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Plasticity in eucaryotic 20S proteasome ring assembly revealed by a subunit deletion in yeast

2004; Springer Nature; Volume: 23; Issue: 3 Linguagem: Inglês

10.1038/sj.emboj.7600059

1460-2075

Irina Velichutina, Pamela L. Connerly, Cassandra S. Arendt, Xia Li, Mark Hochstrasser,

Glycosylation and Glycoproteins Research

Article22 January 2004free access Plasticity in eucaryotic 20S proteasome ring assembly revealed by a subunit deletion in yeast Irina Velichutina Irina Velichutina Search for more papers by this author Pamela L Connerly Pamela L ConnerlyCurrent address: Department of Microbiology, Miami University, 32 Pearson Hall, Oxford, OH 45056, USA Search for more papers by this author Cassandra S Arendt Cassandra S ArendtCurrent address: Department of Biochemistry & Molecular Biology, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA Search for more papers by this author Xia Li Xia Li Search for more papers by this author Mark Hochstrasser Corresponding Author Mark Hochstrasser Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT, USA Search for more papers by this author Irina Velichutina Irina Velichutina Search for more papers by this author Pamela L Connerly Pamela L ConnerlyCurrent address: Department of Microbiology, Miami University, 32 Pearson Hall, Oxford, OH 45056, USA Search for more papers by this author Cassandra S Arendt Cassandra S ArendtCurrent address: Department of Biochemistry & Molecular Biology, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA Search for more papers by this author Xia Li Xia Li Search for more papers by this author Mark Hochstrasser Corresponding Author Mark Hochstrasser Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT, USA Search for more papers by this author Author Information Irina Velichutina‡, Pamela L Connerly‡, Cassandra S Arendt, Xia Li and Mark Hochstrasser 1 1Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT, USA ‡These authors contributed equally to the work *Corresponding author. Department of Molecular Biophysics & Biochemistry, Yale University, 266 Whitney Avenue, PO Box 208114, New Haven, CT 06520, USA. Tel.: +1 203 432 5101; Fax: +1 203 432 5175; E-mail: [email protected] The EMBO Journal (2004)23:500-510https://doi.org/10.1038/sj.emboj.7600059 Current address: Department of Microbiology, Miami University, 32 Pearson Hall, Oxford, OH 45056, USA PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The 20S proteasome is made up of four stacked heptameric rings, which in eucaryotes assemble from 14 different but related subunits. The rules governing subunit assembly and placement are not understood. We show that a different kind of proteasome forms in yeast when the Pre9/α3 subunit is deleted. Purified pre9Δ proteasomes show a two-fold enrichment for the Pre6/α4 subunit, consistent with the presence of an extra copy of Pre6 in each outer ring. Based on disulfide engineering and structure-guided suppressor analyses, Pre6 takes the position normally occupied by Pre9, a substitution that depends on a network of intersubunit salt bridges. When Arabidopsis PAD1/α4 is expressed in yeast, it complements not only pre6Δ but also pre6Δ pre9Δ mutants; therefore, the plant α4 subunit also can occupy multiple positions in a functional yeast proteasome. Importantly, biogenesis of proteasomes is delayed at an early stage in pre9Δ cells, suggesting an advantage for Pre9 over Pre6 incorporation at the α3 position that facilitates correct assembly. Introduction Most of the regulated degradation of intracellular proteins in eucaryotes occurs through the ubiquitin–proteasome system (Pickart, 2001; Glickman and Ciechanover, 2002). Substrates are polyubiquitinated, and the tagged proteins are then degraded by the 26S proteasome, which is composed of a proteolytically active 20S proteasome core bound at each end by a 19S regulatory complex (Baumeister et al, 1998; DeMartino and Slaughter, 1999). The latter confers energy- and ubiquitin-dependence on substrate proteolysis. 