-amido-4-methylcoumarin (Affinity) and Ac-Gly-Pro-Leu-Asp 7-amido-4-methylcoumarin (Bachem) as substrates, respectively. The assay conditions were described previously (19).

Structure of the ␤7/Pre4 C-terminal Tail and Its
Interactions with ␤1/Pre3-The overall architecture of the 20 S proteasome and the general structures of the ␣and ␤-subunits are conserved between T. acidophilum (5), S. cerevisiae (6), and mammals (11). One striking structural difference between the bacterial and the eukaryotic proteasome is confined to the ␤7 subunit. Inspection of the crystal structure of the yeast proteasome reveals a long C-terminal domain of ␤7/Pre4. This tail is composed of 19 amino acid residues and extends from one half of the proteasome to the other where it ends between the subunits ␤1/Pre3 and ␤2/Pup1, the former of which carries the active site for post-acidic peptidase activity (Fig. 1A). Both the carboxyl group of the last residue Ile 225 and the side chain of Gln 223 of ␤7/Pre4 form hydrogen bonds with the side chain of Arg 185 of ␤1/Pre3 (residue numbers refer to the processed subunits according to Ref. 6). Arg 185 within Pre3 together with Gly 219 of ␤7/Pre4 form hydrogen bonds stabilizing Asp 32 of Pre3, which itself forms a hydrogen bond with Leu 34 . The latter two residues are direct neighbors of Lys 33 , which is part of the post-acidic catalytic site, suggesting that this network of hydrogen bonds may be important for post-acidic activity (Fig.  1B). A comparison of the C-terminal sequence of ␤7 subunits from various species (Fig. 1C) revealed that its extension is only moderately conserved among eukaryotes.
The C-terminal Extension of ␤7/Pre4 Facilitates the Formation of 20 S Particles from Two Precursor Complexes-During our studies on the ump4-1/pre4-3 mutant, in which a residue of the ␤7/Pre4 subunit was exchanged that is close to the site where its C-terminal extension emanates from one ␤-ring to reach out to the other, we found that proteasome precursor complexes accumulate. 2 Prompted by this finding we asked whether a defined deletion of the C-terminal tail of ␤7/Pre4 would be inhibitory to the formation of 20 S proteasomes from two 15 S half-proteasome precursor complexes. These precursor complexes are characterized by the presence of the propeptide-bearing precursor forms of the active ␤-type subunits, ␤1/Pre3, ␤2/Pup1, and ␤5/Pre2, and the proteasome maturation factor Ump1 (19). We generated a strain in which the C-terminal 19 amino acid residues of ␤7/Pre4 were deleted (pre4-⌬C19). In the same strain, a double ha epitope tag was linked to the C terminus of ␤2/Pup1. To examine whether the C-terminal truncation of Pre4 caused changes in the intracellular ratios of proteasomal precursor complexes and mature proteasomes, we analyzed cell extracts of the pre4-⌬C19 mutant by gel filtration on a Superose 6 column. As for the wild type, the half-proteasome precursor complex of the pre4-⌬C19 eluted with a peak in fractions 22 and 23 (Fig. 2). The ratio of this precursor complex to mature proteasomes, however, is significantly higher in the mutant (Fig. 2, middle panel) resulting also in a spreading of the immunoblot signal from fractions 21 to 25. As described previously (19), the mature form of ␤2/Pup1 is present in earlier fractions containing proteasomal peptidase activity due to the presence of assembled and active 20 S and 26 S proteasomes. Noteworthy is also the observation that the mature form of ␤2/Pup1 is in addition detected as a free subunit in fractions 29 and 30 of pre4-⌬C19 mutant extracts (Fig. 2). In contrast, no free mature ␤2/Pup1 was detected in these fractions of wild-type extracts. These findings suggest that the free processed subunits observed in the pre4-⌬C19 are derived from matured 20 S proteasomes that fell apart, either in vivo or in vitro.
Similar experiments were performed with wild-type and pre4-⌬C19 strains expressing ␤1/Pre3 tagged at the C terminus with two ha epitopes. Proteins extracted from these strains were fractionated by gel filtration on Superose 6. Again, we observed a strikingly increased amount of ␤1/Pre3 precursor form in fractions 22 and 23 in the pre4-⌬C19 mutant in comparison with the wild type (Fig. 3). In contrast to the ␤2/Pup1 precursor, however, the ␤1/Pre3 precursor form is in addition present in a wide range of fractions ranging from those containing the free subunit to those with 20 S and 26 S proteasomes. Particularly interesting was the simultaneous presence of ␤1/Pre3 mature and precursor forms in the fractions containing 20 S and 26 S proteasome suggesting a defect in ␤1/Pre3 maturation.  (Table I). wt, wild type.  (Table  I). wt, wild type.
