Regulated protein turnover: snapshots of the proteasome in action.

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Regulated protein turnover: snapshots of the proteasome in action Sucharita Bhattacharyya1, Houqing Yu1,2, Carsten Mim1 and Andreas Matouschek1,2

Abstract | The ubiquitin proteasome system (UPS) is the main ATP-dependent protein degradation pathway in the cytosol and nucleus of eukaryotic cells. At its centre is the 26S proteasome, which degrades regulatory proteins and misfolded or damaged proteins. In a major breakthrough, several groups have determined high-resolution structures of the entire 26S proteasome particle in different nucleotide conditions and with and without substrate using cryo-electron microscopy combined with other techniques. These structures provide some surprising insights into the functional mechanism of the proteasome and will give invaluable guidance for genetic and biochemical studies of this key regulatory system.

Department of Molecular Biosciences, Northwestern University, Evanston, Illinois 60208, USA. 2 Department of Chemistry and Biochemistry, The University of Texas at Austin, Austin, Texas 78712, USA. Correspondence to A.M. e-mail: matouschek@austin. utexas.edu doi:10.1038/nrm3741 1

The concentrations of proteins in the cell are set by their rates of synthesis and degradation1. Since the discovery of a eukaryotic intracellular protein degradation pathway in the 1970s, it has become increasingly clear that degradation is regulated intricately, even if this regulation is not as well understood as that of gene expression2–6. Most of this degradation is controlled by the ubiquitin proteasome system (UPS) in the cytosol and nucleus of eukaryotic cells6. At its centre is a proteolytic machine, the proteasome, which is almost as large as the ribosome with its molecular weight of 2.5 MDa. The proteasome is able to degrade any protein in the cell and does so with high specificity. The proteasome can even extract individual subunits out of complexes, leaving the rest of the complex intact 7–10. Specificity is achieved in part by a tagging system that uses the 8.5 kDa protein ubiquitin as a post-translational modifier 11. Proteins are typically targeted to the proteasome by chains of at least four ubiquitins, in which the first ubiquitin is attached to a Lys residue in a target protein, the second ubiquitin is attached to a specific Lys in the first ubiquitin, and so on. Ubiquitin tags are also used to control other cellular events such as membrane trafficking and various steps in signalling cascades11–16. Indeed, one of the first-discovered functions of ubiquitin was as a chromatin modifier attached to histone tails17,18. For the nonproteolytic processes that use ubiquitin signals, the tags consist of a single ubiquitin or chains in which the ubiquitin moieties are linked to each other through different Lys residues from those used for proteolysis11,19. In vitro degradation experiments indicate that ubiquitin tags alone are not enough to target folded proteins for proteolysis, and proteasome substrates must also contain an

unstructured region at which the proteasome can initiate degradation20. As degradation begins, the proteasome runs along the polypeptide chain of the substrate, hydrolysing ATP to ADP and cutting the substrate protein sequentially 21 into small peptides22,23, while the ubiquitin tag is cleaved off and recycled24,25 (FIG. 1). This enables the proteasome to remodel protein complexes by degrading only the subunit at which it first initiates degradation7,9,10,26. It also ensures that the activity of the target protein is completely removed. Many regulatory proteins are composed of domains or modules, and leaving one of these undegraded could create an unwanted activity. Nonetheless, there are a handful of cases in which the proteasome sculpts proteins by degrading them partially: for example, to remove an inhibitory domain or an activating domain in response to a signal27–31. Thus, the proteasome is a powerful protease, the destructive potential of which is finely tuned. In this Review, we highlight recent advances in understanding the structure of the proteasome in terms of how it is arranged as a network of subunit interactions, the resulting implications for substrate recognition and its mechanism of action as an ATP-dependent machine.

A complex of two halves The proteolytic core particle. The proteasome is a complex of ~33 different proteins that are arranged in an elongated particle composed of a central core with cap structures at one or both ends. At the centre is the ~700 kDa core particle, sometimes referred to as the 20S proteasome, which is made up of 28 subunits and contains the proteolytic active sites. By the early 2000s,

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19S regulatory particle

Polyubiquitin chain Substrate unstructured region

Rpn13

Deubiquitylation Rpn10

Ub Ub Ub

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Ub Rpn8– Rpn11

Rpn1 Substrate recruitment

Engagement

Unfolding and translocation Proteolysis

ATPase ring 20S core particle

Figure 1 | Steps of proteasomal degradation. The proteasome recognizes ubiquitin tags in substrates through its Naturedegradation Reviews | Molecular Cell Biology receptors (here regulatory particle non-ATPase 10 (Rpn10) and Rpn13) and then initiates at an unstructured region in the substrate. As the ATPase motors pull the substrate into the degradation channel, the ubiquitin chain is cleaved off, the substrate unfolds and is finally cleaved into peptides.