20S proteasomes are found in all three branches of life, and all eucaryotic species examined have this protease (Baumeister et al, 1998; Kruger et al, 2001). Its subunits assemble into a cylindrical stack of four seven-subunit rings (Figure 1A). β subunits comprising the inner rings bear the active sites, which are exposed to the interior chamber, and substrates enter through a narrow annulus in the outer α rings (Löwe et al, 1995; Arendt and Hochstrasser, 1997; Groll et al, 1997). Whereas the proteasome of the archaeon T. acidophilum is composed of homomeric α and β rings (Löwe et al, 1995), in the yeast Saccharomyces cerevisiae and other eucaryotes there are seven distinct subunits in each α and each β ring (Heinemeyer et al, 1994; Chen and Hochstrasser, 1995; Groll et al, 1997). Figure 1.Composition and function of pre9Δ 20S proteasomes. (A) Schematic of subunit positions in wild-type proteasomes. The 28-subunit complex has C2 symmetry. (B) Matα2 repressor degradation rate in pre9Δ (MHY1069) and wild-type (MHY501) cells measured by pulse–chase analysis at 30°C. (C) βgal activity assays for Deg1-βgal, Leu-βgal, and Ub-Pro-βgal from pre9Δ and wild-type cells. Download figure Download PowerPoint Each subunit of the eucaryotic proteasome is present twice per particle in specific dyad-related positions in the complex. This organization is conserved from yeast to mammals and is thought to be identical in all eucaryotes (Groll et al, 1997; Unno et al, 2002). All subunits have a comparable tertiary fold. Sequence identities among subunits within a species are usually ∼20–40%, but orthologous subunits from different species often have identities in the 55–95% range. Current data suggest that subunit duplication and diversification from simpler homoheptameric ring-forming subunits occurred very early in eucaryotic evolution (Kruger et al, 2001). A limited set of subunit replacements in the β ring of vertebrate proteasomes has also been documented. Specifically, each of the three β subunits that harbor the catalytic centers can be replaced by a γ-interferon-inducible subunit that is ∼60–70% identical. These subunit replacements are important for MHC class I antigen processing (Kruger et al, 2001). How can multiple, structurally similar polypeptides assemble into a large and stereotypical structure with such high fidelity? This problem is not unique to 20S proteasomes. Other protein complexes have subunits arranged in rings or stacks of rings that are composed of different but related polypeptides. An example is the eucaryotic class II chaperonin, a protein-folding catalyst (Archibald et al, 1999). Often eucaryotic ring complexes are related to similar assemblages in other species that have just one or a few different subunits. Ring structures with related but distinct subunit composition sometimes exist in the same cells, and these alternative rings usually have different functions. Examples include Sm/Lsm RNA-processing oligomers and exosomes, which are complexes of ribonuclease subunits (Pannone and Wolin, 2000; Raijmakers et al, 2002). For all these complexes, the mechanism of assembly in vivo remains an important but unanswered question. Archaeal 20S proteasomes self-assemble from purified α and β subunits, but eucaryotic proteasome assembly requires both intramolecular and exogenous chaperones (Maurizi, 1998; Kruger et al, 2001). Certain β-subunit propeptides, particularly that of the β5 subunit, promote assembly by mechanisms that are still obscure (Chen and Hochstrasser, 1996; Kruger et al, 2001). A single-turnover chaperone, Ump1, has also been shown to facilitate proteasome biogenesis (Ramos et al, 1998). Assembly of eucaryotic and eubacterial proteasomes appears to take place via a half-proteasome intermediate, which contains one full α ring and a β ring with unprocessed precursors (Yang et al, 1995; Chen and Hochstrasser, 1996; Nandi et al, 1997; Schmidtke et al, 1997; Zühl et al, 1997). To begin dissecting the rules by which proteasome subunits associate and assemble, we have investigated the molecular basis for an observation made over a decade ago with yeast (Emori et al, 1991). Of the 14 genes encoding the 14 different 20S proteasome subunits in S. cerevisiae, all but one are essential for viability (Emori et al, 1991; Heinemeyer et al, 1994). The lone exception is Pre9/α3, but neither structural nor previous biochemical studies gave any indication as