Structural transitions in the stepwise assembly of proteasome core particles

Domain: biology
Published: 2026-03-24
Editorial outcome: minor_revision

Abstract
20S catalytic core particles (CP) of eukaryotic 26S proteasomes are composed of two identical halves comprising 14 distinct subunits. 15S precursor complexes (PC) represent detectable half-CP assembly intermediates lacking the β7-subunit but containing assembly chaperones Ump1 and Pba1-Pba2. Incorporation of β7 drives 15S-PC dimerisation and further CP maturation. Our cryo-EM structures of the yeast 15S-PC and all 13S-PC-derived intermediates suggest that assembly in yeast is not restricted to a single trajectory, but instead involves alternative, and potentially simultaneous pathways. Comparison of the intermediates reveal how Ump1 and β-subunits become structured with each additionally incorporated β-subunit, and how this prepares peptidase sites for auto-activation. We identify two transient interactions of Pba1 with the α-ring, which are important for an ordered progression of maturation. Pba1 loop 81-117 intercalates between subunits α3 and α4 in 13S-15S-PCs and is displaced upon 15S-PC dimerisation. The second interaction involves the α1 N-terminus, deletion of which leads to a defect in Pba1-Pba2 release. These findings indicate how changes in α-ring subunit conformations coordinate CP maturation with Pba1‑Pba2 release.

Introduction
The 26S proteasome is a central and essential component of the ubiquitin/proteasome system in eukaryotic cells1. Because selective protein degradation by the proteasome is of critical importance, both in quality control and regulation of proteins, the proteasome is involved in a multitude of cellular processes and has emerged as a drug target in the treatment of certain types of cancer2. The eukaryotic proteasome is composed of two 19S regulatory particles (RP) and the 20S catalytic core particle (CP)3. CPs are composed of 28 (2×14 distinct) subunits and assemble independently from RPs in a process involving various dedicated chaperones that are unique to eukaryotic proteasome assembly pathways4. The central chamber of the CP is formed by two heptameric rings of β-subunits that are sandwiched between the two distal α-rings. The CP harbours the peptide-cleaving catalytic activities, which are mediated by active sites in three of the seven β-subunits (β1, β2, and β5) of each ring5. The active sites of each of these subunits are characterized by N-terminal threonine residues, which are exposed after autocatalytic removal of propeptides6.

In contrast to 13 of the 14 genes encoding CP subunits, the genes encoding assembly chaperones are not essential for viability in yeast6,7. Their absence, however, causes distinct defects in proteasome assembly with biochemical and phenotypic consequences. Most dramatic are the defects of yeast cells lacking the assembly chaperone Ump1, which display poor growth and hypersensitivity to proteotoxic stress8. Ump1 is found in early assembly intermediates known as 13S precursor complexes (13S-PC) that contain a complete ring of seven α-subunits plus subunits β2, β3, and β49–11. In ump1Δ cells, proteasome biogenesis is impaired, leading to CPs with incompletely matured β-subunits and reduced activity8. In mammals, UMP1, alias POMP, is essential for cell viability12. The 13S-PC, in addition to Ump1 and the above-mentioned α- and β-subunits, contains the heterodimeric chaperone Pba1-Pba2, which is bound to the distal α-ring surface13. Pba1-Pba2 (or its mammalian ortholog PAC1-PAC2) facilitates precursor assembly and promotes proper proteasome maturation by blocking off-pathway association of α-rings14–16. Deletion mutations (pba1Δ or pba2Δ) cause only mild phenotypes in yeast but display genetic interactions with other mutations affecting proteasome regulation or assembly (rpn4Δ, ump1Δ, pre9Δ)11,15,16. Lack of Pba1-Pba2 function, however, causes subtle changes in the composition of proteasomal complexes, with an increase in Blm10-associated CPs17. In mice, knock-out of the Pba1 ortholog PAC1 is embryonically lethal18, underlining the importance of this chaperone for proper proteasome biogenesis in higher eukaryotes. During the assembly process, Pba1-Pba2 remains associated with nascent CPs until their final maturation17,19,20. The latter process involves removal of β-subunit propeptides and the degradation of Ump1, which becomes encased upon formation of CPs by the β7-driven dimerisation of two so-called 15S-PCs8,19,21,22. An additional chaperone heterodimer, Pba3-Pba4, acts in earlier assembly steps preceding formation of 13S-PCs23,24.

15S-PCs are derived from 13S-PCs by the addition of β1, β5, and β6 in an order that has been still unclear. Previous studies reported 13S-PC structures containing both β5 and β613,21, implying that association of β5 and β6 occurs before β1 incorporation (Table S1). The 15S-PC bears all 14 CP subunits but β711,25, and its structure has not been solved. Half-proteasomes, consisting of β7-bound 15S-PCs, have been purified from recombinant insect cell systems and human cells (Supplementary Table 1)21–23,26, but remain undetectable in yeast cells11,25. Dimerisation of 15S-PCs by addition of β7 leads to a complete but still immature PC, which we refer to as late-PC11,19,25. This complex is characterized by the presence of two Pba1-Pba2 molecules that are attached to α-ring surfaces, two molecules of Ump1 encased within the structure, and unprocessed β-subunits19,21–23,26. Conformational changes in the β-rings upon dimerisation trigger autocatalytic processing, probably initiating with β2 activation19. Mature β2 promotes further maturation steps by shortening of other propeptides. The active β-subunits then degrade Ump1. These maturation steps not only go along with structural changes in the β-rings but also in the α-rings, which lead to a release of Pba1-Pba2, an indicator of the completion of CP maturation17,19. Because CP assembly occurs rather fast, most assembly intermediates, such as the 13S-PC and the late-PC, are very short-lived and therefore present at only very low steady state concentrations in wild-type (WT) cells. Therefore, purification and characterization of such complexes often employ cells with mutations in α- or β-subunits that slow down assembly or maturation steps13,19,22. Downsides of these otherwise helpful approaches are that these mutations go along with structural changes. In contrast to the above-mentioned PCs, the 15S-PC is detectable at steady state in WT cells, albeit at relatively low levels8. In this work, we have increased the accumulation of PCs by mutating the β7-subunit, which is not a component of 13S- or 15S-PCs, in order to purify such PCs without any mutations in the incorporated α- or β-subunits. This approach allowed us to perform a cryo-EM-based structural characterization of 13S- and 15S-PCs, as well as multiple structurally uncharacterised intermediates between them. This analysis hints for unexpected alternative routes of β-subunit incorporation and reveals a number of previously unrecognised details regarding structural transitions in subunits and chaperones that underlie the stepwise assembly and maturation of CPs.

Results
Purification of in vivo-formed authentic CP assembly intermediates from S. cerevisiae

Human and yeast PC structures were reported in previous cryo-EM studies, some of which either employed reconstitution of the complexes in insect cell systems or utilized yeast strains with mutated β-subunits, respectively13,21,23,26. These approaches revealed early assembly intermediates such as the 13S-PC and a 13S-PC associated with β5 and β613,21, implying that association of β5 and β6 occurs before β1 incorporation (Table S1), but they could not resolve the structure of a fully assembled 15S-PC. In addition, due to the usage of β1 mutant strains, the timing of β1 incorporation during assembly of yeast CPs remained unclear. We set out to accumulate native early-PCs in yeast by using a strain lacking a C-terminal extension of the β7 subunit (β7-ΔCTE)27. Absence of the β7-CTE significantly impairs dimerisation of 15S-PCs, resulting in their accumulation. Because β7 is not a part of 15S-PCs or earlier assembly intermediates, C-terminal truncation of β7 has no impact on the structures of these complexes. One consequence of the accumulation of 15S-PCs, however, is a shortage of the Pba1-Pba2 chaperone, because it is only recycled when the assembly pathway reaches the final steps of CP maturation20. Consequently, Pba1-Pba2-deficient α-ring surfaces associate with Blm10. To compensate, we concomitantly overexpressed Pba1 and Pba2 in a blm10Δ background20 thereby enriching authentic Pba1-Pba2-capped 15S-PCs and earlier intermediates, which facilitated their affinity purification via N-terminally FLAG-6xHis-tagged Ump1 (Supplementary Fig. 1).

