Beth M. Stadtmueller1and Christopher P. Hill1,*
1Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, UT 84112-5650, USA
Proteasomes degrade a multitude of protein substrates in the cytosol and nucleus, and thereby are essential
structure, activators are required to facilitate substrate access. Structural and biochemical studies of two
activator families, 11S and Blm10, have provided insights to proteasome activation mechanisms, although
the biological functions of thesefactors remain obscure. Recent advances have improved our understanding
of the third activator family, including the 19S activator, which targets polyubiquitylated proteins for degra-
dation. Here we present a structural perspective on how proteasomes are activated and how substrates are
delivered to the proteolytic sites.
Protein turnover is anessential and highly regulated process that
is performed in cytosolic and nuclear compartments of eukary-
otic cells primarily by proteasomes. This abundant protein com-
plex is also found in archaea and in some eubacteria, and its
activity is central to many cellular functions including protein
quality control, DNA repair, transcription, cell-cycle regulation,
signal transduction, and antigen presentation (Pickart and
Cohen, 2004). ‘‘Proteasome’’ can refer to a variety of complexes
whose cores comprise the cylindrical 20S proteasome (aka core
particle/CP), which houses proteolytic sites within a central
chamber. Because of this closed architecture, the 20S protea-
some is an inherently repressed enzyme, although essentially
any protein that enters the catalytic chamber will be degraded.
20S proteasomes are known to associate with three different
families of activators that provide access to the central proteo-
lytic chamber. The most broadly conserved family includes the
eukaryotic 19S activator (aka regulatory particle/RP/PA700)
and its archaeal PAN and eubacterial ARC/Mpa homologs.
both ends of a 20S proteasome to form a 26S proteasome,
recognizes polyubiquitylated substrates, and may be comprised
other two activator families are the 11S complexes (aka PA28,
REG, PA26) and PA200/Blm10. These factors are less broadly
conserved than the ATP-dependent activators, and their
substrates and biological functions are less clear, although the
mechanisms they use to activate proteasomes have been better
We begin by reviewing the architecture of the 20S protea-
some, including features of the substrate entrance gate that
maintain the default, closed conformations. We then discuss
activation mechanisms, highlighting the insights provided by
structures of proteasome-activator complexes. We compare
and contrast the effects of activator binding on 20S proteasome
structure and present a unified model for how activators open
the entrance pore. Finally, we discuss models for how the
ATP-dependent activators process substrates by unfolding
and translocating them into the proteasome, and illustrate the
emerging view that conformational changes make multiple
contributions to 19S activator function.
The 20S Proteasome
Proteasomes comprise four stacked heptameric rings, two
a type surrounding two b beta type, in an a7b7b7a7pattern, to
form a 28 subunit, barrel-like structure (Groll et al., 1997; Kwon
et al., 2004; Lowe et al., 1995) (Figure 1A). The proteasome
contains an interior chamber (Figure 1B), and the inner surface
promotes protein unfolding (Ruschak et al., 2010). Although
the a and b subunits share sequence and structural similarity,
there are functionally important differences associated with their
distinct N termini. The a subunit N-terminal residues form a gate
at the center of the a ring that restricts substrate entry in the
absence of an activator (Figure 1C) (Groll et al., 2000). The
b subunit N termini contribute to the proteolytic active sites,
which belong to the N-terminal nucleophile hydrolase family,
and use a threonine side chain as the attacking nucleophile
and the free N-terminal amine to activate an ordered water
molecule that is incorporated into the product during hydrolysis
(Figure 1D) (Brannigan et al., 1995; Seemuller et al., 1995).
Archaea and eubacteria generally express just one a type and
one b type subunit, resulting in proteasomes that have seven-
fold symmetry. In contrast, eukaryotic proteasomes comprise
seven distinct a subunits (a1–a7) and seven distinct b subunits
(b1–b7) that each occupy unique positions in the respective
rings, resulting in asymmetric structures with only approximate
seven-fold symmetry. Just three of the seven eukaryotic
b subunits, b1, b2, and b5, possess proteolytic sites, and these
exhibit preference for cleavage following acidic, basic, or hydro-
phobic residues, respectively.
(b1i, b2i, b5i) (Figure 1A), whose expression can be upregulated
in interferon g-inducible cells. These subunits replace their
conventional counterparts to form immunoproteasomes that,
consistent with optimal binding of peptides to MHC-I molecules,
show reduced cleavage following acidic residues and enhanced
Higher eukaryotes also encode a thymus-specific catalytic
b subunit, b5t, which replaces b5 to form thymoproteasomes,
Molecular Cell 41, January 7, 2011 ª2011 Elsevier Inc.
which are thought to function in the positive selection of MHC
class I restricted T cells because they have reduced ability to
cleave after hydrophobic residues and thus produce peptides
having weak affinity for MHC-I molecules (Murata et al., 2007).