to why this subunit is uniquely dispensable. We report that proteasomes purified from pre9Δ cells have replaced the missing Pre9 subunit with an additional copy of the Pre6/α4 subunit. Remarkably, a Pre6 ortholog from the plant Arabidopsis thaliana is also able to fill this position. The Pre6 subunit therefore can take two different slots within the hetero-oligomer, and this capacity is evolutionarily conserved. Analysis of proteasome assembly intermediates in pre9Δ cells revealed an accumulation of free proteasome subunits and a strong reduction in the level of half-proteasome intermediates and mature proteasomes. This suggests that correct subunit arrangement is achieved at least in part through more efficient incorporation of Pre9 relative to Pre6 at the α3 position during an early stage in proteasome assembly. These data have implications for our understanding of both proteasome assembly and evolution, and are also probably relevant to other ring-shaped protein assemblies. Results Loss of Pre9/α3 has only modest effects on proteasome function Deletion of PRE9 causes only minor phenotypic abnormalities, in contrast to deletion of any of the remaining 20S subunit genes (Emori et al, 1991; Fu et al, 1998). Cell doubling time in our strain background was 8% slower for pre9Δ cultures in rich medium at 30°C relative to wild type. Haploid pre9Δ cells were more severely impaired for growth when incubated at 37°C or when exposed to the amino-acid analog canavanine, and they were more resistant than wild type to cadmium (not shown, but see Figure 2C). These traits are hallmarks of weak proteasomal mutants (Arendt and Hochstrasser, 1997). We also measured the degradation of several proteasome substrates in pre9Δ cells. Degradation of Matα2, an endogenous substrate, showed only an approximately two-fold slowdown (Figure 1B). When we measured β-galactosidase (βgal) activity levels of several different short-lived βgal-based test proteins, at most very small increases (∼2.5-fold) were seen in pre9Δ compared to wild-type cells (Figure 1C), suggesting very minor changes in the degradation rates of these substrates. Figure 2.Two extra copies of the Pre6/α4 subunit in each pre9Δ proteasome. (A) Purified 20S proteasomes analyzed by gradient SDS–PAGE and Coomassie blue staining. (B) Average ratio of amino-acid levels in the indicated species (all are 20S proteasome subunits) from panel (A) in pre9Δ relative to wild-type proteasomes. (C) Partial suppression of pre9Δ by increased dosage of PRE6. Cells were spotted in 10-fold serial dilutions on minimal medium (SD) or SD containing 1 μg/ml canavanine. Download figure Download PowerPoint 20S proteasomes from wild-type and pre9Δ strains were partially purified on glycerol gradients. As previously observed (Emori et al, 1991), basal peptidase activities derived from several of the catalytic centers were higher in the mutant particles than in wild type (not shown). Wild-type particles have relatively weak peptidase activity because the proteasome channel in the α ring is predominantly closed (Osmulski and Gaczynska, 2000). The N-terminus of Pre9/α3 provides a key part of the ‘gate’ controlling this channel, consistent with the higher apparent activity of pre9Δ proteasomes (Groll et al, 2000). In summary, changes in proteasome function are observed in vitro and in vivo when the Pre9 subunit is missing, but the alterations are relatively subtle. Purified pre9Δ proteasomes have two extra copies of Pre6/α4 Several hypotheses could explain the high level of proteasome function in the pre9Δ mutant. The remaining α subunits could arrange themselves into a six-membered ring analogous to the homohexamer formed by the proteasome-related hslV protease in bacteria (Baumeister et al, 1998). Alternatively, a gap could exist in the α ring lacking Pre9, which might be stabilized by contacts among the remaining subunits. Finally, the position normally occupied by Pre9 could be taken by another protein, such as another proteasome subunit. We purified 20S proteasomes from wild-type and pre9Δ cells and compared their subunit composition (Figure 2A). At least 11 distinct species (of the total of 14 expected) were resolved. One band of the size predicted for Pre9 