This strategy yielded sufficient material to determine high-resolution cryo-EM structures of six early PCs ranging from 13S-PC to 15S-PC, differing primarily in the β-ring composition (Fig. 1, Supplementary Fig. 2, Table 1). The 13S-PC structure, resolved at 3.3 Å resolution (Supplementary Fig. 3A), comprises a fully assembled α-ring, β2-4, and the assembly chaperones Pba1-Pba2 and Ump1. It overlays well with the structure of a yeast 13S-PC isolated from a pre3-1 strain (β1-G34D)(PDB 7LSX, Cα rmsd 1.03 Å, over the entire structure)13. As the 13S-PC represents the earliest of the structurally characterised intermediates, all later intermediates contain all of its subunits. Unlike prior studies, we captured a 13S-PC with an additional β1-subunit, termed 13S+β1-PC, at 3.2 Å (Supplementary Fig. 3B), and identified 13S-PCs containing the subunits β1-5 (13S+β1+β5-PC) at 4.2 Å (Supplementary Fig. 3C), modelled as a Cα structure due to low resolution of the 3D reconstruction. A previously uncharacterized 13S+β5-PC, containing β2-5, was found in a 13S-13S+β5 dimer, in which a 13S-PC and a 13S+β5-PC are wedged together (Supplementary Fig. 4A). As for a complex that we termed the 13S-15S dimer (Supplementary Fig. 4B), the 13S-13S+β5 dimer was exclusively detected in FLAG-elutions after centrifugal filtration. Both complexes dissociate upon dilution before grid preparation, indicating that they are non-physiological artefacts arising from the high complex concentration needed for cryo-EM sample preparation. The 13S-13S+β5 dimer was resolved to 3.1 Å. In the 13S-13S+β5 dimer, both halves associate to form an artificial antiparallel β2-β2’ interface, positioning the β4 and β5’ subunits close but too far apart for interaction (Supplementary Fig. 5). The β2-subunits are hydrogen-bonded by a parallel β-sheet (encompassing β2 and β2’ residues 118-121), while β4 and β5’ lack contact. Intriguingly, these findings explain previously detected long-distance cross-links in our study involving early PCs from the same genetic background (Supplementary Table 2)20. Like earlier studies involving yeast and human PCs13,20,21,26, we identified a PC species containing β2-6 (13S+β5+β6-PC), resolved at 3.3 Å (Supplementary Fig. 3D), which closely matches a recently published structure from pre3-1/β1 background (PDB 7LS6, rmsd 1.00 Å over the entire structure)13. Finally, we obtained a 3.1 Å reconstruction of the native 15S-PC, comprising β1-6 (Supplementary Fig. 3E). Among all intermediates in our dataset, the 15S-PC accounts for 13.7% of particles, substantially less than the 13S-PC (37%) and 13S+β5-PC (25.6%), but almost twice that of all other complexes (Fig. 1).

β-subunit incorporation during assembly of 15S-PCs does not follow one fixed order

Neither recent cryo-EM studies on human complexes21,23,26, nor a study on PCs isolated from yeast cells bearing the pre3-1 mutation13 detected any 13S+β1-PC species (Supplementary Table 1). These observations suggested that incorporation of β5 and β6 takes place before that of β1. Our structures of the 13S-, 13S+β5+β6- and 15S-PCs as well as the 13S+β5-PC in the 13S-13S+β5 dimer support this order of events. However, we also obtained structures of a 13S+β1-PC and a 13S+β1+β5-PC (Fig. 1) suggesting an additional, alternative assembly pathway, in which β1 is incorporated before β5 and β6 (Fig. 2A). An alternative possibility is that the observed complexes might have arisen not as genuine assembly intermediates but as dissociation products formed during purification. To test whether β1 can assemble with 13S-PCs before β5 and β6 in vivo, we generated strains that allowed to selectively shutdown expression of β5 or β6 by placing the galactose-inducible and glucose-repressible PGAL1 promoter in front of the endogenous gene copies of the respective genes (PRE2 and PRE7)28. To verify efficient shutdown of β5 and to follow incorporation of this subunit into PCs, the 3’ end of the PRE2 gene was, in addition, fused to a sequence encoding a C-terminal 2xHA-tag to enable detection of the β5 subunit with high sensitivity. Upon shift to glucose media, β5-2xHA was undetectable in a native-PAGE/Western blot analysis of proteasomal complexes in extracts of the β5-shutdown (SD) strain (Supplementary Fig. 6). Simultaneous probing of the same western blot membrane for the presence of α7 showed that β5-SD caused a drastic reduction in 20S CP formation with a simultaneous accumulation of early PCs. The latter effect was similarly observed for the β6-SD strains, which is consistent with the essential role of both subunits for the completion of CP assembly. To assess β1 incorporation, we introduced an N-terminal FLAG-tag upstream of the β1 propeptide (β1pro), enabling selective pull-down of β1-containing PCs. In contrast to an untagged control, FLAG-tagged β1 pulled down 13S+β1-PCs without β5 (upon β5-SD), and 13S+β1+β5-PCs (upon β6-SD), thereby proving that neither incorporation of β5 nor of β6 is a requirement for β1 assembly with 13S-PCs (Fig. 2B). These in vivo data support our structural observations and indicate an alternative assembly pathway in which β1 assembles with 13S-PCs before β5 and β6 (Fig. 2A). However, the higher abundance of β5-containing versus β1-containing complexes in our EM dataset (Fig. 1), suggests that β5 incorporation before β1 is the predominant assembly route in yeast cells (bottom pathway in Fig. 2A).

Stepwise restructuring of S3/S4 β-hairpin loops prepares nascent CPs for activation

In the mature CP structure, S3/S4 β-hairpin loops of all β-subunits are located at the contact surfaces with their right-hand neighbours within the same β-ring and are juxtaposed to subunits in the opposing β-ring5,19. These structural features may help to stabilise nascent assembly intermediates by promoting the binding of incoming subunits and fixing them in the correct position by a restructuring event21. Asp17 and Lys29 of the catalytic subunits β1, β2, and β5, which are part of the catalytic triad, are located in the S3/S4 loops, positioning these residues in close proximity to the third residue of the triad, the catalytic Thr1. This structural arrangement indicates that the S3/S4 loops have an important function in properly positioning the catalytic residues for autocatalytic activation.

The early PC structures presented here allowed us to analyse the roles of all S3/S4 loops without interfering mutations. In the 13S+β5+β6- and 15S-PCs, the S3/S4 β-hairpin loops of most β subunits appear to be flexible as they are not resolved (Supplementary Table 3). Only the S3/S4 loops of β4 and β5 are resolved in these PCs (Supplementary Figs. 7 and 8). Residues 25–28 of β4 and β5, which later form sheet S4 in the mature CP, align with β-sheets of their neighbouring subunits in an antiparallel orientation. Specifically, residues 25-28 in β4 align with sheet S10 in β5 (residues 125–129), and residues 25-28 in β5 align with sheet S9 in β6 (residues 135-138) (Supplementary Figs. 7 and 8). In contrast, this alignment is not detectable in the 13S+β1+β5-PC, indicating that β6 incorporation promotes this structural transition. The S4 strands of β4 and β5 furthermore interact with helices H3 of β5 and β6, respectively, pulling them closer to the core of these subunits when the S3/S4 loops restructure during late assembly stages. For β4, the position of the S3/S4 loop overlays well with that in the pre1-1 late-PC19, while the loop in β5 is up to 4.7 Å offset towards the β5 core in the 15S-PC to accommodate the different orientation of β6.

The S3/S4 loops of β1, β2 and β3 remain unstructured in the 15S-PC, most likely due to insufficient conformational constraint. While β1pro and the β1 S3/S4 loop are flexible in all early β1-containing PCs (Figs. 1, 3, and Supplementary Fig. 9), their folding is only observed after β7 incorporation and subsequent dimerisation. Similarly, β2 S3/S4 loop folding is primarily driven by dimerisation. However, the β2 propeptide (β2pro) remains attached to Thr1 in early precursors (Fig. 1), its cleavage in the pre1-1 late-PC coincides with S3/S4 loop formation (Fig. 3 and Supplementary Fig. 9). This indicates that dimerisation is the key trigger for autoactivation of β2. In the absence of its direct neighbour (β7), the S3/S4 loop in β6 remains flexible across all structures analysed here. These observations reveal that β-hairpin loop restructuring, active site residue positioning, propeptide cleavage, and CP maturation are tightly coupled. This is expected, as the formation of hydrogen-bond networks - specifically between Lys29, Thr1, Ser129 and Asp166 in β1, and Lys29, Thr1, Ser131 and Asp168 in β5- is essential for the autocatalytic activation of these proteolytic subunits29. For autoactivation, these residues must be in close proximity and correctly oriented. In the 15S-PC, only the β2 catalytic site is sufficiently formed for all involved residues to show Cα-backbone density (Fig. 3). In contrast, β1 and β5 have flexible loops between residues 165-173 (Supplementary Table 4), leaving Asp166/Asp168 mispositioned to protonate Thr1 via Ser129/Ser131 for autoactivation29. The β2 loop is stabilised upon β1-association, as it is resolved in all β1-containing early PCs. Intriguingly, it is also folded in both halves of the 13S-13S+β5 dimer, suggesting that interactions with a binding partner in the position of β1 are crucial for its folding.