atomic-resolution crystal structures of the 20S proteasome from several different sources, including yeast and mammals, had been solved32–34. These structures showed that the subunits of the core particle are arranged into four seven-subunit rings: two rings consisting of related α-subunits and two consisting of related β-subunits. The rings are stacked on top of each other to form a compact cylinder with two β-subunit rings in the centre that are topped at each end by a ring of α-subunits. The particle is hollow and contains a set of chambers running along its central axis that are accessible only through a narrow pore at each end of the core particle32–35. The proteolytic sites of the proteasome are located on three of the β-subunits in the central cavity, resulting in a total of six proteolytic sites. They show relatively weak preferences for specific target amino acids, and together they can cleave almost any sequence22,23,36. These proteolytic sites pose no danger for cellular proteins, as the pores at the entrance to the core particle are so narrow that folded proteins cannot pass through them without being unfolded32,33,35. Therefore, a major determinant of the specificity of the proteasome lies in its quaternary structure: the proteolytic activity is sequestered and access to it is sterically controlled. This strategy of controlling catalytic activity by restricting access to reactive sites is similar to the approach used by subcellular compartments such as mitochondria, except that the proteasome forms the compartment itself from a protein shell37.

and enable largely unstructured proteins or peptides to pass through the degradation channel6. The 19S regulatory particle, by contrast, recognizes ubiquitin tags and hydrolyses ATP to unfold its substrates and translocate them into the core particle. Most proteasomes in yeast are thought to contain one or two 19S regulatory particles in combination with a 20S core particle, and this form is referred to as the 26S proteasome6. Hybrid proteasomes that comprise a 19S regulatory particle on one end of the core particle and a different activator at the opposite end also exist, but it is not yet understood how the different caps act together 6,43–46. Recently, it was discovered that another form of the proteasome might exist in cells, in which the AAA+ protein CDC48 (also known as p97 or VCP) serves as an additional ATPase cap47,48. The 11S cap was the first to be understood structurally through a high-resolution crystal structure of a yeast core particle bound to an 11S cap from Trypanosoma brucei 6,38,41. The 11S cap consists of a ring of seven identical subunits, which dock onto the core particle through their carboxyl termini. Activation loops, from each of the 11S cap subunits, insert into cavities that exist between the α-subunits of the core particle and induce rearrangements in the α-subunits to open the central pore38,49. It is known from biochemical experiments that the 19S regulatory particle docks onto the core particle by a similar mechanism50–52.

Controlled access through regulators. The pores at the entrance to the degradation channel on the core particle are gated. They are generally closed in the free core particle and can open after activators or caps dock to the core particle6,35,38–41. There are three or perhaps four different types of activators6,39,42. The 11S cap (proteasome activator 28 (PA28; also known as REG) in mammals) and bleomycin-sensitive 10 cap (Blm10; also known as PA200 in humans) stimulate protein degradation without ATP hydrolysis and without recognizing ubiquitin6. Presumably, they have relatively specialized functions

The 19S regulatory particle. The 19S regulatory particle is an ~900 kDa complex of at least 19 subunits and two catalytic activities: ATP hydrolysis and a specialized proteolytic cleavage that removes ubiquitin chains from substrate proteins. Biochemical experiments have organized the regulatory particle into two subcomplexes, the ‘base’ and the ‘lid’53. The base consists of six ATPases (regulatory particle triple A protein 1 (Rpt1)–6), two large organizing subunits (regulatory particle nonATPase 1 (Rpn1) and Rpn2) and two established ubiquitin receptors (Rpn10 and Rpn13). The lid consists

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REVIEWS of the deubiquitylating enzyme Rpn11, which is a JAB1– MPN–MOV34 (JAMM; also known as MPN) domain metalloproteinase, its homologous but non-catalytic binding partner Rpn8 and seven scaffolding subunits (Rpn3, Rpn5, Rpn6, Rpn7, Rpn9, Rpn12 and Rpn15 (also called Sem1))6. The scaffolding subunits, except for Rpn15, contain protein–protein interaction motifs called PCI (proteasome–CSN (COP9 signalo some)–eIF3 (eukaryotic translation initiation factor 3)) domains6. In addition to the stoichiometric proteasome sub units, a large number of proteins associate with the 19S regulatory particle to modulate degradation. Some of these, such as the UBL (ubiquitin-like) and UBA (ubiquitin-associated) domain-containing proteins Rad23 (radiation sensitive 23), Dsk2 (dominant suppressor of Kar2) and Ddi1 (DNA damage-inducible 1), serve as alternative ubiquitin receptors54–62. Others, such as Ubp6 (ubiquitin-specific protease 6) in yeast (also known as USP14 (ubiquitin-specific protease 14) in mammals) and UCH37 (ubiquitin C-terminal hydrolase 37) in mammals, trim ubiquitin chains, whereas proteins such as yeast Hul5 (HECT ubiquitin ligase 5) extend the ubiquitin chains of substrates on the proteasome63–69. New structural understanding. Images of the eukaryotic 26S proteasome in its entirety came from electron micro scopy studies, often combined with mass spectrometry and biochemical methods such as protein–protein crosslinking 53,70,71, and by 2010 the first subnanometreresolution structure of the complete 26S proteasome emerged72. These structures started to place individual subunits within the particle, but the locations of many of them, including the substrate receptors, remained ambiguous. Meanwhile, atomic-resolution structures of homologous ATPase subunits from archaeal proteasomes provided insights into how substrates are likely to be translocated by the proteasomal ATPases73–75. Ultimately, the substrate receptors were localized within the eukaryotic 26S proteasome structure76. It was only in the past 2 years that a series of papers from several groups revealed the structure of the 19S regulatory particle and of the entire yeast and human 26S proteasome at almost atomic resolution77–82. The initial set of reports presented proteasome structures that were prepared in the presence of saturating ATP levels and without substrate77–80. The more recent studies show proteasome structures in the presence of a substrate81 or with a slowly hydrolysable ATP analogue (ATPγS)82. These studies combined high-resolution electron microscopy structures of the entire particle with crystal structures and homology models of isolated subunits, and a wealth of biochemical, proteomic and genetic data to determine the structure of the 26S proteasome83,84. Together, these approaches place all of the known stoichiometric subunits within the particle and in many cases orient their crystal structures well enough to interpret information at atomic resolution in meaningful ways77–80,85. The substrate-free proteasome structures from the different organisms mostly agree with one another, which highlights that the proteasome structure is largely conserved from yeast to mammals77–80. The substrate- and ATPγS-bound proteasome