was missing from the pre9Δ particles, as expected. More interestingly, the band immediately above the Pre9 band consistently stained more intensely in pre9Δ samples than in wild-type ones. This band from an SDS gel-separated pre9Δ proteasome preparation was excised and subjected to matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) mass fingerprinting. The protein was identified as the proteasome subunit Pre6/α4. Pre6 and Pre9 share only ∼33% identity, which is comparable to the similarity of Pre9 to other α subunits. The increased levels of Pre6 in pre9Δ proteasomes might reflect the ability of the Pre6 subunit to occupy two positions in each α ring, which would predict a two-fold increase in Pre6 subunits per proteasome in the pre9Δ mutant relative to wild type. To quantitate the actual increase, equivalent amounts of purified pre9Δ and wild-type proteasomes were resolved by SDS–PAGE, and subunits were subjected to quantitative amino-acid analysis. The Pre6 bands and two control 20S proteasome bands were analyzed. As shown in Figure 2B, the amount of Pre6 was indeed approximately two-fold higher in the pre9Δ proteasomes relative to wild-type particles. In contrast, for the two control subunits, little or no increase was observed in the mutant. For both wild-type and pre9Δ proteasomes, comparison of the experimentally determined amino-acid content of the Pre6 and Pre10 (control 1) species to the values predicted from their known sequences showed strong agreement. This provided independent evidence for the initial mass spectrometric identification of Pre6, and indicated that there was minimal contamination of these bands by other proteins in the gel (see Materials and methods). We conclude that in pre9Δ cells, most or all 20S proteasomes contain twice the wild-type number of Pre6 subunits. The Pre6 substitution could explain why only modest proteolytic abnormalities characterize the pre9Δ mutant. Disulfide engineering indicates that Pre6 occupies the α3 position The simplest model for subunit arrangement in the pre9Δ proteasome would place the extra Pre6 subunit in the position normally occupied by Pre9 in the wild-type particle, next to Pre8/α2 (Figure 1A). If Pre6 occupation of the α3 position is not favored, then overexpression of Pre6 might partially suppress phenotypic anomalies associated with pre9Δ. In fact, an increased dosage of PRE6 could modestly suppress the poor growth of pre9Δ cells on canavanine plates (Figure 2C). Little or no suppression was seen with high-copy expression of two other α subunits, Doa5/α5 or Pre10/α7. We observed extremely weak but reproducible growth suppression of pre9Δ with high-copy PRE8, suggesting that higher levels of Pre8/α2 might also facilitate slightly the incorporation of its predicted new neighbor, Pre6. To obtain direct physical evidence for juxtaposition of Pre6 and Pre8 in the pre9Δ proteasome, we attempted to crosslink cysteine residues that were introduced into the two subunits at positions predicted to be in close proximity (Figure 3 cartoon; Figure 4A). [Attempts to crystallize pre9Δ proteasomes have so far failed (M Groll and M Hochstrasser, unpublished).] The feasibility of subunit crosslinking was first tested by engineering a disulfide bond between Pre8 and its normal neighbor, Pre9. From the yeast proteasome structure (PDB entry 1RYP), we identified Pre9 residues that contacted Pre8 and, by sequence alignment, were conserved in Pre6. The β carbons of Pre8-Lys160 and Pre9-Leu56 are within 4.9 Å of each other, close to β-carbon separations seen in natural disulfide bonds (Skiba et al, 1999), so these residues were changed to cysteines. Figure 3.Juxtaposition of Pre6 and Pre8 in pre9Δ proteasomes revealed by disulfide engineering. (A) Control disulfide crosslinking in aqueous iodine of Pre8-HF(His/Flag)–Pre9-T7 for wild-type proteasomes analyzed by anti-Flag immunoblot. +DTT, reducing agent added prior to electrophoresis. (B) Anti-Flag immunoblot of CuCl2-induced crosslinking of Pre8-HF to Pre6 in extracts from strains MHY2863 (lanes 5–9), MHY2865 (lanes 10–12), and MHY2867 (lanes 13–15). A parallel Pre8-HF–Pre9 (MHY1839; lanes 1–4) crosslinking reaction allowed direct comparison of crosslinking efficiency. Asterisk, an