Proteasomes can be valuable therapeutic targets. For
example, bortezomib, a boronic acid peptide, inhibits the
proteolytic sites by forming specific interactions between the
boronate and the b subunit Thr1 side chain hydroxyl and main
chain amine (Figure 1D). Bortezomib has high specificity for
the human b5 proteolytic site and is used for the treatment of
multiple myeloma and mantle cell lymphoma (Groll et al.,
2009). Inhibitors targeting proteasome variants are also prom-
ising candidates for drug development, such as the recently
described inhibitor PR-957, which specifically inhibits the b5i
immunoproteasome subunit and shows promise for the treat-
ment of autoimmune diseases (Muchamuel et al., 2009). More-
over, relatively large differences that exist between eukaryotic
and eubacterial proteasome active sites offer the potential to
develop antimicrobial drugs including those targeting Mycobac-
terium tuberculosis proteasomes (Lin et al., 2009).
The 20S Proteasome Gate
Substrates enter the proteasome through a gated pore in the
center of each a subunit ring (Wenzel and Baumeister, 1995),
and their passage is restricted by two structural elements. One
obstacle is a narrow channel known as the a-annulus that is
located slightly below the surface of the a ring (Figure 1B).
Because this appears to be a fixed opening of 13 A˚diameter
(?20 A˚in eubacteria), it ensures that substrates are substantially
unfolded before they can enter the proteasome. The other
obstacle is a closed gate formed from N-terminal residues of
the a subunits, although the gate closure mechanism is different
gate adopts an ordered, asymmetric conformation that is
defined by amino acid sequences that are unique to each
a subunit (Figure 1E). This structure restricts entry of even small
Figure 1. Structure of the 20S Proteasome
(A) Surface representation of the S. cerevisiae 20S proteasome (Groll et al., 1997). Individual subunits are labeled. b subunits shown to the right indicate that
alternative counterparts can be expressed for some of the subunits. The bovine liver proteasome is closely superimposible to this structure (Unno et al.,
2002). Archaeal and eubacterial 20S proteasomes are very similar but are typically comprised of one type of a and one type of b subunit, and are therefore
(B) Cutaway side view cartoon representation of the S. cerevisiae 20S proteasome. The region surrounding an active site is indicated with a box. The closed gate
region is colored gray. The a annulus, just interior from the gate, is an opening formed by loops (red) in the a subunits.
(C) Top view surface representation of the S. cerevisiae 20S proteasome. The gate region is indicated with a circle.
(D) Close-up of the S. cerevisiae b5 active site in complex with the inhibitor bortezomib (Groll et al., 2006). Corresponds to the boxed region in (B).
(E) Top view cartoon of S. cerevisiae helix H0 and N-terminal residues that form the closed gate. This eukaryotic gate is sealed primarily by a2, a3, and a4, whose
N-terminal residues are well ordered and participate in numerous hydrogen bonds and van der Waals contacts. Corresponds to the region circled in (C).
(F) Same as (E) for an archaeal gate (Religa et al., 2010). Residues shown in white are highly mobile.
(G) Same as (E) for a eubacterial gate (Li et al., 2010). This gate is ordered but is quite different from the eukaryotic structure. The seven subunits are chemically
identical but adopt a total of three different conformations at their N termini, as indicated by the different shades. A phenylalanine side chain from each of the
medium shade subunits contributes to the closed gate structure and is shown near the center of this panel.
Molecular Cell 41, January 7, 2011 ª2011 Elsevier Inc.
substrates; its importance is indicated by its precise conserva-
tion from yeastto mammals (Unno etal.,2002) andbythe finding
that a mutation disrupting this structure causes defects in the
survival of yeast upon prolonged starvation (Bajorek et al.,
2003). In contrast, while the 12 N-terminal residues of archaeal
proteasome a subunits are also located at the entrance pore
and can extend through the a-annulus to impede access of
unfolded proteins, they are highly flexible (Benaroudj et al.,
2003; Forster et al., 2003; Religa et al., 2010) (Figure 1F), thereby
explaining why archaeal proteasomes display a relatively high
level of activity toward small peptide substrates. It was initially
thought that the eubacterial entrance pore resembled the
unstructured archaeal gate, but recent structure determinations
suggest instead that the N-terminal residues form a closed
conformation that is ordered but nevertheless very different
from that of eukaryotic proteasomes (Figure 1G) (Li et al., 2010).