Together, our findings suggest that the S3/S4 β-hairpin loops and the loops spanning residues 166-173 in the catalytic β-subunits progressively fold during 15S-PC assembly, as conformational freedom is restricted by neighbouring subunits in the same or opposite β-ring following half-CP dimerisation.

Progressive Ump1 (re)structuring during PC assembly coincides with successive β-subunit addition and dimerisation into a U-turn

Isolated Ump1 is intrinsically unstructured30,31. Its complete structure was only resolved in late-PCs containing all proteasomal subunits and associated chaperones19,21,32. The structures presented here emphasize that Ump1 progressively folds upon interaction with proteasomal subunits. In the 13S-PC, residues 1-31, 43-47, and 128-130 of the 14 amino acid long protein are unfolded (Fig. 1 and Supplementary Table 5). Addition of β1 to the PC does not improve folding of Ump1, since helices H5 and H6 of Ump1 are already in close contact with α7 and α1 in the area where β1 joins the complex (Fig. 4A). Binding of β5, on the other side, promotes folding of residues 43-47 into helix H3 of Ump1. This interaction is stabilised by contacts between Ump1-Leu34 and the backbone of β5-Gln89, as well as Ump1-Ser35 and the backbone of β5-Lys91 in the 15S-PC (Fig. 4B). When comparing 13S+β1+β5-PC with 15S-PC or 13S+β5+β6-PC, it seems that residues 21-31 fold upon addition of β6 to the complex, although β6 does not directly interact with Ump1. We and others have previously shown that the β5 propeptide (β5pro) interacts with the N-terminus of Ump1 and with β6 in the late-PC19,32. Hence, folding of residues 21–31 of Ump1 in the PCs might depend on interactions between β6 and β5 pro, which should already be in close proximity in these complexes (Fig. 4C). However, residues 21-31 of Ump1 are already folded in the 13S-13S+β5 dimer, despite the absence of β6, indicating that folding of this region is caused by restriction of available space rather than an interaction between specific subunits. Structural overlay of the 15S-PC with the pre1-1 late-PC reveals several interesting details. Firstly, incorporation of β7 into the nascent proteasome pushes subunits β1 and β6 up to 10 Å out of their position in the 15S-PC towards β2 and β5, respectively (Fig. 4C). At the same time, the N-terminus of Ump1 folds due to spatial constraints imposed by β7, β1, and β5 pro, as seen in our study of the pre1-1 late-PC19. Secondly, we observe a small (~2 Å) compression of Ump1 along the central axis of the complex (Fig. 4C), possibly induced by the dimerisation and spatial restraints in the newly formed antechamber. This compression goes along with a shift of β2 pro towards Ump1 and an upward movement of α1. Thirdly, this restructuring of Ump1 leads to a loss of contact between Ump1-Asp136 and α3-Arg114 (Fig. 4B).

We conclude that β7-driven 15S-PC dimerisation enables folding of the Ump1 N-terminus, restricting the available space in the antechamber in such a way that initial Ump1 interactions with the 20S CP subunits are abolished. These changes also induce autoactivation of β2 and β5 as highlighted in previous studies19,21.

Pba1 loop inserts between α3 and α4 in early PCs in a similar way as Rpt2 in active 26S proteasomes

So far, three important interactions between Pba1-Pba2 and α-subunits have been described. First, the C-terminal HbYX motif of Pba1 inserts into the α5/α6 pocket. Second, the HbYX motif at the C-terminus of Pba2 inserts into the α6/α7 pocket16,33. Third, the N-terminus of Pba1 extends through the α-ring pore, interacting with all α-subunits as well as Ump113.

The early PC structures presented here reveal an additional interaction between Pba1 residues 93-109 and the α3/α4 pocket (Fig. 5A). Human PAC1 lacks this loop; instead, its N-terminus extends into a U-turn directed towards the α3/α4 pocket21. There are several hydrogen bonds between this Pba1 loop and the α3/α4 pocket. The Pba1 residues Ser93 and Asn99 form hydrogen bonds with α3-Ser31/Glu27 and α3-Ser154, respectively. In addition, a network of hydrogen bonds is established between Arg30 and Asn79 of α4 and Pba1-Glu103 (Fig. 5A). The specificity and importance of these interactions is highlighted by the fact that Pba1-Pba2 is unable to associate with PCs in a strain lacking α3 (pre9Δ), where a second α4 subunit is incorporated into the CP in place of the missing α3 (Supplementary Fig. 10). Modelling of this Pba1 loop into the α4/α4 pocket of the pre9Δ CP34 indicates spatial and electrostatic hindrance of binding. Intriguingly, the side chain of Pba1-Glu103 roughly occupies the space of Tyr436 in the HbYX motif of Rpt2 in the substrate-engaged yeast 26S proteasome (Fig. 5B)35. It also interacts with α4-Arg30. Thereby, the Pba1 loop intercalates between α3 and α4 in a similar way as the Rpt2 HbYX motif in the 26S proteasome during substrate processing, creating a gap between α3 and α4 (Fig. 5C, D). In comparison with the position in the mature CP, the Pba1 loop in the 15S-PC keeps α4 from reaching its final position in the α-ring. Notably, the C-terminal regions of Pba1 and Pba2 in the 15S-PC align well with the pre1-1 late-PC structure, but differ from the conformation observed in a reconstituted Pba1-Pba2-20S structure33 (Supplementary Fig. 11), supporting our recent hypothesis that Pba1-Pba2 adopt distinct binding modes in PCs compared to mature CPs19.

The N-terminus of α1 is involved in a signaling cascade to dissociate Pba1-Pba2 from proteasomal PCs

Within all the early PCs in this study, the α1 N-terminal residues 3-16 interact with Pba1, Pba2, α2, and α3 (Fig. 5E and Supplementary Fig. 12A). In the pre1-1 late-PC19, however, the N-terminal residues of α1 appear rearranged after 15S-PC dimerisation, altering several interactions between α1 and its contacting subunits. While this relocation disrupts interactions between α1 and α2 as well as Pba2, it creates new contacts between α1 and the N-terminus of α7. Intriguingly, this movement is accompanied by a more than 2 Å shift of the Pba1 N-terminus in the pore towards α7 and a small shift of the α3 N-terminus towards α2. Although the resolution of our maps does not allow for exact interpretation of side chain locations in this area, we hypothesize that relocation of the α1 N-terminus might destabilise the α3 N-terminus. The α3 N-terminus, in turn, needs to occupy a space that is taken by the Pba1 N-terminus in the PCs to close the α-ring pore in the mature CP. We speculate that the signal of relocation could be transmitted via α1-Ser8, α3-Arg4 and -Arg5, α4-Arg6 to Pba1-Glu11 (Fig. 5E), which is the residue that appears to cause the shift of Pba1 through interactions with α3 and α4 (Fig. 5E). This structural transition might prepare for the pulling back of the Pba1 N-terminus and closure of the α-ring gate by the α-subunits’ N-termini.