unknown cross-reacting band. (C) Crosslinking (CuCl2) of two engineered His6-tagged Pre6 subunits in pre9Δ proteasomes visualized by anti-His immunoblotting. Strains used were MHY2900 (lanes 1–3), MHY2901 (lanes 5–6), MHY2896 (lanes 7–8), and MHY2897 (lanes 9–10). Download figure Download PowerPoint Figure 4.A network of salt bridges important for Pre6 subunit occupation of the α3 position in pre9Δ proteasomes. (A) RasMol figures (PDB 1RYP) showing subunit interfaces. For the predicted interface between Pre8 and Pre6 in the pre9Δ proteasome (right panel), the Cα backbone of Pre6 was fitted onto the Pre9 structure. (B) Summary of key suppression tests with engineered complementary side-chain changes at Pre6 interfaces in the pre9Δ proteasome. Salt bridges are indicated by red lines and charge clashes by blue stars. (C) Suppression of the growth defect associated with pre9Δ pre6-K37E,D56K by pre8-K38E (see Figure 4Biii). The pre6 allele was on a high-copy (2μ) plasmid. MHY1603 cells expressing the indicated pRS425 plasmid-borne alleles were grown on FOA medium (30°C, 5 d) to evict the PRE6/URA3 plasmid originally present. (D) Suppressor analysis on FOA of pre9Δ pre6-K37E,D56KCEN and pre9Δ pre6-K37ECEN with high-copy (2μ) pre8-K38E and/or doa5-E60K. High-copy plasmid-borne alleles present (empty plasmids not listed): 1, pre8-K38E; 2, PRE8; 3, doa5-E60K; 4, DOA5; 5, pre8-K38E+doa5-E60K; 6, pre8-K38E+DOA5; 7, PRE8+doa5-E60K; 8, PRE8+DOA5; 9, none; 10, pre8-K38E; 11, PRE8; 12, doa5-E60K; 13, DOA5; 14, pre8-K38E+doa5-E60K; 15, none (*but carries PRE6, not pre6-K37E). 1–8, 30°C for 7 d; 9–15, 30°C for 4 d. Download figure Download PowerPoint Incubation of extracts from pre8-K160C pre9-L56C yeast cells under oxidative conditions resulted in the time-dependent formation of a novel larger species that was detected when the samples were run on nonreducing SDS gels and subjected to immunoblot analysis with an antibody against Flag epitope-tagged Pre8 (Figure 3A, lanes 1–4). Consistent with the inference that this is a disulfide-linked Pre9–Pre8 species, the low mobility band disappeared when the reducing agent dithiothreitol (DTT) was added to a crosslinked sample prior to electrophoresis (lane 5). Although Pre9 was tagged with a T7 epitope, it reacted very poorly with anti-T7 epitope antibodies. To verify the presence of Pre9 in the crosslinked species, we carried out an identical oxidative time course using a Pre9 allele lacking the Cys substitution (Figure 3A, lanes 6–9). The low mobility band was no longer observed. Crosslinking also required the K160C substitution in Pre8 (not shown). These data indicate that intersubunit contacts can be monitored by disulfide engineering. We tested whether crosslinking could be detected between Pre8 and Pre6 specifically when proteasomes lacked Pre9 (Figure 3B). In pre9Δ proteasomes, Pre6-Leu54 should be in a position similar to that of Pre9-Leu56 in wild-type particles if Pre6 occupied the α3 position (Figure 4A). For the pre9Δ proteasome with Pre8-Lys160 and Pre6-Leu54 mutated to Cys, we could not know exactly how close the mutant residues would be to one another, so crosslinking might not occur as readily as in the control experiments (Figure 3A and B). Nevertheless, a time-dependent accumulation of a DTT-sensitive crosslinked species was observed in pre6-L54C pre8-K160C pre9Δ cell extracts (Figure 3B, lanes 5–9). In Pre9+ proteasomes, the Pre6–Pre8 crosslinked species was no longer detected (lanes 10–12), and it was also lost if Pre6 lacked Cys54 (lanes 13–15) or Pre8 lacked the Cys160 mutation (not shown). A second prediction for Pre6 substitution at the Pre9 position in pre9Δ proteasomes would be that two Pre6 subunits would abut one another (Figure 3C). We tested this by substituting cysteines at a distinct set of Pre6 residues, Asn79 and Ile155, which should be in close proximity across the predicted Pre6–Pre6 interface. As shown in Figure 3C, efficient Pre6–Pre6 crosslinking occurred in pre9Δ (lanes 4–6) but not PRE9 cells (lanes 1–3). Crosslinking did not occur in pre9Δ cells unless Asn79 and Ile155 of Pre6 were both replaced with Cys (lanes 7–10). We conclude that the extra copies of Pre6 in the pre9Δ proteasome can take