The ␤7/Pre4 C-terminal Tail Is Essential for ␤1/Pre3-mediated Post-acidic Peptidase Activity-Previous work by others (23) had shown that a mutation in the PRE4 gene that led to a C-terminal shortening of ␤7/Pre4 by 15 residues resulted in a loss of the post-acidic peptidase activity, which is mediated by the ␤1/Pre3 subunit. Consistent with that report, we observed a significant accumulation of ␤1/Pre3 precursor forms in assembled proteasomes in our mutant pre4-⌬C19 (Fig. 3) together with a complete loss of post-acidic activity (Fig. 4A). We therefore asked whether the defect in post-acidic activity in this mutant was due to a defect in ␤1/Pre3 processing. To answer this question, we generated a congenic set of strains carrying on one side either the pre4-⌬C19 allele or its wild-type counterpart, and on the other side a gene expressing a mature form of ␤1/Pre3 (Pre3-⌬pro) or wild-type Pre3. The ␤1/Pre3 propeptide is dispensable for proteasome assembly in vivo (20,24), but its deletion inactivates the post-acidic activity since its unprotected N terminus is subject to N␣-acetylation (20). Inactivation of the NAT1 gene encoding a subunit of Nat1-Ard1 N␣-acetyltransferase, however, restores near wild-type levels of post-acidic activity to the pre3-⌬pro strain (20). Therefore all strains used in the experiment shown in Fig. 4 in addition lacked the NAT1 gene. As shown in Fig. 4D, none of the mutations affected the stability of ␤7/Pre4 as its ratio to subunit ␣7/Pre10 was not affected. Indistinguishably from the pre4-⌬C19 single mutant, the pre4-⌬C19 pre3-⌬pro double mutant did not display any post-acidic peptidase activity (Fig. 4A). In extracts of the pre3-⌬pro single mutant, in contrast, wild-type activity levels were detected (Fig. 4A). We conclude that the C-terminal extension of ␤7/Pre4 is essential for post-acidic activity of the proteasome independent of an apparent role in mediating efficient autocatalytic processing of Pro-␤1/Pro-Pre3.
In contrast to its effect on post-acidic activity, the pre4-⌬C19 mutation resulted only in a slight reduction of proteasomal chymotryptic and tryptic activity (Fig. 4, B and C). These data suggest that the C-terminal extension of ␤7/Pre4, aside of its role in the dimerization of half-proteasome precursors, has a specific role in the formation of the post-acidic peptidase site. Consistent with previous studies (8,9,24), the post-acidic activity does not appear to be required for the activation of the chymotryptic and tryptic peptidase sites.
Dissection of the Role of the ␤7/Pre4 C-terminal Extension in Post-acidic Activity-To further understand the involvement of the C-terminal extension of ␤7/Pre4 in the activity of the postacidic peptidase site, we prepared a set of mutants expressing ␤7/Pre4 variants with truncated C termini (Fig. 5). We asked in particular whether truncations that included the residues Ile 225 and Gly 219 affected post-acidic activity of the proteasome. These two residues are involved in a network of hydrogen bonds formed with residues flanking the Lys 33 in the catalytic center of ␤1/Pre3 (see above; Fig. 1B). A deletion of only the last amino acid residue Ile 225 (pre4-⌬C1) resulted in a drop of postacidic activity in crude extracts by 20% in comparison with the wild type consistent with a significant role of this residue in the post-acidic activity (Fig. 5A). A further deletion into the C terminus that included residue Gly 219 resulted in a loss of about 90% of the post-acidic activity. None of the deletions affected the stability of the ␤7/Pre4 subunit as its ratio to ␣7/Pre10 was unchanged (Fig. 5B). Taken together these data are consistent with a model (depicted in Fig. 1B) in which the C-terminal extension of ␤7/Pre4 engages in hydrogen bonds that are essential to stabilize an active conformation of the catalytic site in ␤1/Pre3. This function of the ␤7/Pre4 C-terminal extension can be separated from its role in half-proteasome precursor dimerization (see below).