structures show some intriguing differences compared with the substrate-free ATP structures. The two sets of structures might represent different states of the proteasome, perhaps a resting state for the substrate-free ATP structure and activated states for the substrate-bound and ATPγS structures81,82.

A fresh view of regulation The initial models of the proteasome showed that the base of the regulatory particle docked onto the core particle through a ring of ATPase subunits. The base was adjacent to a seemingly more loosely attached lid53,70,71. The first surprise from the new structures is that they show the regulatory particle as a cohesive structure with a network of interactions within the cap and with the core particle to form an integrated 26S proteasome77–80. The six ATPases of the base, Rpt1–6, form a ring that docks onto the core particle, as was observed in the earlier structures, and the subunits are arranged in the order determined in biochemical studies86. Each ATPase subunit is composed of extended α‑helices near the amino terminus, followed by an oligonucleotide- and oligosaccharide-binding (OB) domain and AAA+ domains before the C terminus73,75. The AAA+ domains and the OB domains each form rings with central pores77–80. The AAA+ domains contain the elements that are required to bind and hydrolyse ATP and to dock onto the core particle50–52,87. The C‑terminal tails of three of the AAA+ domains insert into cavities at the surface of the core particle to open the pore to the degradation channel; this gates the passage to the proteolytic sites, reminiscent of the 11S cap structure with the core particle50–52. On the opposite side of the AAA+ domains, the OB domain ring resides next to the AAA+ ring but does not pack tightly against it 77–80. Finally, the N‑terminal α‑helices form coiled-coils with neighbouring ATPase subunits, such that three pairs of α‑helical coils protrude from the base towards the outer ends of the proteasome cylinder. Thus, the ATPase subunits form an assembly that reaches over 100 Å from the core particle through about two-thirds of the regulatory particle and participate in a network of interactions that might enable communication between substrate receptors and the ATPase motor. The organizing subunit Rpn1 binds to one outer side of the ATPase ring near the core particle, and Rpn2 stacks on the top of the ATPases at the distal end of the proteasome. The ubiquitin receptors Rpn10 and Rpn13 are located at the periphery of the proteasome particle and near its distal end, where they seem to be appropriately positioned to capture substrates77–80. Rpn13 binds to Rpn2 and is almost as far away from the centre of the proteasome as possible. Rpn10 binds near Rpn2 to the JAMM domain protein Rpn11, about a quarter turn away from Rpn13 and slightly closer to the ATPase ring 77–80. Therefore, the substrate-binding and -processing activities are lined up along the long axis of the proteasome, in the order in which they act on a substrate on the way to the proteolytic sites88. The lid binds to the side of the base and forms a wide network of interactions through a large surface, almost like a hand holding onto the side of the particle (FIG. 2).



REVIEWS Substrate receptor Rpn13 Rpn2 Deubiquitylating enzyme Rpn11 and non-catalytic partner Rpn8

Substrate receptor Rpn10

ATPase subunits PCI domain-containing subunits

Core particle

Figure 2 | A scaffold of PCI domain-containing subunits integrates regulatory Nature Reviews | MolecularinCell particle subunits and the core particle. The structure of the proteasome the Biology presence of ATP, but without substrate, shows that the six PCI (proteasome–CSN (COP9 signalosome)–eIF3 (eukaryotic translation initiation factor 3)) domain-containing subunits (shown in yellow) form a U‑shaped structure that interacts with many subunits in the regulatory particle, and reveals a ring of α‑subunits in the core particle (shown in dark grey). These interactions probably stabilize the regulatory particle and its interaction with the proteolytic core. They might also provide a mechanism for cooperative interaction of the different biochemical activities of the various subunits. The six ATPase subunits are shown in blue, the ubiquitin receptors Rpn10 (regulatory particle non-ATPase 10) and Rpn13 are shown in red, the organizing subunit Rpn2 is shown in orange and the metalloproteinase subunits (the Rpn8–Rpn11 heterodimer) are shown in light purple. The figure was generated from the electron microscopy structure (European Molecular Biology Laboratory’s European Bioinformatics Institute code: EMD‑1992) from REF. 77 and the atomic coordinates from REF. 80 (Protein Data Bank (PDB) accession: 4B4T). All models were generated and visualized with the University of California, San Francisco Chimera package168. The crystal structures (PDB accession: 4B4T) were fitted into the experimental electron microscopy map (EMD‑1992) with the algorithms provided by Chimera and corrected manually. The Segger package was used for the segmentation of the electron density map169.