the positions normally occupied by Pre9 in the α rings and that the new Pre6 interfaces with surrounding subunits are very similar to those used by Pre9. This conclusion was confirmed by the genetic data in the next section. A network of salt bridges is important for Pre6 positioning in pre9Δ proteasomes We previously developed structure-guided pseudoreversion strategies to probe the functional significance of specific subunit interactions within the proteasome (Chen and Hochstrasser, 1996; Arendt and Hochstrasser, 1997). An analogous approach was designed to investigate interactions of the Pre6 subunits in pre9Δ cells. Several Pre9 residues that contact the adjacent Pre8 subunit in wild-type proteasomes are conserved in Pre6. When Pre6 is located in the α3 position in the pre9Δ proteasome, these Pre6/‘α3’ residues could make similar contacts with Pre8. We focused on a salt bridge between Pre9-Glu58 and Pre8-Lys38; Pre6 has an Asp residue (D56) at the position corresponding to Pre9-E58 (Figure 4A). In pre9Δ cells, where Pre6 is in both the α3 and α4 slots, D56 of Pre6/‘α3’ could salt bridge with Pre8-K38 while D56 of Pre6/α4 might pair with K37 of the Pre6/‘α3’ subunit (Figure 4Bi). We reasoned that mutations in the Pre6 subunit that affect the putative Pre8-K38–Pre6-D56 salt bridge might be deleterious, which in turn might be alleviated by a compensatory mutation in the adjacent Pre8 subunit that might restore the salt bridge. However, mutation of Pre6-Asp56 to Asn or Lys had little if any effect on growth (i.e., on essential proteasome functions) in either a PRE9 or pre9Δ background (not shown). In contrast, the pre6-K37E mutation was strongly deleterious but only when PRE9 was deleted (Figure 4D and not shown). This suggested that loss of Pre9 sensitized the proteasome to perturbations of certain neighbor-interacting residues of the duplicated Pre6 subunit. Specifically, the pre6-K37E mutation might cause charge clashes with both Pre6-D56 in the α4 position and with Doa5-E60 at the α5 position. To alleviate the predicted clash of Pre6-E37 in the α3 position with Pre6-D56 at α4, we engineered a D56K substitution into pre6-K37E, creating the pre6-K37E,D56K double mutant (Figure 4Bii). However, the double mutant grew worse, not better, than pre6-K37E when combined with pre9Δ. When on a low-copy (CEN) plasmid, pre6-K37E allowed very slow growth but pre6-K37E,D56K was lethal (Figure 4D); weak growth was only seen if pre6-K37E,D56K was overproduced (Figure 4C, ‘vector’). Poor growth could reflect the fact that in pre6-K37E,D56K pre9Δ proteasomes, the mutant K56 of Pre6/‘α3’ should appose Pre8-K38 (Figure 4Bii). We therefore introduced a high-copy pre8-K38E allele into pre6-K37E,D56K pre9Δ cells to see if growth would be enhanced (Figure 4B iii). Indeed, significant suppression of the pre6-K37E,D56K pre9Δ growth defect was observed at 30°C (Figure 4C). Importantly, the suppression effects were both allele- and gene-specific (Figure 4C). A pre8 allele encoding a Lys-to-Glu mutation at residue 177 (which affects the surface facing the 19S cap) did not suppress, nor did wild-type PRE8. A Lys-to-Glu mutation of a residue equivalent to pre8-K38 in another α subunit, Pre10-K41, also failed to suppress. While the pre8-K38E mutation restored Pre8/α2–Pre6/‘α3’ intersubunit contact, the Pre6/α4–Doa5/α5 interface would still be defective in the pre8-K38E pre6-K37E,D56K pre9Δ mutant (Figure 4Biii). We therefore asked if simultaneous introduction of high-copy pre8-K38E and doa5-E60K alleles into a pre6-E37,K56CENpre9Δ strain could suppress the original lethality. This should effectively restore all the targeted salt bridges linked to the Pre6 subunits (Figure 4Biv), although the polarity is reversed and impaired expression/folding of the multiple mutated subunits might occur. In fact, more vigorous growth was observed with the two suppressors than with pre8-K38E alone (Figure 4D, strain 5 versus 6). This suppression was largely dependent on doa5-E60K (strains 3,7). Tellingly, however, when suppression of the pre6-K37E single mutant was evaluated (Figure 4D, 9–14), enhanced growth was only seen with high-copy doa5-E60K and not with both pre8-K38E and doa5-E60K (strain 12 versus 14), consistent