Activation of the Post-acidic Site and Its Role in Proteasome Assembly Are Not Correlating Functions of the ␤7/Pre4
Tail-Because the deletion of only seven residues of the ␤7/Pre4 tail resulted in a drop of the post-acidic activity to less than 10%, we asked whether the formation or stability of the assembled 20 S proteasome was also affected by this shorter tail truncation. To answer this question, we analyzed the distribution of an epitope-tagged version of ␤2/Pup1 (Pup1-ha) in crude extracts of pre4-⌬C7 mutant cells fractionated by gel filtration. In this mutant, the amount of precursor complexes containing unprocessed proPup1-ha was far less strikingly increased than in the pre4-⌬C19 mutant (Fig. 2). Only a slight increase of such precursor complexes was detected in pre4-⌬C7 in comparison to wild type. We conclude that a truncation of the ␤7/Pre4 Cterminal extension that nearly abolishes post-acidic activity only moderately affects proteasome assembly supporting a model assigning two distinct functions to this domain.
Truncation of ␤7/Pre4 C Terminus and Deletion of UMP1 Result in a Synthetic Growth Defect-Despite the observed defects in proteasome assembly (see above), the pre4-⌬C19 mutant cells showed no significant growth defects at 30 or 37°C when compared with congenic wild-type cells (Fig. 1D and data not shown). This observation is in accordance with the finding that the overall amount of active proteasomes in the cell, as judged by the chymotryptic and tryptic activities in this mutant, was very similar to that of wild type (see above). These data suggested that the reduced efficiency of assembly of 20 S proteasomes from its precursors is compensated by a moderate increase in the expression of proteasome subunit genes. We have shown previously that maturation factor Ump1 is required for efficient assembly of 20 S proteasomes from two half-proteasome precursors (19), similar to what is described above for the C-terminal extension of ␤7/Pre4. To test whether the ump1-⌬ mutation and the pre4-⌬C19 mutation have a synthetic effect, we crossed strains with these two mutations and analyzed the progeny by tetrad dissections. Consistent with the observation that both mutations affect the dimerization of half-proteasome precursor complexes, the double mutant showed a strong synthetic growth defect (Fig. 1D). To test whether the observed synthetic growth inhibition by the pre4-⌬C19 and ump1-⌬ mutations is due to an imbalance of proteasome subunits, e.g. due to a more rapid turnover of the mutant Pre4-⌬C19 subunit, we compared the levels of subunits ␤7/Pre4 and ␣7/Pre10 by immunoblotting. The results shown in Fig. 1E demonstrate that the overall levels of these subunits are similar in the single mutants and in the double mutants supporting the notion that the synthetic phenotype is due to a strong inhibition of proteasome assembly. Similar to what we observed previously for the processing of the active subunits ␤1, ␤2, and ␤5 (19), the ump1-⌬ mutation also inhibited the processing of ␤7/Pre4 (Fig. 1E, lanes c and d).
The Deletion of the ␤2/Pup1 C-terminal Extension Is Lethal-Another prominent feature that distinguishes eukaryotic ␤-subunits from those in the Thermophilus "Urproteasome" is a ϳ30-residue C-terminal extension of the ␤2 subunit (Fig. 6C). Analysis of the crystal structure of the yeast proteasome (Fig.  6A) revealed that the extension of ␤2/Pup1 wraps around the ␤3/Pup3 subunit within the same ring of ␤-subunits. To study the relevance of this extension to the biogenesis of functional proteasomes, we replaced one copy of the PUP1 gene in a diploid yeast strain with an otherwise identical copy in which the codon for Tyr 232 (corresponds to Tyr 203 in the processed subunit of the known crystal structure reported in Ref. 6; underlined in Fig. 6C) was replaced by a TAA translation termination codon followed by the T CYC1 terminator. The resulting pup1-⌬C30 allele encodes a Pup1 protein that is truncated at the C terminus by 30 residues. To test for the effect of this truncation on the physiology of S. cerevisiae, we analyzed tetrads of ascospores derived from the resulting heterozygous strain JD329 (Table I). As shown in Fig. 6B, each tetrad yielded only two viable spore clones, all of which were tryptophanauxotrophic, indicating that they lacked the TRP1-marked pup1-⌬C30 allele. Microscopic inspection of the inviable spore clones revealed that the spores had germinated and undergone a few cell divisions before they stopped dividing (data not shown). The tetrad analysis therefore demonstrated