Because of this striking relocation of the α1 N-terminus, we asked what the effects of a deletion of this peptide would be on CP assembly and maturation. To this end, we generated yeast mutants expressing α1/Scl1 lacking residues 2-16 (Scl1-Δ2-16). Two such mutant alleles were stably introduced into the genome of otherwise WT strains expressing epitope-tagged versions of either Pba2 or Ump1. Native gel analyses of the proteasomal complexes present in these mutants showed that the deletion of the α1 N-terminus causes a drastic accumulation of larger assembly products, which appeared to be late-PCs still associated with the Pba1-Pba2 chaperone, as indicated by the presence of the Pba2-HA-tag (Supplementary Fig. 12B). In contrast to the pre1-1 late-PC, this scl1-Δ2-16 late-PC does not contain detectable Ump1, indicating that the α1/scl1-Δ2-16 mutation does not interfere with degradation of this chaperone. We conclude that deletion of the α1 N-terminus causes a CP maturation defect hindering the release of Pba2-HA. Our structural analysis suggests that the N-terminus of α1, which is in bonding distance to the loop segment encompassing residues 121–138 of Pba2 and the N-termini of α2 and α3, prevents the latter from prematurely closing the central pore in the α-ring, which apparently would result in trapping of the Pba1-N-terminus. To test this interpretation, we have compared a set of strains, in which the N-termini of α1 and α3 were truncated either alone or in combination. Truncation of the α3 N-terminus, in contrast to truncation of α1, had no effect on the release of Pba1-Pba2 (Fig. 5F). Truncation of the α3 N-terminus, however, abrogated the defect of Pba1-Pba2 release caused by the α1 truncation. These data indicate that the N-terminus of α1 has a critical role in maintaining the arrangement of the α-subunit N-termini in the assembling CP in a way that is compatible with Pba1-Pba2 release by keeping the α3 N-terminus in an open conformation as long as the Pba1 N-terminus is located in the α-ring pore.

Methods
Protein expression, purification and analysis

Yeast strains used for purification and biochemical analyses of proteasomal precursor complexes (PCs) are listed in Table S6. Strain MO27 (pre4-ΔC19 FLAG-6xHis-UMP1 blm10-Δ PGAL1-PBA1 PGAL1-PBA2) was used for purification of early PCs, taking advantage of the 6xHis- and FLAG-tags20. MO27 cells were grown in 4 liter complete medium supplemented with galactose as a carbon source. The N-terminal FLAG-6His tag on Ump1 allowed purification of Ump1-containing PCs. Overexpression of Pba1-Pba2 and deletion of the BLM10 gene were important to increase the yield of Pba1-Pba2-capped complexes. A tandem affinity purification protocol taking advantage of the 6His and FLAG tags was performed as follows. Cells were pelleted and frozen in liquid nitrogen. The cells were ground to powder using a Mixer Mill MM400 (Retsch) for 1 min at 30 Hz. Proteins were extracted by incubating the powder with FLAG buffer (50 mM Tris pH 7.5, 5 mM MgCl2, 2 mM ATP, 150 mM NaCl, and 15% (v/v) glycerol) supplemented with 10 mM imidazole and protease inhibitors (cOmplete, EDTA-free, Cat. No. 11836170001, Roche Diagnostics), in the proportion 2 ml per g wet weight, for 30 min at 4°C with rotation. Cell debris was removed by centrifugation at 30,000 xg for 30 min at 4°C, and the supernatant was then passed through a 0.2 μm filter. The filtrate was incubated with Ni-NTA Superflow resin (Cat. No. 30430, Qiagen) equilibrated in FLAG buffer for 1 hour at 4°C with agitation. The resin was washed 4 times with 10 column bed volumes of FLAG buffer supplemented with 10 mM imidazole. Bound complexes were eluted in two steps with FLAG buffer supplemented with 50 mM and 100 mM imidazole, respectively. Eluted fractions were diluted 2.5 times in FLAG buffer and further incubated with EZview Red ANTI-FLAG M2 Affinity Gel (Cat. No. F2426-1ML, Batch SLCL4336, Sigma-Aldrich) for 90 min at 4°C with rotation. The beads were washed 3 times with 20 column volumes of FLAG buffer. Bound proteins were eluted three times (E1-E3) with one column volume each of FLAG buffer without ATP and without Glycerol containing 0.3 mg/ml FLAG peptide (Cat. No. F3290, Sigma-Aldrich). Eluted material was then concentrated using Vivaspin 6 columns 50,000 MWCO (Cat. No. GE28-92-2318, Sigma-Aldrich). Protein concentration of the eluted material was about 0.4 mg/ml. MO24 (PRE1-FLAG-6His) was used to purify wild-type 20S CPs as a control in native PAGE analysis using the same two-step procedure, except that cells were grown in 1 liter of complete medium with glucose (YPD). Protein concentration of the purified material was about 0.4 mg/ml. Samples were diluted with cold sample buffer (240 mM Tris-HCl, pH 8.8, 0.04% (w/v) bromophenol). Purified complexes were analysed under native conditions in 4-16% Bis-Tris Gels (Cat. No. BN1002 Box, Invitrogen), for which the samples were diluted with cold sample buffer (240 mM Tris-HCl, pH 8.8, 0.04% (w/v) bromophenol). Electrophoresis was performed on ice at 16 mA/gel for around 1.5 hours. The proteins in the gel were visualized by silver staining.

Constructs (pJD844/845 and pJD847) for the generation of α1/scl1 and α3/pre7 (pJD847) mutant yeast strains, respectively, were generated by overlap extension PCR to delete internal parts of the SCL1 or PRE7 ORFs (primers used are listed in Table S7). Two variants were created for α1/Scl1, one deleting amino acid residues 2-16 (α1ΔNT(MT)), and another replacing these residues with a glycine residue (α1ΔNT(MGT)); for α3/pre7, one version deleting residues 2-10 (α3ΔNT) was generated. The PCR products were cloned into the integrative yeast vector YIplac21140. Using a two-step gene transplacement strategy, the mutant versions were then introduced stably into the genome of yeast strains expressing 2xHA-tagged versions of Ump1 or Pba241. The resulting yeast mutant strains (Supplementary Table 6) were grown to an OD600nm = 1 in 25 mL cultures. Cells were washed in H2O and resuspended in 75 µL lysis buffer (50 mM Tris pH 7.5, 5 mM MgCl2, 2 mM ATP, 1 mM DTT, 15% (v/v) glycerol and 1x protease inhibitor (cOmplete, EDTA-free, Cat. No. 11836170001, Roche Diagnostics)). Crude lysates were generated by vortexing in the presence of glass beads. After a clearing spin (15 min 30,000 xg at 4°C), 8 µg of total protein were analysed by native PAGE in 3–12% Bis-Tris gel (Cat. No. BN1001 BOX, Invitrogen), western blotting onto PVDF membranes (Cat. No. 03010040001, Roche), and detection using peroxidase-coupled rat monoclonal (clone 3F10) anti-HA antibody (Cat. No. 12013819001, Merck/Sigma-Aldrich, at 1:2500 dilution) enhanced chemoluminescence (ECL) reagent SuperSignal West Femto (Cat. No. 34095, Thermofisher) and X ray film.

To investigate the order of β-subunit assembly in vivo, we generated a set of strains in which the β1 subunits carry an N-terminal FLAG-tag (using a two-step gene transplacement strategy and vector YIplac211)40. For this purpose, a DNA segment was created by overlap extension PCR that encodes FLAG-tagged Pre3/β1, and cloned into YIplac21140 after cleavage with SacI and HindIII (Supplementary Table 7). FLAG-tagged Pre3 was introduced either into strains, in which the β5-encoding gene PRE2 or the β6-encoding gene PRE7 were placed under the control of the glucose-repressible PGAL1 promoter using a one-step gene transplacement strategy and the oligos listed in Supplementary Table 728,42. FLAG-pro-β1-containing complexes were collected and analysed as follows. Cells were grown in 50 mL minimal media containing galactose till OD 0.5 to 0.6 and then collected, resuspended in 200 mL minimal media containing glucose, and incubated further for 15 hours. Cells were harvested, frozen, and kept at -80°C. Pelleted cells were ground into powder in a mortar in the presence of liquid nitrogen. Tris buffer (50 mM, pH 7.4) supplemented with 150 mM NaCl and protease inhibitors was then added in the ratio 1 mL per gram fresh cells. Cell debris was removed by centrifugation at 30,000 xg for 30 min at 4°C. 400 µl crude extract were incubated with 50 µl EZview red anti-FLAG M2 affinity gel (see above) for 1 hour at 4°C with mild shaking. Flow-through was removed and the resin was washed twice with 40 column volumes of extraction buffer. Bound complexes were eluted in 100 µl extraction buffer containing 300 µg/ml FLAG peptide (see above). A strain without a FLAG tag served as a control. Total extract proteins (input control) and eluted complexes were separated by native PAGE in 3-12% Bis-Tris gels (see above). Protein complexes were transferred to a 0,2 µm Amersham nitrocellulose membrane (Cat. No. 10600001, Avantor/VWR) during 2 hours at 0.8 mA/cm2 in a semidry blot transfer chamber. Membranes were incubated overnight with rabbit polyclonal antibodies anti-Prs1 (1:1000)27 and rat monoclonal (clone 3F10) anti-HA antibody (Cat. No. 11867431001, Lot-No. 62572200, Merck/Sigma-Aldrich, at 1:2500 dilution). Membranes were washed 4x with PBS and incubated for one hour with the secondary goat anti-Rabbit, Alexa Fluor Plus 800-coupled IgG (Cat. No. A32730, Lot-No. VL313935, Thermo Fisher Scientific) and the goat anti-Rat, Alexa Fluor 680-coupled secondary IgG (Cat. No. A-21096; Lot-No. 2409044, Thermo Fisher Scientific). Signals were scanned with the Li-Cor Odyssey Infrared Imager and processed with Image Studio version 5.2.