with enhanced interaction of doa5-K60–pre6-E37 (at α4) but a deleterious interaction between pre8-E38 and the normal pre6-D56 residue (at α3). These structure-based pseudoreversion analyses strongly support the conclusion that the extra Pre6 subunits in the pre9Δ proteasome sit between Pre8/α2 and Pre6/α4 in each α ring. More importantly, they make a compelling case for a network of salt-bridging interactions between neighboring α subunits making substantial contributions to proper proteasome subunit arrangement, at least for the alternative pre9Δ proteasome. Evolutionary conservation of Pre9/α3 replacement by α4 Previously, we had found that several yeast 20S proteasome subunits, including the α subunits Doa5/α5 and Pre9/α3, could be replaced by their orthologs from A. thaliana (Fu et al, 1998). In Figure 5 (top left), it can be seen that the plant α4 subunit, PAD1, could also replace its yeast counterpart, Pre6. We then tested whether PAD1 shared the ability of Pre6 to replace the α3 subunit in the yeast pre9Δ proteasome. Indeed, PAD1 supported the growth of a pre6Δ pre9Δ strain as well (Figure 5). Complementing activity was specific to the PAD1 gene. This finding was especially remarkable given that pre9Δ cells were extremely sensitive to mutations in PRE6 (Figure 4 and not shown). Pre6 and PAD1 are ∼58% identical over 249 residues. Thus, even though 42% of the residues between the orthologous subunits are different, the plasticity of α4 subunit placement has been conserved, at least in the context of the yeast proteasome. Pre6 residues, which when mutated lead to synthetic growth defects with pre9Δ, for example Lys37, are similar or identical in PAD1 and α4 orthologs from most other species. Figure 5.The A. thaliana Pre6/α4 ortholog PAD1 can replace Pre6 in both pre6Δ and pre6Δ pre9Δ cells. Genes encoding the indicated proteins were expressed from TRP1 plasmids in MHY1600 (left) and MHY1603 (right), which both originally also carried a PRE6/URA3 plasmid. Cells were spotted in 10-fold serial dilutions and incubated at 23°C; growth on FOA requires loss of the PRE6/URA3 plasmid. AtPAC1 and AtPAE1 were shown previously to complement deletion of their yeast orthologs, Pre9/α3 and Doa5/α5 (Fu et al, 1998). Download figure Download PowerPoint Mutant pre9Δ cells have a defect early in proteasome assembly The ability of the Pre6/α4 subunit to assume the α3 position raises the question of why Pre6 is not observed in this position in wild-type cells in at least a fraction of proteasomes. We reasoned that Pre9 might have some advantage over Pre6 in incorporating stably into the assembling particle during proteasome biogenesis. A prediction of this hypothesis is that assembly would be delayed in cells lacking Pre9. We evaluated this by gel filtration, which allows the resolution of 26S and 20S proteasomes from unincorporated subunits and precursor subparticles such as the 15S intermediate (‘half-proteasome’) that is bound to the Ump1 chaperone (Ramos et al, 1998). Congenic wild-type and pre9Δ strains were generated that expressed Ump1 and Pup1/β2 proteins as HA2-epitope-tagged derivatives. Size-fractionated extracts from these cells were analyzed by immunoblotting (Figure 6). Figure 6.An early proteasome assembly defect in pre9Δ cells. (A) Anti-HA immunoblots of Superose 12 column fractions from extracts of wild-type and congenic pre9Δ cells. Both the Pup1 and Ump1 proteins have C-terminal HA epitope tags. Right, whole-cell extract (WCE). Pgk1 was used as a loading control. (B) Immunoblot analysis of the same fractions run on separate gels using the anti-α subunit monoclonal antibody MCP231. The antibody recognizes a common epitope in multiple α subunits (arrows), reacting most strongly with Pre10/α7 and Pre5/α6. Download figure Download PowerPoint In wild-type cells, the Pup1 precursor, proPup1, was detected primarily in the 15S intermediate and to a lesser extent as free subunit (Figure 6A, top panel). Pup1 is proteolytically processed at a late stage in proteasome assembly to its mature form (mPup1). The Ump1 chaperone was also primarily detected in the 15S intermediate, as expected (Ramos et al, 1998). Strikingly, when Pre9 was absent from cells, a very different profil