The PCI domains in the scaffolding subunits Rpn3, Rpn5, Rpn6, Rpn7, Rpn9 and Rpn12 form an U‑shaped structure from which arms of α‑helical repeats radiate out. This allows them to form contacts from the top of the 19S activator all the way down to the first ring of the core particle. The PCI-containing domain proteins connect to the organizing subunit Rpn2, the deubiquitylating sub unit Rpn11, the ATPase ring and the α-subunits of the core particle77,78,89. The closed bottom of the U‑shaped structure faces towards the core, and the opening points towards the distal end77–80. The deubiquitylating subunit Rpn11 and its binding partner Rpn8 are near the open end of the U and almost connect the two arms. Rpn11 is

also in contact with Rpn2 at the top of the structure. Two of the lid subunits Rpn5 and Rpn6 near the bottom of the U contact the core particle directly 77–80 (FIG. 2). These contacts form the hinge for a rotation of the regulatory particle when a substrate binds81. Hence, the lid seems to help attach the regulatory particle to the core and to stabilize the entire structure89. This interpretation of the structure is supported by genetic data that show that lid subunits are required for proper proteasome assembly 89–91. The network of contacts formed by the lid might also integrate and modulate the biochemical functions of the different proteasome subunits and provide an interface for allosteric regulation77–80. Indeed, biochemical studies have shown that the binding of ubiquitylated substrates to the proteasome can increase ATP and peptide hydrolysis rates, which suggests that the proteasome motor might operate in different ‘gears’, as has been demonstrated for related bacterial proteolytic machines92–97. A puzzling aspect of the substrate-free ATP proteasome structures is that they lack a clear, continuous channel through which substrates can reach the proteolytic sites. The OB ring of the ATPase subunits, the AAA+ domain ring and the adjacent ring of the core particle α-subunits each form central channels, but the rings are tilted and shifted relative to one another so that the channels do not align77,80 (FIG. 3a). The AAA+ domain ring and the entrance to the 20S core particle α-ring are offset by as much as 10 Å77. However, in the substrate-bound (FIG. 3b) and ATPγS (FIG. 3c) proteasome structures, subunits in the regulatory particle shift and rotate such that all of the rings become coaxially aligned to form a continuous substrate channel81,82. The central pore in the AAA+ subunits widens considerably as compared to the substrate-free ATP structures, and this is consistent with biochemical results that measure increased degradation rates of peptides in the presence of ATPγS instead of ATP81,82,98 (FIG. 3b,c).

Degradation by concerted effort Recognizing ubiquitin chains. The degradation signal (also known as the degron) that targets proteins for destruction by the proteasome consists of a polyubiquitin chain as the proteasome-binding tag and an unstructured region as the proteasome initiation site. The polyubiquitin chain has to be at least four ubiquitin moieties long to tightly bind to the proteasome99 and can be much longer in vivo100. In the canonical degron, the ubiquitins are linked through Lys48 and less frequently through Lys11. However, the distinction is not strict, and Lys63-linked chains or multi ple shorter ubiquitin chains can also target proteins for degradation11,101. The proteasome in turn contains at least two intrinsic ubiquitin receptors (Rpn10 and Rpn13) and acts with several additional detachable ubiquitin receptors56,102,103. These receptors recognize ubiquitin through small domains: a Pru (pleckstrin-like receptor for ubiquitin) domain in Rpn13, one (yeast) or two (mammalian) UIM (ubiquitin-interacting motif) domains in Rpn10 and one or two UBA domains in the detachable ubiquitin receptors. Each of these domains recognizes only one or perhaps two ubiquitin moieties; thus, it is not immediately obvious how the proteasome recognizes longer



REVIEWS ubiquitin chains102,104–106. The substrate-free proteasome structures show that Rpn10 and Rpn13 are about 90 Å apart from each other 77–80 (FIG. 4). Polyubiquitin chains linked through Lys48 form relatively compact but flexible structures. A chain of four ubiquitin moieties should be able to take up a structure that is long enough to bind to the Rpn10 and Rpn13 receptors simultaneously11,76–78,107–109. By requiring two receptors to bind substrates simultaneously, the proteasome would be able to discriminate between the monoubiquitin tags (used sometimes as protein interaction signals) and longer polyubiquitin chains (used in degrons)78,110. However, it is unlikely that receptor spacing would discriminate between Lys48‑linked chains and the Lys63‑linked chains typically associated with cellular processes that do not involve the proteasome. Lys63‑linked chains are quite flexible111,112 and should be able to bind to both receptors at the same time. Indeed, abolishing ubiquitin binding by Rpn10 or Rpn13 affects yeast growth only minimally, so that substrate binding by both intrinsic ubiquitin receptors simultaneously is at least not a strict requirement for proteasomal degradation76,102. Perhaps the receptors on the proteasome by themselves do not distinguish effectively between ubiquitin chains with different linkages. Engaging substrates. It is not known how the proteasome binds the unstructured region of the degron, as these details are not resolved in the substrate-bound proteasome structure81. Using archaeal and bacterial ATPdependent proteases as a guide, the receptor is likely to be formed by a series of loops in the central pore of the AAA+ domains near the bottom of the ATPase ring; these loops probably function as ‘paddles’ that move the substrates through the degradation channel (the ATPase motor is discussed in more detail below). The ubiquitin receptors have to place the substrate such that the unstructured regions can reach the AAA+ domain paddles and be engaged by the translocation motor 26. The ubiquitin-binding site of Rpn10 is near the N‑terminal ends of two of the ATPase subunits, but it is still ~90 Å away from the motor loops, whereas Rpn13 is even further away 77,80. The unstructured regions themselves might bridge some of this distance, but they have to be at least long enough to reach from the narrow entrance to the substrate channel formed by the OB domain ring to the motor loops. In vitro experiments show that the unstructured regions must be a certain minimum length and placed appropriately to enable effective proteasome-mediated protein degradation20,113–118. Therefore, the length and positioning of unstructured regions relative to the ubiquitin tag might contribute to substrate selection. Versatile design. So far, we have discussed how the proteasome might avoid the unintended degradation of proteins that have not been targeted for destruction. However, the proteasome also faces the opposite challenge: it must be able to degrade all of the different regulatory proteins and damaged or misfolded proteins that are targeted for destruction, for instance during the cellular stress response. Part of the solution to this