Electron microscopy sample preparation and data acquisition

Before plunge freezing, 6 µl of the concentrated flag elution (~8.2 mg/mL) was supplemented with 2 µl of 0.4% CHAPSO. Immediately afterwards, 5 µl of the resulting solution was applied to a freshly glow-discharged Quantifoil UltrAuFoil grid and incubated for 45 s at 4°C and 85% humidity. Excess liquid was manually blotted away for 11 s, and the grid was plunge frozen in liquid ethane using a manual plunge freezing device (Neptune fluid flow systems). Screening of cryo-EM conditions was done on a Talos F200C transmission electron microscope. High-resolution cryo-EM data were collected on a Titan Krios G3i transmission electron microscope (Thermo Fisher Scientific) operated at 300 kV equipped with a BioQuantum GIF post-column energy filter (Gatan) and a K3 direct electron detector (Gatan). 28,764 micrographs were recorded in counting mode at a pixel size of 0.834 Å. The defocus range was set between -0.8 and -2.8 µm. Each micrograph was dose-fractionated to 37 frames with a total exposure time of 1 s and a total electron dose of roughly 65 e-/Å2. Automated data collection was done using EPU (Thermo Fisher Scientific).

Cryo-EM image analysis

EM-data processing was done using cryoSPARC version 4.5.343. Movie and frame alignment and dose weighting were performed with MotionCor2, and contrast transfer functions were determined via CTFFIND444,45. All refinements used gold‑standard Fourier shell correlation (FSC) calculations, and reported resolutions are based on the FSC=0.143 criterion of mask‑corrected FSC curves. All refinement jobs were done without applying any symmetry. The processing strategy is visualized in Supplementary Fig. 2. In short, low‑quality micrographs with an estimated resolution of over 5 Å, an estimated defocus of -3.0 µm, or a relative ice thickness over 2.0 were discarded. Particle picking was done template-based using 2D templates generated from particles picked via cryoSPARCs blob‑picker. Low‑quality particles were then removed through three iterative rounds of 2D classification, followed by subset selection. Afterwards, three ab initio models were generated, and the particles were assigned to these models via heterogeneous refinement. The particle stack corresponding to the 13S‑13S+β5 dimer was further cleaned by 3D classification and reconstructed using non‑uniform refinement in cryoSPARC. Following heterogeneous refinement of the initial particle stack, the isolated early PCs and the 13S-15S dimer were separated by one round of 2D classification and subset selection. Particles representing the 13S‑15S dimer were used for ab initio reconstruction, followed by 3D reconstruction via non‑uniform refinement. The early PCs were separated by masked 3D classification and refined individually using masked non‑uniform refinement. Particle numbers, applied symmetry settings, and global resolutions of the reconstructed complexes are summarized in Table 1 and Supplementary Fig. 2.

Molecular modelling

All pdb-models were built using relevant chains (α1-7, β1-6, Ump1, Pba1 and Pba2) from PDB entry 8RVL19 as a starting model. The resulting initial pdb-models were fitted into the electron densities using UCSF ChimeraX46. The N‑terminal FLAG‑6xHis tag attached on Ump1 was manually build using COOT47 (Supplementary Fig. 13). The final pdb-models were built in iterative cycles of manual building with COOT and real-space refinement in PHENIX48,49. The data collection and model statistics are summarised in Table 1.