that the C-terminal extension of ␤2 is essential for viability of S. cerevisiae cells. We conclude that this extension is essential for the generation of functional proteasomes. To investigate whether the Pup1-⌬C30 subunit is capable of assembling into proteasomal complexes and to follow the fate of the resulting complexes, we generated a diploid strain that expressed an epitope-tagged version of the truncated Pup1 (Pup1-⌬C30-ha). Extracts from the resulting strain JD325 (see Table I) were analyzed by gel filtration and immunoblotting and compared with an otherwise identical wild-type strain expressing Pup1-ha. As shown in Fig.  6D, unprocessed Pup1-⌬C30-ha was detectable in increased amounts in fractions 22 and 23 that typically contain the Ump1-containing half-proteasome precursor complex (19) and moreover was also present in the fractions 19 -21 comprising larger assemblies similar in size to the 20 S particles. When compared with a wild-type strain, however, only minute quantities of processed Pup1-⌬C30-ha were detectable in fractions 15-19 that typically contain 26 S proteasome (CP⅐R 2 ) and the 20 S particle (CP) with one regulatory particle (CP⅐R) (Fig. 6D). Similarly, we observed a striking reduction of Pup1-⌬C30-hacontaining CP⅐R or CP⅐R 2 complexes when we analyzed crude extracts from the mutant strain by native PAGE (Fig. 6E). Note that the experiments with the Pup1-⌬C30-ha variant were performed with a diploid strain carrying also a wild-type PUP1 gene copy. The small amounts of larger assemblies containing processed forms of Pup1-⌬C30-ha therefore may depend on the presence of a functional Pup1 subunit in the other half of the complex. We conclude that Pup1-⌬C30 assembles into proteasomal complexes but that formation of 26 S proteasomes containing this truncated subunit is impaired. DISCUSSION Several proteasomal ␤-subunits of eukaryotic proteasomes are distinguished from their counterparts in bacteria by striking C-terminal extensions. In this report we show that the C-terminal extension of the yeast ␤7/Pre4 subunit serves two distinct functions in proteasome biogenesis and activity. First, it functions as a molecular clamp mediating and stabilizing the interaction of two half-proteasome precursor complexes. The second function that can be assigned to the seven C-terminal residues of Pre4 is to keep the post-acidic site in an active conformation. Another finding of this study is that the Cterminal extension of ␤2/Pup1 serves an essential function in proteasome biogenesis.
The C-terminal Tail of ␤7/Pre4 Acts as a Molecular Clamp Stabilizing the 20 S Proteasome-The C-terminal tail of ␤7/ Pre4, which extends from one half of the proteasome to the other (Fig. 1A), facilitates the formation of 20 S particles from two half-proteasome precursor complexes. In the pre4-⌬C19 mutant lacking this extension, we detected an accumulation of half-proteasome precursor complexes (Figs. 2 and 3). The overall proteasomal chymotryptic or tryptic activities detected in this mutant, however, were only slightly reduced (less than 5 and 9%, respectively) when compared with wild type (Fig. 4, B and C). Similar to effects observed previously in mutants with FIG. 5. Effects of C-terminal truncations of ␤7/Pre4 on post-acidic peptidase activity. A, shown are the relative proteasomal post-acidic activities in crude extracts from cells with wild-type (wt) Pre4 (set to 100%) or from strains with truncated Pre4 shortened at the C terminus by one (⌬C1), seven (⌬C7), or 19 residues (⌬C19). The strains used were, respectively, JD284-6D, JD310, JD311, and JD300-3D (Table I). The bars represent the mean of nine measurements (triplicates on three independent extracts each). Standard deviations are indicated. B, ␤7/Pre4 (top) and ␣7/Pre10 (bottom) levels in crude extracts from wild-type (wt), pre4-⌬C7, pre4-⌬C1, and pre4-⌬C19 cells, respectively. wt*, strain expressing Pre10-ha. Immunoblotting conditions were as for Figs. 1E and 4D.  (Table I)  proteasome defects (19), the assembly defect in the pre4-⌬C19 mutant therefore seems to be partly compensated by a slightly increased expression of proteasome subunits (Fig. 4D). As a result, the pre4-⌬C19 mutant strain does not display any obvious growth defects or phenotypes. When we combined the pre4-⌬C19 with the ump1-⌬ mutation, however, we observed a striking synthetic inhibition of growth (Fig. 1D). Similar to the data presented here for pre4-⌬C19, the ump1-⌬ mutation was shown previously to impair the assembly of 20 S proteasomes from two half-proteasome precursor complexes (19).