Figure 3 | Structural rearrangements on substrate and ▶ nucleotide analogue binding. Cutaway views of the central channels of the proteasome in the substrate-free (a), substrate-bound (b) and slowly hydrolysable ATP analogue (ATPγS)-bound (c) conditions, showing the approximate locations of regulatory particle non-ATPase 11 (Rpn11), the AAA+ ring and the oligonucleotide- and oligosaccharide-binding (OB) ring. Under substrate-free condition, the OB domain ring (dark blue), the AAA+ domain ring (light blue) and the entrance to the core particle (grey) are all slightly out of alignment (a). Rpn11 (green) sits to the side of the OB domain ring. However, on substrate binding (b) or in the presence of ATPγS (c), the rings shift and tilt such that the central pore of each of the rings is aligned coaxially, which results in the formation of a continuous central channel. Rpn11 moves directly over the OB ring in both the substrate-bound and ATPγS conditions, aligning its catalytic active site with the translocation channel and possibly preventing substrate escape. Following substrate binding, the central pore of the AAA+ domain ring expands considerably (b): the cutaway view, looking directly into the translocation channel from the top of the proteasome, shows that the central pore of the AAA+ ring expands on substrate binding to enable substrate entry. The figure was generated from the electron microscopy maps from REF. 77 (European Molecular Biology Laboratory’s European Bioinformatics Institute code: EMD‑1992), REF. 81 (EMD‑5669) and REF. 82 (EMD‑2348). All models were generated and visualized with the University of California, San Francisco (UCSF) Chimera package. The electron density map from REF. 82 (EMD‑2348) was provided by A. Martin (University of California, Berkeley, USA) and further segmented using the Segger package168,169.

challenge could be that ubiquitin tags can be attached to several Lys residues in proteins, and the ubiquitin chains themselves vary in length and geometry 6,11,119. By binding to different polyubiquitin chains in a substrate or to different ubiquitin moieties in a long polyubiquitin chain, the proteasome would be able to find a productive binding mode to feed the unstructured region of the substrate into the translocation channel. The presence of several ubiquitin receptors on the proteasome might enable the proteasome to choose the appropriate receptors to position structurally diverse substrates optimally for degradation. In addition, the large organizing subunit Rpn1 serves as the docking site for the detachable ubiquitin receptors. These receptors bind the proteasome and substrates through UBL and UBA domains, respectively 56,120,121. The UBL and UBA domains are connected through flexible linkers117,122, and the flexible design of the UBL–UBA receptors might provide even more plasticity in orienting substrates to place their initiation region into the degradation channel. Finally, it is possible that the UBL–UBA proteins by themselves, or in cooperation with Rpn10 and Rpn13, bind to several ubiquitin chains on a substrate simultaneously 106. Genetic experiments indicate that the known ubiquitin receptors on the proteasome have partially redundant roles and that any one receptor is sufficient for yeast



REVIEWS a Substrate-free Rpn11

OB domain ring AAA+ large domain ring

survival, at least under normal culture conditions76,102. Indeed, even abolishing the ability of Rpn10 and Rpn13 to bind ubiquitin at the same time as deleting the three known UBL–UBA proteins is not lethal in yeast. This suggests that other proteasome subunits could contribute to substrate binding or perhaps even that the initiation region in degrons might be sufficient to target some proteins for degradation under certain circumstances123. Thus, Rpn1 and its UBL–UBA receptors, together with Rpn10, Rpn13 and the pore in the ATPase ring, build a versatile and partially redundant interaction platform that can engage a wide range of substrates through several recognition surfaces. Removing ubiquitin chains. The proteasome does not degrade the ubiquitin tag on a substrate. Instead, a protease activity of Rpn11 cleaves the isopeptide bond between the substrate and the first ubiquitin to release the entire polyubiquitin chain24,25. Rpn11 resembles conventional metalloproteinases, but in the context of the proteasome, ubiquitin cleavage by Rpn11 is dependent on ATP. The simplest explanation for this is that ubiquitin removal is coupled to the ATP-dependent process of substrate translocation into the degradation channel24,25. The proteasome structures place Rpn11 close to the entrance of the degradation channel, but in the substrate-free ATP structures, the proteolytic site of Rpn11 faces away from the direct path of Rpn10 towards the channel entrance77–80. In the substrate-bound structure, Rpn11 is shifted and rotated so that its catalytic groove aligns with the path to the central channel through the protease81. Hence, translocation of the substrate into the degradation channel would position the attachment point of the polyubiquitin chain at the active site of the deubiquitylating enzyme. Other deubiquitylating enzymes, such as Ubp6 in yeast (also known as USP14 in mammals) or UCH37 in mammals, associate with the proteasome and progressively trim polyubiquitin chains from the distal end of the chains6,25,64,124. Their action is counteracted by ubiquitinprotein ligases such as Hul5 or UBE3C, which extend ubiquitin chains on the proteasome64,67. These different enzymes seem to contribute another layer to the substrate selection, although the mechanism is not entirely understood, and their positioning in the proteasome structure is not known77,83. One possibility is that an ubiquitylation and deubiquitylation cycle fine-tunes degradation and reduces the risk of the proteasome clogging up with degradation-resistant proteins64. The ubiquitin ligases might also increase proteasome processivity by reubiquitylating substrates as the proteasome progresses along the polypeptide chain125. Finally, Ubp6 also affects degradation by allosterically activating the proteasome63,93.