Data visualisation

Structure visualization and comparison was done using UCSF ChimeraX46. All contacts are calculated using UCSF ChimeraX with VDW overlap ≥ -0.4 Å, ignoring interactions between residues < 2 apart in sequence. Hydrogen bonding distances and angle criteria are relaxed by initial default settings (distance tolerance 0.4 Å, angle tolerance 20°) as recommended by ChimeraX.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript by Mark et al, several structures of proteasomal core particle subcomplexes are reported. Some of these\ncomplexes are known assembly intermediates, and some are self-reported by the authors to be purification/concentration\nartifacts or previously unresolved assembly products that represent alternative assembly pathways. Based on analyses of\nthese structures and a single biochemical experiment, the authors propose: i) multiple beta subunit assembly sequences\nexist in yeast and possibly humans; ii) that S3/S4 beta hairpin loops serve both to stabilize incoming beta subunits and to\norient residues in the catalytic triad for propeptide removal; and iii) a network of interactions that, when disrupted, result in\nthe release of Pba1-2 from the maturing CP.\nOverall, the structures are a helpful complement to a large number of structures already published containing various CP\nintermediates, and they reveal what are likely additional regulatory mechanisms that, if true, would expand our\nunderstanding of CP biogenesis. The manuscript is clearly written, and the figures are generally clear. However, there are\nsome concerns that the mechanisms proposed rely entirely on the structures, and are not validated via any secondary\nmechanisms (save for a sole biochemical experiment). Thus, the manuscript would be strengthened significantly if the\nevidence for the mechanisms proposed weren’t based entirely on structures.\nMajor comments:\n1. As mentioned above, the major limitation of the manuscript is that, with the exception of the novel interaction proposed for\nthe Alpha1 N-terminus, the authors largely assume that the functions and events inferred from their structures occur as they\nstate, without explicit validation (e.g., via mutagenesis or other experiments).\nA good example of this is that, even in the abstract, the authors explicitly claim that structures harboring distinct,\noverlapping subsets of beta subunits is “showing that alternative assembly pathways to the 15S-PC exist in yeast,” rather\nthan considering the possibility that they are partially disassembled species or off-pathway assembly products that resulted from\ndepletion of the catalytic amounts of Pba1-2 that occurs in response to Beta7 mutation (the authors state this occurs on pg. 4,\nlines 131-133). Although the authors cite some circumstantial evidence from previous work that is consistent with their\nobservations, those studies also required potentially damaging genetic perturbations or substantial manipulation of\nprecursors that could have induced subunit dissociation.\nProvision of some direct evidence that these are likely to be assembly intermediates rather than dissociation products is\nrequired to substantiate this claim. As an example for the alternative assembly pathways, can the authors demonstrate that\nBeta5/6 dissociates from the 15S-PC more readily than Beta1 upon increasingly harsh buffer conditions as would be\nexpected for such a pathway? Can the authors directly demonstrate addition of Beta5 and/or Beta6 to 13S-PCs containing\nBeta1, or demonstrate that Beta1 directly adds to the 13S-PC containing only Beta2-4?\n2. The repositioning of the N-terminus of Alpha1 to promote Pba1/2 dissociation is intriguing, but the mode of transmission\nproposed by the authors (Alpha1-\nSer8   Alpha3-Arg4/Arg5   Alpha4-Arg6   Pba1-Glu11) is unlikely, given that proteasomes lacking the N-terminal 10 residues\nof Alpha3 appear to assemble and mature normally. As more meaningful support for this model, disruption of the residues in\nthis proposed cascade aside from solely Scl1/Alpha1 should be assayed for an effect on Pba1/2 similar to that shown for\nAlpha1.\nMinor comments:\n1. In Figure 5F, a loading control showing that Pba2-HA is expressed in the (otherwise) WT strain should be included.\n2. Pg. 2, line 73 the authors used “und” instead of “and.”\n3. Pg. 3., line 109: “b-subunits” should be “β-subunits” to match the rest of the manuscript.\n4. Pg. 5, line 200: reference #21 appears to be in a different format that the rest of the manuscript.\n5. Pg. 5, lines 210 and 211: the appears to be a space missing between the word residues and the residue numbers.\n6. Pg. 8, line 336: hypothesize is misspelled.", "reviewer_2": "The manuscript by Mark et al. reports on the cryo-EM structure analysis of the yeast 15S-PC and several intermediates\nreflecting stepwise assembly of beta-subunit to the CP precursor complex 13S-PC. I found the work is structurally interesting\nto the proteasome field and bridges an important missing gap in understanding the biogenesis pathway of the proteasome\ncore particle. Nonetheless, there are a number of structural studies published in the past several years that have made\nnotable progress toward understanding the structural basis of the proteasome biogenesis, which overshadows the present\nmanuscript and makes it less appealing and more like an incremental advance. In addition, there remains a certain level of\nuncertainty or potential risk of over-interpretation in the present form, given the lack of additional biochemical tests. I think\nthis paper is a potentially good candidate for publication in Nature Communications, if the authors can address the following\nissues in a revision.\n(1) While the paper provides ample structural models, there is no data to either validate or support that the observed 13S to\n15S-PC intermediate complexes are indeed on the pathway of proteasome biogenesis. Instead, the authors took it for\ngranted that they have observed “all intermediates resulting from step wise addition of beta-subunits …”, without any\nadditional evidence from complementary methods like functional analysis, mutagenesis, and/or mass spectrometry etc.\nWhat if these complexes were from partially disassembled intermediate of 15S-PC in a poorly controlled, disfavored\ncondition, therefore representing off-pathway intermediates? What if these complexes can only survive in the in vitro\ncondition, but non-existential in vivo? Believing that they are the stepwise assembly intermediate is foremost a hypothesis\nor speculation if no other evidence can help consolidate their true functional identity. If the authors cannot fully justify\ntheir conclusion, they should instead turn down the tone and address necessary caveats and limitations of this study.\n(2) Another way to help justify the hypothesis of claiming that the solved structures are on-pathway assembly intermediates\nis to make thorough structural comparison of these structures with other CP assembly intermediates previously published,\nwhenever feasible. These comparative studies should be presented as part of main figures or supplementary figures to\nsupport the description in pages 4-5 and will at least help verify which part of the assembly is rationally expected or\ntemporally compatible with the existent structural models in the literature. If there are apparent incompatible features in the\ncomparison, the authors should seek complementary experiment to make necessary reconciliation.\n(3) The local EM densities of S3/S4 beta-hairpin loops and Pba1 loop that inserts into the alpha pocket must be presented in\neither main figures or supplementary figures to support the authors’ claim that they indeed solved these loops to high\nenough resolution that allowed their accurate atomic modeling and conclusion. Similarly, the local EM densities of the N-\nterminal of alpha1 must be presented for visual assessment to support their atomic and functional interpretation. More\ngenerally, any “previously unrecognized details” must be supported or justified by clear presentations of high-resolution\nEM densities in either main figures or supplementary figures.\n(4) In Table S1, why is the percentage of disallowed residues all above 90%? This is either done wrong, or the table\ncontains typo.\n(5) The introduction could be improved in the clarification of what CP assembly intermediates have been structurally solved,\nwhat is yet to be solved, and why the present study is important. The discussion section may also be improved with a\nsummary on the key differences between the newly solved structures and the previously published CP assembly\nintermediate structures.\n(6) It would be very helpful to add a new main figure to summarize the main findings on the assembly mechanism. The\nlogic flow of the paper is a little bit brittle and lacks focus of a central story. This may be improved with better mechanistic\ndiscussion and conclusion paragraphs at the end of the paper.\nMinor issues:\n(1) Please present gel filtration or size-exclusion chromatographic results of the purified complexes in supplementary\nfigures.\n(2) There are numerous places (e.g. line 70, 73, 74, 109, 114, etc.) where Greek alpha is written as “a”, and beta is written as\n“b”.\n(3) Table S1 may have to be moved to main text as a main Table."}, "author_response": "Point-by-Point Response to Reviewer Comments\nWe sincerely thank both reviewers for their insightful, constructive and appreciative feedback, which has served as a\nvaluable catalyst for additional experiments and significant revisions to our manuscript. We believe that the resulting\nimprovements have substantially strengthened the clarity and overall quality of the manuscript.\nReviewer #1\nIn this manuscript by Mark et al, several structures of proteasomal core particle subcomplexes are reported. Some of these\ncomplexes are known assembly intermediates, and some are self-reported by the authors to be purification/concentration\nartifacts or previously unresolved assembly products that represent alternative assembly pathways. Based on analyses of\nthese structures and a single biochemical experiment, the authors propose: i) multiple beta subunit assembly sequences\nexist in yeast and possibly humans; ii) that S3/S4 beta hairpin loops serve both to stabilize incoming beta subunits and to\norient residues in the catalytic triad for propeptide removal; and iii) a network of interactions that, when disrupted, result\nin the release of Pba1-2 from the maturing CP.\nOverall, the structures are a helpful complement to a large number of structures already published containing various CP\nintermediates, and they reveal what are likely additional regulatory mechanisms that, if true, would expand our\nunderstanding of CP biogenesis. The manuscript is clearly written, and the figures are generally clear. However, there are\nsome concerns that the mechanisms proposed rely entirely on the structures, and are not validated via any secondary\nmechanisms (save for a sole biochemical experiment). Thus, the manuscript would be strengthened significantly if the\nevidence for the mechanisms proposed weren’t based entirely on structures.