Why do eukaryotic proteasomes require a C-terminal extension of ␤7 to mediate efficient assembly of proteasomes while T. acidophilum ␤-subunits lack such a feature? In this archaea, the interaction between half-proteasome precursor complexes is apparently stabilized by other features. One such feature of T. acidophilum ␤-subunits at the interface of the two ␤-rings could be a small extension in form of the side chain of Arg 165 . This residue is located at the end of helix H4 that protrudes from the core of the ␤-subunit and is 38 residues distant from its C terminus (5). These seven small protuberances from each ␤-ring extend to the respective other ring into grooves between the ␤-subunits (5). Collectively, these zipper-like interactions may serve a similar holding function as the long C-terminal extension of ␤7 in eukaryotes. Despite a significant sequence divergence and a reduction in length by four amino acid residues in comparison to its S. cerevisiae counterpart (Fig. 1C), the C-terminal region of the bovine ␤7 subunit occupies a similar location between the ␤1 and ␤2 subunits in the crystal structure. The smaller number of hydrogen bonds that the bovine ␤7 engages in, however, may result in a more flexible contact allowing for an easier replacement of housekeeping subunits by interferon-induced immunosubunits ␤1i and ␤2i (11). The shorter extension of bovine ␤7 appears to be compensated by an additional extension of the ␤1 subunit (11). These two extensions of the neighboring subunits ␤1 and ␤7 seem to form a short "zipper." The C-terminal Seven Residues of ␤7/Pre4 Are an Important Structural Component of the Post-acidic Peptidase Site-Previous work by others (23) had already shown that a shortening of the ␤7/Pre4 C terminus (pre4-⌬C15) results in a loss of the post-acidic peptidase activity mediated by the ␤1/Pre3 subunit. Consistent with these findings, we observed almost a total loss of post-acidic peptidase activity in our pre4-⌬C19 mutant (Fig.  4A). Because we also observed a defect in the processing of pro␤1/Pre3 (Fig. 3) in this strain, we asked whether the Cterminal extension of ␤7/Pre4 is important to trigger autocatalytic processing of the propeptide of ␤1/Pre3 or whether it is an essential structural component of the post-acidic peptidase active site per se. That the latter is true was concluded from the lack of post-acidic activity in a strain expressing a mature form of ␤1/Pre3 (Ub-Pre3-⌬pro in Fig. 4) and ␤7/Pre4 lacking its C-terminal extension. We conclude that the ␤7/Pre4 C-terminal extension is an essential component of the post-acidic site by providing a network of hydrogen bonds that stabilizes an active conformation of ␤1/Pre3 as illustrated in Fig. 1B. Although one of the shorter truncations removing only seven residues (pre4-⌬C7) resulted in a severe reduction in post-acidic activity (by ϳ90%; Fig. 5), it had relatively little effect on the dimerization of two half-proteasome precursor complexes (Fig. 2). We interpret this result as evidence that these two functions of the C-terminal extension of ␤7/Pre4 are distinct and separable. A comparison of the C-terminal extensions of the orthologous ␤7 subunits of S. cerevisiae and other species revealed that it is not highly conserved in sequence (Fig. 1C). This observation suggests that this domain in the S. cerevisiae ␤7/Pre4 is specifically adapted to its function in post-acidic activity in the context of the budding yeast 20 S proteasome.
The C-terminal Extension of ␤2/Pup1 Is Essential for Proteasome Biogenesis-In comparison with ␤-subunits of the T. acidophilum proteasome, subunit ␤2 of eukaryotic proteasomes is characterized by a C-terminal extension of ϳ30 amino acid residues (Fig. 6C). As shown in Fig. 6A, this extension wraps around the neighboring subunit ␤3/Pup3 in the same ␤-ring of the S. cerevisiae proteasome (6). A similar structural arrangement was found for the subunits ␤2 and ␤3 in the bovine proteasome (11). We show that a deletion of this extension is lethal to yeast cells (Fig. 6B). Our experiments with a diploid strain that expressed wild-type ␤2/Pup1 in addition to an epitope-tagged variant of the truncated Pup1 (pup1-⌬C30-ha) showed that the latter is incorporated into proteasomal complexes. Propeptide processing of the Pup1-⌬C30-ha variant and the formation of 26 S proteasomes containing this subunit, however, were very inefficient (Fig. 6). One possible explanation is that subunit ␤3/Pup3, which seems to be held in place by the ␤2/Pup1 C-terminal tail, cannot be incorporated properly in the absence of this tail leading to an inhibition of the assembly and maturation of 26 S proteasomes.