Core particle

b Substrate-bound

c ATPγS

Action of the ATPase motor The proteasome is processive21 and releases partially degraded proteins only rarely 27–31,126–128. Therefore, the motor that drives the proteasome along the polypeptide chain of a substrate has to be able to act on many different sequences while unfolding any domains that it encounters. How this happens is an intriguing question Nature Reviews | Molecular Cell Biology


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Figure 4 | Canonical ubiquitin receptors and their Nature Reviews | Molecular Biology locations on the proteasome. The locations Cell of the two well-characterized ubiquitin receptors on the proteasome — Rpn10 (regulatory particle non-ATPase 10; blue) and Rpn13 (cyan) — are highlighted, together with the organizing subunit Rpn1 (orange) and the deubiquitylating enzyme Rpn11 (purple). The remaining proteasome subunits are shown in grey. The locations of Rpn10 and Rpn13 might enable a polyubiquitin chain to bind both receptors simultaneously, or the receptors might bind substrate individually or in combination with accessory proteins. Rpn1 provides a docking site for UBL (ubiquitin-like) domain- and UBA (ubiquitin-associated)containing proteins such as Rad23 (radiation sensitive 23) that deliver substrates to the proteasome. The figure was generated from the electron microscopy structure (European Molecular Biology Laboratory’s European Bioinformatics Institute code: EMD‑1992) from REF. 77 and the atomic coordinates from REF. 80 (Protein Data Bank accession: 4B4T) using the University of California, San Francisco Chimera package168. The Segger package was used for the segmentation of the electron density map169.

of molecular recognition and protein mechanics. At the core of the motor is a ring of six distinct ATPase subunits — Rpt1–6. The ATPases belong to the AAA+ family, which is a subset of the much larger class of phosphate-binding loop (P-loop) nucleoside triphosphatases (NTPases)87. AAA+ proteins have various functions, including protein degradation, nucleic acid helix unwinding and membrane protein disassembly, but despite this diversity, common sequence and structural features characterize the family 87. The ATPase site lies between a large AAA+ domain, which corresponds to the nucleotide-binding domain of all P-loop NTPases, and a small AAA+ domain that is specific to AAA+ proteins87,129,130. The ATP binding, hydrolysis and ADP + inorganic phosphate (Pi) release cycle is accompanied by rearrangements of the AAA+ domains relative to each other. These changes drive the different reactions catalysed by AAA+ proteins and often enable the protein to carry out mechanical work. In many AAA+ proteins, the ATPase subunits

are arranged in ring-like structures, such as closed rings, open rings or spirals; these proteins give some insight into how the ATPase motor of the proteasome might function87,129,130. Bacteria and archaea have ATP-dependent proteases that are functionally analogous to the eukaryotic proteasome. They share a common architecture of cylindrical particles that are built from rings of subunits stacked on top of each other. Their ATPase caps also consist of six AAA+ subunits, although the subunits are identical130,131. The bacterial and archaeal degradation systems typically do not use an ubiquitin tagging system for substrate selection and the protease caps thus lack the proteins involved in ubiquitin recognition and processing. The biochemical mechanism of the AAA+ protease ATPase motor has been most extensively studied in the bacterial and archaeal systems. The central pore formed by their ATPase subunits is lined with loops that protrude from the large AAA+ domains130. These loops interact with substrate proteins and undergo conformational changes driven by the ATP hydrolysis cycle132–134. One of the loops contains an aromatic-hydrophobic-Gly motif and is thought to function as a paddle that drives the polypeptide chain of the substrate through the channel into the proteolytic core particle72,132,133,135–139. Indeed, caseinolytic peptidase XP (ClpXP) can generate pulling forces in the range of 20 to 40 pN on its substrate97,140,141. Structures of the bacterial and archaeal proteases indicate how conformational changes in the ATP subunits might move the loops to carry out mechanical work130. For example, in Escherichia coli ClpXP, the large AAA+ domain of one ATPase subunit and the small AAA+ domain of the neighbouring ATPase subunit form rigid intersubunit modules or rigid bodies142,143. The ATPase cycle results in conformational changes at the interface of the large and small AAA+ subdomains within one subunit, which are propagated throughout the ring by the rigid units formed by the AAA+ domains of neighbouring subunits142,143. Crystal structures of ClpX and other related bacterial protease ATPase caps show that the rings can be asymmetric, so that some subunits are in conformations that are not bound to nucleotides130,142. The structures are consistent with biochemical studies of many AAA+ proteases, which show that only a maximum of four nucleotides are bound within the ring at one time98,144–147. It is therefore unlikely that the conformational changes that drive the mechanical unravelling of substrates happen in parallel in all subunits142,147. How the activity of the ATPase subunits around the ring is coordinated to propel substrates through the translocation channel remains a key question, and the mechanism might differ between AAA+ proteases from the three domains of life, Bacteria, Archaea and Eukarya. Coordinated ATP hydrolysis around the ring has been demonstrated for a mitochondrial homologue of the AAA+ protease filamentous temperature sensitive H (FtsH), and evidence for the archaeal proteasome shows at least some degree of coordinated ATP hydrolysis activity 98,148. In ClpX, ATP hydrolysis does not need to occur sequentially, as the motor can function even