\nMajor comments:\n1. As mentioned above, the major limitation of the manuscript is that, with the exception of the novel interaction proposed\nfor the Alpha1 N-terminus, the authors largely assume that the functions and events inferred from their structures occur as\nthey state, without explicit validation (e.g., via mutagenesis or other experiments).\nA good example of this is that, even in the abstract, the authors explicitly claim that structures harboring distinct,\noverlapping subsets of beta subunits is “showing that alternative assembly pathways to the 15S-PC exist in yeast,” rather\nthan considering the possibility that they are partially disassembled species or off-pathway assembly products that\nresulted from depletion of the catalytic amounts of Pba1-2 that occurs in response to Beta7 mutation (the authors state\nthis occurs on pg. 4, lines 131-133). Although the authors cite some circumstantial evidence from previous work that is\nconsistent with their observations, those studies also required potentially damaging genetic perturbations or substantial\nmanipulation of precursors that could have induced subunit dissociation.\nProvision of some direct evidence that these are likely to be assembly intermediates rather than dissociation products is\nrequired to substantiate this claim. As an example for the alternative assembly pathways, can the authors demonstrate\nthat Beta5/6 dissociates from the 15S-PC more readily than Beta1 upon increasingly harsh buffer conditions as would be\nexpected for such a pathway? Can the authors directly demonstrate addition of Beta5 and/or Beta6 to 13S-PCs containing\nBeta1, or demonstrate that Beta1 directly adds to the 13S-PC containing only Beta2-4?\nOur Response: We thank the reviewer for the overall positive evaluation of our work. We acknowledge the reviewer's\nreasonable concern that some of the precursor complexes structurally characterized in this study may not constitute\nphysiologically relevant precursors in the biogenesis pathway leading to the mature 20S proteasome. Because it is\ntechnically next to impossible to biochemically follow the 20S assembly in real time in vivo in wild-type cells, we have\nopted to accumulate 13S-15S precursors by slowing down downstream dimerisation of 15S PCs using a strain lacking C-\nterminal residues of the β7 subunit. We chose this mutation because the β7 subunit itself is not part of these complexes.\nThe impaired 15S-PC dimerization kinetics resulting from this mutation lead to a depletion of the Pba1-Pba2 chaperone\ndue to a reduced efficiency of its recycling, which occurs only upon final maturation of the proteasome. This problem was\naddressed by the simultaneous overexpression of both subunits of this chaperone. As intended, our isolated pool of\nproteasomal precursors is devoid of precursor complexes lacking Pba1-Pba2. To our knowledge, our study is unique in\nisolating native yeast 13S-15S complexes bearing no mutations and with sufficient chaperones present to prevent off-\npathway assemblies. In principle, however, a general limitation of purification protocols is the inherent risk of disrupting\nfragile protein complexes. As the reviewer posits, the emergence of the unique 13S+β1-PC species from later-stage PC\nassemblies might occur by a non-physiological dissociation of the β5 or β5/β6 subunits from those complexes. The isolated\nproteasomal pool contains as much 13S+β5-PC as all subsequent PCs combined, while 13S+β1-PC is underrepresented. We\ntherefore suspect that β5 binds better to PC or is more readily available than β1. Hence a scenario where β5 or β5/β6\nsubunits dissociate non-physiologically seems unlikely, as it would require the selective dissociation of β5, but not β1 from\nonly a small subset of the PC intermediates. Still, since both reviewers expressed similar concerns about our conclusions\nregarding the alternative assembly pathway starting with β1 addition to 13S-PCs, we addressed these concerns\nexperimentally using an in vivo approach. Specifically, we created a strain that allowed to selectively shut down expression\nof the β5 subunit in a strain with a β1 subunits carrying an N-terminal FLAG tag appended to its propeptide. To verify\nefficient shut down of β5, this subunit carried a C-terminal 2xHA tag to enable detection of the subunit with a high\nsensitivity. The N-terminal FLAG tag on β1 subunits allowed us to pull down PCs that had incorporated this subunit. As\nshown in the new figure 2B, in contrast to an untagged control, FLAG-tagged β1 pulled down 13S-PCs in the absence of\neither β5 or β6. These findings clearly establish that β1 has the propensity to assemble with 13S-PCs without prior addition\nof β5 and/or β6 and therefore suggest that the observed complexes containing β1 but not β5 and/or β6 are likely assembly\nproducts and not the result of complexes resulting from dissociation of β5 and/or β6. These in vivo data are therefore in\nline with our conclusion based upon our isolation and structural characterization of 13S+β1-PCs, and thus provide further\nsupport for an alternative assembly pathway, in which β1 is assembling with 13S-PCs before the addition of β5 and β6.\nThe observation that all intermediates of this alternative pathway were identified in our structural analysis (and supported by\nthe above-mentioned genetic approach), furthermore suggests that these complexes are not results of non-productive off-\npathways. While the above-mentioned biochemical and structural evidence supports the feasibility of an alternative\nassembly route, the observed relatively larger amounts of 13S+β5-PC compared to 13S+β1-PC suggest that β5 more often\nis the first subunit to assemble with 13S-PCs. We have adjusted our wording of this issue in the abstract, main text and\ndiscussion, and adjusted the figure 2A to highlight (by a stronger arrow) that this pathway has a higher prevalence than the\nalternative pathway, in which β1 appears to be the first subunit to join the 13S-PC.\n2. The repositioning of the N-terminus of Alpha1 to promote Pba1/2 dissociation is intriguing, but the mode of\ntransmission proposed by the authors (Alpha1-Ser8 à Alpha3-Arg4/Arg5 à Alpha4-Arg6 à Pba1-Glu11) is unlikely, given that\nproteasomes lacking the N-terminal 10 residues of Alpha3 appear to assemble and mature normally. As more meaningful\nsupport for this model, disruption of the residues in this proposed cascade aside from solely Scl1/Alpha1 should be assayed\nfor an effect on Pba1/2 similar to that shown for Alpha1.\nOur Response: We thank the reviewer for the critical feedback on this part of our study, which prompted us to perform\nadditional experiments to scrutinize and further develop our mechanistic explanation for the results obtained with N-\nterminally truncated α1. Our structural analysis suggests that the N-terminus of α1, which is in bonding distance to the\nloop segment aa121- 138 of Pba2 and the N-termini of α2 and α3, prevents the latter from prematurely closing the central\npore in the α-ring such that the Pba1-N-terminus is trapped. To test this interpretation, we have created and analyzed a set\nof strains, in which the N-termini of α1 and α3 were truncated either alone, or in combination. Consistent with the\nreviewer’s notion, truncation of the α3 N-terminus, in contrast to truncation of α1, had no effect on the release of Pba1-\nPba2. Strikingly, truncation of the α3 N-terminus abrogated the defect of Pba1-Pba2 release caused by the α1 truncation.\nThese data, shown in the new figure 5F, support our interpretation that the N-terminus of α1 has a critical role in\nmaintaining the arrangement of α-subunit N-termini in the assembling core particle in a way compatible with Pba1-Pba2\nrelease by keeping the α3 N-terminus in an open conformation as long as the Pba1 N-terminus is located in the α-ring pore.\nPlease also see our response to reviewer 2 (1).\nMinor comments:\n1. In Figure 5F, a loading control showing that Pba2-HA is expressed in the (otherwise) WT strain should be included.\nOur Response: The new figure 5F includes a lane showing that Pba2-HA is only detectable in 13S-15S precursors in wild-\ntype cells, but not in late-PCs as is observed in strains with N-terminally truncated α1.\n2. Pg. 2, line 73 the authors used “und” instead of “and.”\n3. Pg. 3., line 109: “b-subunits” should be “β-subunits” to match the rest of the manuscript.\n4. Pg. 5, line 200: reference #21 appears to be in a different format that the rest of the manuscript.\n5. Pg. 5, lines 210 and 211: the appears to be a space missing between the word residues and the residue numbers.\n6. Pg. 8, line 336: hypothesize is misspelled.\nOur Response: Thank you for pointing out these typos and formatting issues, which we now have fixed.\nReviewer #2:\nThe manuscript by Mark et al. reports on the cryo-EM structure analysis of the yeast 15S-PC and several intermediates\nreflecting stepwise assembly of beta-subunit to the CP precursor complex 13S-PC. I found the work is structurally\ninteresting to the proteasome field and bridges an important missing gap in understanding the biogenesis pathway of the\nproteasome core particle. Nonetheless, there are a number of structural studies published in the past several years that\nhave made notable progress toward understanding the structural basis of the proteasome biogenesis, which overshadows\nthe present manuscript and makes it less appealing and more like an incremental advance. In addition, there remains a\ncertain level of uncertainty or potential risk of over-interpretation in the present form, given the lack of additional\nbiochemical tests. I think this paper is a potentially good candidate for publication in Nature Communications, if the\nauthors can address the following issues in a revision.\n(1) While the paper provides ample structural models, there is no data to either validate or support that the observed 13S\nto 15S-PC intermediate complexes are indeed on the pathway of proteasome biogenesis. Instead, the authors took it for\ngranted that they have observed “all intermediates resulting from step wise addition of beta-subunits …”, without any\nadditional evidence from complementary methods like functional analysis, mutagenesis, and/or mass spectrometry etc.\nWhat if these complexes were from partially disassembled intermediate of 15S-PC in a poorly controlled, disfavored\ncondition, therefore representing off-pathway intermediates? What if these complexes can only survive in the in vitro\ncondition, but non-existential in vivo? Believing that they are the stepwise assembly intermediate is foremost a hypothesis\nor speculation if no other evidence can help consolidate their true functional identity. If the authors cannot fully justify\ntheir conclusion, they should instead turn down the tone and address necessary caveats and limitations of this study.\nOur Response: We thank the reviewer for considering our work as structurally interesting to the proteasome field and\nbridging an important missing gap in understanding the biogenesis pathway of the proteasome core particle. We\nacknowledge the reviewer's reasonable concern regarding the lack of biochemical validation of our interpretations and\nwould like to refer to our answer to major comment 1 of reviewer 1. As mentioned above, our new biochemical data show\nthat complexes that contain β1 but lack β5 and/or β6 are not just the result of in vitro disassembly of later intermediates,\nbut can form in vivo in cells lacking either β5 or β6 due to an expression shutdown (new Figures 2B and Extended figure 3).