REVIEWS when individual subunits are inactivated149. However, recent studies of ClpXP suggest that its translocation mechanism is characterized by some coordination between the ClpX subunits 97. Finally, dramatic conformational rearrangements of the ring structure also do not seem to be required for the degradation cycle of AAA+ proteases, as covalent crosslinks that keep the ring closed during the entire ATPase-degradation cycle affect degradation only minimally 143. Rearrangements of the ATPase subunits. The eukaryotic proteasome is more processive than the bacterial and archaeal proteases and, unlike the analogous machines in non-eukaryotes, its ATPase motor operates as a heterohexamer 150,151. The different ATPase subunits have specific, non-redundant roles in the gating of the trans location channel and even in translocation50–52,92,152–156. All six ATPase subunits contain the aromatic-hydrophobicGly motif that is found in the bacterial protease translocation paddles. However, the arrangement of the ATPase subunits in the proteasome is noticeably different to what has generally been observed for the bacterial proteases, and the arrangement changes with the nucleotide bound and the presence or absence of substrate. Substrate-free

Rpt1

Rpt3

Rpt6 Rpt2

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Rpt5

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Figure 5 | The ATPase arrangements in different conditions. A schematic Nature | Molecular Biology representation of the arrangement of the six ATPases of Reviews the proteasome and Cell the aromatic-hydrophobic-Gly motif loop (loop from each ATPase structure) for each of the conditions in which proteasome structures were determined. The large and small domain are shown for each ATPase, and different colours are used for each subunit. Vertical lines at the bottom of Rpt2 (regulatory particle triple A protein 2), Rpt3 and Rpt5 represent the carboxy-terminal tails that open the core particle gate. The spiralled arrangement of the AAA+ domains is obtained in both the substrate-free and slowly hydrolysable ATP analogue (ATPγS) conditions, although the subunits rearrange in the ATPγS condition so that Rpt5 ‘bridges’ the spiral. In the substrate-bound condition, the ATPase domains rearrange to a level plane, but the aromatic-hydrophobic-Gly loops are still arranged in a spiral owing to the different tiltings of the ATPase domains. The figure is adapted, with permission, from REF. 155 © (2013) Macmillan Publishers Ltd. All rights reserved.

In the substrate-free proteasome structures, the large AAA+ domains are arranged along a spiral staircase such that there is a substantial offset between the lowest and highest subunit in the ring 77,80. The offset is bridged by one of the subunits, Rpt6, hence closing the ring. In the ATPγS proteasome structure, the spiral is shifted by half a turn so that Rpt5 becomes the bridging subunit 82. The bridging subunit is in a conformation that seems to prevent ATP binding 82. The large AAA+ domains in the substrate-bound structure of the proteasome rearrange to form a more planar ring, reminiscent of the more uniform arrangement of ATPase subunits in the bacterial proteases81. This arrangement enables the large AAA+ domains to form rigid body units with the small AAA+ domains of neighbouring ATPase subunits, as seen in ClpX81,142. However, each individual ATPase subunit is tilted differently relative to the plane of the ring, which places the aromatic‑hydrophobic-Gly motif loops in a spiral81 (FIG. 5). It is not clear what role the ATPase subunit arrangements have during the degradation cycle in substrate translocation; however, the structural studies discuss several possible mechanisms77,79–82. The spiral conformation of the substrate-free ATP proteasome structure might represent a low-energy or standby state of the machine77,79,81,82. Substrate binding could then switch the structure into an active flat ring arrangement that can drive translocation77,79,81. The switching of the ring conformation to the translocation active state might be induced when the ATPases engage the initiation region of a substrate81. Indeed, binding of ubiquitylated substrates to the proteasome increases the ATP and peptide hydrolysis activities of the proteasome 92–95,154. The planar ATPase ring could then function similarly to the ClpX mechanism, in which the ATPase cycle drives conformational changes of rigid intersubunit bodies and their aromatic-hydrophobic-Gly motif loops142. The arrangement of the loops in a spiral might enable the proteasome to interact with a longer stretch of the substrate through local motions of the pore loops. Alternatively, the ATPase subunits might use larger motions by moving through the different tilt angles to thread substrates to the core particle81. This does not mean that the ATPase subunits contribute equally to translocation and, in fact, mutations in the aromatic-hydrophobicGly motif loops and other conserved regions in the different ATPase subunits affect protein degradation and ATPase activity in distinct ways92,153–156. A different model is that the arrangements of the AAA+ domains observed in the different conditions represent distinct intermediates in the ATPase cycle and that the active proteasome cycles through spiral conformations as it moves substrates through the substrate channel. The aromatic-hydrophobic-Gly motif loops shift positions between the ATPγS and substratefree ATP structures, and the movement is different for each subunit. Some loops seem to shift primarily vertically (along the long axis of the cylindrical particle), and these changes might be responsible for substrate translocation, whereas other loops move largely horizontally (in the plane of the ATPase ring), and these



REVIEWS loops might be responsible for holding the substrate in place as the machine resets during the ATP hydrolysis cycle82. Both types of mechanisms are thought to occur in other large ATP-dependent protein machines, and comparison of these with the proteasome might help us to understand the mechanics of the proteasome– substrate interaction during translocation.

Mechanistic insights from other translocases AAA+ helicases and recombinase A (RecA)-type helicases (another subgroup of P‑loop NTPases) are two families of ATP-dependent machines for which translocation mechanisms have been elucidated by structural studies. Some of these helicases also contain rings of ATPase subunits that adopt various conformational states to interact with their substrates77,79,157–160. The Drosophila melanogaster helicase minichromosome maintenance (Mcm; an AAA+ ATPase) and the bacterial Rho transcription termination factor helicase (a RecA-type ATPase) switch between an inactive conformation with a helical ATPase ring and an active conformation with a flat ATPase ring 157–160, reminiscent of the ATPase ring conformations shown for the proteasome. Rho termination factor interacts with its RNA substrate through hairpin loops that project from each ATPase subunit into a central pore, much like the aromatic-hydrophobic-Gly loops in the proteasome are thought to interact with its substrates. In the active flat ring structure, the loops are arranged in a spiral, and they cycle through the different positions of the spiral as the subunits hydrolyse ATP160,161. A similar arrangement is seen in the bovine papilloma virus replicative helicase E1 (also an AAA+ protein)160,161. Hence, these helicases function by a mechanism that is similar to one that envisages the substrate-free ATP structure of the proteasome as representing a resting state for the machine. In another RecA-type helicase, DnaB, the ATPase subunits are arranged in a spiral, but in this instance the spiral structure represents the active state of the enzyme. A crystal structure of DnaB shows it in a spiral staircase conformation with its substrate bound, and the entire ATPase domains are thought to cycle through the different positions of the staircase as part of the translocation mechanism162. Thus, the DnaB structure provides a precedent for a proteasome mechanism in which the ATPase ring undergoes larger structural changes and subunits cycle through the arrangements seen in the different proteasome structures. Matyskiela et al. discuss a mechanism in which the proteasome structures obtained represent ‘dwell’ states of the machine between bursts of translocation activity 81. An example of this type of mechanism has been described for the DNA packaging motor of bacteriophage φ29 (REFS 81,163–165). The packaging motor spends most of its time in a dwell phase, even in the presence of a substrate, and only a small fraction of time in an active translocating phase, which is characterized by rapid structural changes81,165. Singlemolecule studies of ClpXP show that it also translocates its substrate by a mechanism characterized by dwell

phases and bursts of activity 97,140,141. The positions of the ATPase subunits are well defined even in the substrate-bound and ATPγS structures, and it is therefore unlikely that the ATPase subunits are undergoing large conformational changes in most proteasome particles81,82,84. Therefore, the ATPγS and substratebound proteasome structures might represent different aspects of a dwell-phase state81,82. Translocation of the substrate would occur during burst phases of rapid ATP hydrolysis around the ring and could be characterized by the movement of entire AAA+ domains or by local loop motions in a static ring. After degradation of the substrate is complete, ATP hydrolysis in the bridging subunit could reset the ring to a resting state, which would be represented by the substrate-free ATP structure81,82. Hence, the different proteasome structures provide intriguing models for how the proteasome might function, but they do not yet provide a definitive picture of how translocation occurs. Future biochemical and structural studies should define this mechanism.

Conclusions Over the past 2 years, proteasome structures at nearatomic resolution have redefined our understanding of this large protein machine, which lies at the centre of much cellular regulation. They show the proteasome as an integrated structure that enables communication between the different enzymatic activities of the machine. They highlight the versatile substrate binding platform of the proteasome that allows it to act on a wide range of substrates. The structures in the presence or absence of a substrate, and in different nucleotide conditions, show conformational changes, particularly in the motor domains of the proteasome, that are even more marked than expected. The new structures support many aspects of the current view of proteasome action and will be instrumental in generating new hypotheses for how the machine functions during the degradation cycle. The new structures are also likely to influence drug development. The proteasome is directly involved in many diseases, including cancers and neurodegenerative diseases, and thus is an important pharmacological target. The current proteasome drugs are inhibitors that target the proteolytic sites 36,42. The recent structures of the proteasome could enable the design of an entirely new class of drugs that modulate conformational changes in the regulatory particle or target its enzymatic activities to activate or inhibit the proteasome. Proteasome activators could also be useful tools for clearing toxic protein aggregates that accumulate in many neurodegenerative diseases166,167. Indeed, a small molecule inhibitor targeting the deubiquitylating activity of the proteasome has already been established124. Therefore, it is difficult to overestimate the impact that the new structures will have on our understanding of how cellular protein concentrations are regulated, and it seems probable that protein destruction will turn out to be as finely coordinated as protein synthesis.



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Acknowledgements

The authors would like to thank A. Martin and M. Matyskiela for providing segmented electron density maps of their proteasome structures, and members of the Matouschek laboratory, especially J. Renn for critically reading this manuscript. Work in the authors’ laboratory is supported by the Gates Foundation, the Welch Foundation and US National Institutes of Health grants R01 GM63004, R01 GM094479, U54 GM105816 and U54 CA143869.

Competing interests statement

The authors declare no competing interests.


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