\nThese findings therefore underscore the ability of β1 to assemble with 13S-PCs early on, before β5 and β6 have been\nincorporated. As discussed in some detail in the response to reviewer 1, while there is evidence that an early incorporation\nof β5 is likely to be the prominent route of assembly, our findings suggest that there is some flexibility in the system.\nAs we tried to better highlight in the revised manuscript, we strongly believe that our finding, aside from providing more\ncomplete and higher resolution structures of 13-15S PCs than have been previously available (at least for yeast PCs) indeed\nbridge a number of important gaps in our understanding of CP assembly. Specifically, by structurally resolving four\nintermediates on the way from yeast 13S to the 15S-PC (half-CP without β7), that have not been available before (yeast\n13S+β5-PC, 13S+β1-PC, 13S+β1+β5-PC,15S-PC) we were able to monitor how multiple interactions of the Ump1 chaperone\nwith incoming β-subunits not only contribute to their incorporation into the nascent complex, but also how these\ninteractions help to gradually shape Ump1’s final donut-like structure as it is highlighted in Figure 4A. Our structural\nresolution of these intermediates, which are free of mutations, allow to follow the stepwise restructuring of β-hairpin\nloops that prepare the complexes for the later CP activation by autocatalytic processing of β-subunit propeptides. We\nidentify a previously overlooked loop of Pba1 that intercalates between subunits α3 and α4. Furthermore, our structures of\nearly assembly intermediates point to a role of the α1 N-terminus in signalling CP maturation to Pba1-Pba2 release.\n(2) Another way to help justify the hypothesis of claiming that the solved structures are on-pathway assembly\nintermediates is to make thorough structural comparison of these structures with other CP assembly intermediates\npreviously published, whenever feasible. These comparative studies should be presented as part of main figures or\nsupplementary figures to support the description in pages 4-5 and will at least help verify which part of the assembly is\nrationally expected or temporally compatible with the existent structural models in the literature. If there are apparent\nincompatible features in the comparison, the authors should seek complementary experiment to make necessary\nreconciliation.\nOur Response: We have now added a new supplemental table S1 to the manuscript summarizing already published\nstructures of β-ring assembly intermediates found in human and yeast cells (Schnell et al., 2021; Adolf et al., 2024; Zhang\net al., 2024; Han et al., 2025). Apart from the 13S-PC identified by all groups and the 13S+β5+β6-PC identified by Schnell et\nal. and Adolf et al., we also resolved the 13S+β5-PC, 13S+β1-PC, 13S+β1+β5-PC, as well as the 15S-PC. Yeast complexes\nelucidated by several groups are all practically identical at c-alpha level, as suggested by the RMSDs presented in the\nmanuscript (line 127 and line 147). We cannot identify any incompatible features.\n(3) The local EM densities of S3/S4 beta-hairpin loops and Pba1 loop that inserts into the alpha pocket must be presented\nin either main figures or supplementary figures to support the authors’ claim that they indeed solved these loops to high\nenough resolution that allowed their accurate atomic modeling and conclusion. Similarly, the local EM densities of the N-\nterminal of alpha1 must be presented for visual assessment to support their atomic and functional interpretation. More\ngenerally, any “previously unrecognized details” must be supported or justified by clear presentations of high-resolution\nEM densities in either main figures or supplementary figures.\nOur Response: Addressing the reviewers request for visual evidence, we have incorporated local EM densities into our\nfigures: the S3/S4- β-hairpin loops are shown in Figure S4, the Pba1 loop in Extended Figure 5C, and the N-terminus of α1\nin Figure S7.\n(4) In Table S1, why is the percentage of disallowed residues all above 90%? This is either done wrong, or the table contains\ntypo.\nOur Response: This was a typo. Thank you for pointing it out.\n(5) The introduction could be improved in the clarification of what CP assembly intermediates have been structurally\nsolved, what is yet to be solved, and why the present study is important. The discussion section may also be improved with\na summary on the key differences between the newly solved structures and the previously published CP assembly\nintermediate structures.\nOur Response: We revised the introduction to add more clarity with regard to already published PC structures. Specifically,\nwe now mention all PC structures that overlap with our study (lines 58-60 and 74-78), cite further relevant publications and\nhighlight the novelty of the structures presented here (lines 96-98). As mentioned above, we also included a new table\n(Table S1) enabling comparison of overlaps and novelties between already published PCs and structures presented in our\nmanuscript. The C-alpha RMSD values between our structures and already published PCs from yeast are given in the\nmanuscript (lines 127 and line 147). We revised the discussion section according to the reviewer’s suggestion to highlight\nour main novel findings described in our paper, which is supported by the new figure 6 (as outlined below in our response\nto item 6).\n(6) It would be very helpful to add a new main figure to summarize the main findings on the assembly mechanism. The\nlogic flow of the paper is a little bit brittle and lacks focus of a central story. This may be improved with better mechanistic\ndiscussion and conclusion paragraphs at the end of the paper.\nOur Response: We added a new figure (Figure 6) to provide a conceptual summary of the key findings of our study,\nhighlighting the alternative assembly pathways, the gradual folding of Ump1, and the importance of the α1 N-terminus for\nPba1-Pba2 release, which is associated with loosening of the so far uncharacterised loop in Pba1 (residues 81-117)\nintercalated between α3 and α4. This figure includes, as indicated in the legend, six structures of yeast 13-15S-PCs, four of\nwhich have not been described before, including the first structure of a 15S-PC (half-CP without β7; resolution of 3.1A). In\ndetail the figure summarizes 1) that, aside from the previously reported importance of the Pba1 and Pba2 C-termini and\nthe Pba1 N-terminus, we identified a loop in Pba1 (residues 81-117) as a structural feature that is transiently intercalating\nbetween α3 and α4. 2) Furthermore, we discovered a role of the N-terminus of α1 in the control of Pba1-Pba2 release. 3)\nWe observe that β1 has the capacity to be incorporated before β5 and β6, even though the quantity of observed\ncomplexes suggests that β5 is more often the first subunit to be added to 13S-PCs, as is emphasized by the stronger arrows\nin the bottom pathway. 4) and 5) finally, as indicated by the overarching arrow at the bottom, we characterized the\nstepwise structural maturation of Ump1 and of the S3/S4 β-hairpin loops in the β-subunits, which prepare for final\nmaturation upon β7-driven PC dimerization.\nMinor issues:\n(1) Please present gel filtration or size-exclusion chromatographic results of the purified complexes in supplementary\nfigures.\nOur Response: We are unable to provide gel filtration data due to the limitations imposed by extensive sample dilution,\nwhich reduces protein concentration below the threshold for reliable detection. Instead we have utilized native gel\nelectrophoresis (Figure S1) as a more sensitive and less disruptive method for analyzing the assembly intermediates.\n(2) There are numerous places (e.g. line 70, 73, 74, 109, 114, etc.) where Greek alpha is written as “a”, and beta is written\nas “b”.\nOur Response: Thank you for pointing out these inconsistencies. We corrected the typos.\n(3) Table S1 may have to be moved to main text as a main Table.\nOur Response: We have moved table S1 to the main section (now named Table 1)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "In this revised manuscript, Mark et al. included additional data to address a number of concerns raised in the initial round of\nreview, with the foremost being that the Beta1-containing 13S precursor complex was not convincingly shown to be an \"on-\npathway\" assembly product, rather than an artifactual dissociation product or an artifact of limiting amounts of assembly\nchaperones that might result in altered subunit compositions. The authors have addressed this by overproducing Pba1-2 to\nprevent their depletion, and have demonstrated the ability to detect Beta1 in yeast cells expressing vanishingly low levels of\nBeta5 or Beta6, which have traditionally been considered to incorporate prior to Beta1. Although this data does not\nunequivocally prove a Beta1-first assembly mechanism, it does help to further substantiate this possibility and reduce the\nlikelihood of alternative explanations. Text changes to the abstract and main article have softened this claim to a level this\nReviewer believes is appropriate.\nSome minor edits are noted below:\n1. pg. 2, line 57: the term ortholog might be a better word choice than alias for describing the Ump1 ortholog in humans.\n2. Pg. 4, line 148: the word \"too\" should be \"to\".", "reviewer_2": "The revised manuscript has sufficiently addressed all my questions. I recommend its publication in the present form."}, "author_response": "Point-by-Point Response to Reviewer Comments\nReviewer #1 (Remarks to the Author):\nIn this revised manuscript, Mark et al. included additional data to address a number of concerns\nraised in the initial round of review, with the foremost being that the Beta1-containing 13S\nprecursor complex was not convincingly shown to be an \"on-pathway\" assembly product, rather\nthan an artifactual dissociation product or an artifact of limiting amounts of assembly chaperones\nthat might result in altered subunit compositions. The authors have addressed this by overproducing\nPba1-2 to prevent their depletion, and have demonstrated the ability to detect Beta1 in yeast cells\nexpressing vanishingly low levels of Beta5 or Beta6, which have traditionally been considered to\nincorporate prior to Beta1. Although this data does not unequivocally prove a Beta1-first assembly\nmechanism, it does help to further substantiate this possibility and reduce the likelihood of\nalternative explanations. Text changes to the abstract and main article have softened this claim to a\nlevel this Reviewer believes is appropriate.\nSome minor edits are noted below:\n1. pg. 2, line 57: the term ortholog might be a better word choice than alias for describing the Ump1\northolog in humans.\n2. Pg. 4, line 148: the word \"too\" should be \"to\".\nOur response:\nWe thank the reviewer for the positive assessment of our revised manuscript. Thank you for pointing\nout the editorial improvements, which we have made as suggested.\nReviewer #2 (Remarks to the Author):\nThe revised manuscript has sufficiently addressed all my questions. I recommend its publication in\nthe present form."}]

Decision letter