Higher eukaryotes express three 11S isoforms called PA28a,b,g
(aka, REGa,b,g) (Rechsteiner and Hill, 2005). PA28a and PA28b
tamer and appears to be the more ancient variant by virtue of its
expression in most metazoans and in some single cell organ-
isms, including Dictyostelium discoideum (Masson et al.,
2009). The trypanosome Trypanosoma brucei expresses a ho-
gent from the PA28 homologs but nevertheless retains the ability
to activate proteasomes purified from a wide variety of species
(Whitby et al., 2000). Although it is generally reported that 11S
activators stimulate the hydrolysis of model peptide substrates
but not proteins, REGg/PA28g has been implicated in the degra-
dation of some natively unfolded proteins (Mao et al., 2008; Nie
et al., 2010; Suzuki et al., 2009). Many studies have implicated
PA28ab in the production of MHC class I ligands, although the
mechanistic basis for this function remains elusive (Groettrup
et al., 2010).
Biochemical and structural studies of 11S activators are rela-
meric ring that has a central channel 20–30 A˚in diameter running
along its length (Figure 2A) (Knowlton et al., 1997). The
C-terminal residues of each subunit, which provide proteasome
binding energy (Ma et al., 1993), and internal activation loops,
which are critical for stimulation of peptide hydrolysis (Zhang
et al., 1998), are arranged in a seven-fold symmetric array at
the wide end of this torus-shaped molecule. The way in which
these structural elements bind the proteasome and open the
entrance pore was revealed by structures of PA26 in complex
Figure 2. ATP-Independent Activators
(A) Side and top view cartoon representations of PA28a/REGa (Knowlton et al., 1997). A single subunit is colored orange.
(B) Same as (A) for PA26 as observed in proteasome complexes (Forster et al., 2005). Note the diaphragm-like structure formed in the central channel of PA26.
(C) Cutaway cartoon representation of the PA26-S. cerevisiae proteasome complex. This panel was generated by removing subunits, to leave a total of eight
proteasome and four PA26 subunits. The PA26 activation loop (AL) and C terminus (C) are indicated for one of the subunits.
(D) Side view of Blm10 as seen in the proteasome complex (Sadre-Bazzaz et al., 2010). Rainbow colored, blue to red from N to C termini.
(E) Blm10 top view surface representation. An arrow indicates the largest opening through the Blm10 dome, which is not visible in this view.
(F) Close-up of the Blm10 pore viewed in the direction of the arrow in (E).
(G) Cutaway cartoon representation of the Blm10-proteasome complex. The Blm10 C terminus is indicated.
Molecular Cell 41, January 7, 2011 ª2011 Elsevier Inc.
et al., 2003; Whitby et al., 2000) and Thermoplasma acidophilum
(Forster et al., 2005).
As shown in Figure 3, PA26 C-terminal residues insert into
pockets between proteasome a subunits where they form
main-chain to main-chain hydrogen bonds and a salt bridge
between the PA26 C-terminal carboxylate and a highly
conserved proteasome lysine side chain (Lys 66) (Figures 3B
and 3F). A glutamate side chain located in the PA26 activation
loop contacts and repositions a conserved structural element
located above the surface of the proteasome called the Pro17
reverse turn (Figure 3G). Small, 0.5–3.5 A˚movements of each
subunit’s Pro17 reverse turn trigger gate opening by disrupting
packing and hydrogen bonding interactions of the precisely
closed conformation (Figure 3A) of eukaryotic proteasomes,
and by widening the pore opening to a more circular arrange-
ment that allows a belt of intersubunit contacts to form around
the circumference of the opening (Figures 3D and 3E). Curiously,
four a subunit residues that stabilize the open conformation in
both the archaeal and yeast proteasome PA26 complex struc-
tures, Tyr8, Asp9, Pro17, and Tyr26 (Figures 3D and 3E), are
highly conserved, even in species that do not express 11S
activators. It was therefore postulated that this conservation
exists because the same open proteasome conformation is
Figure 3. Mechanism of Gate Opening
(A) Top view of the S. cerevisiae proteasome gate in the closed conformation. Similar to Figure 1E but rotated and including the side chains of Tyr8, Asp9, Pro17,
andTyr26(T.acidophilumnumbering)ofeachsubunit(pink).Theseresiduesstabilize theopenconformationand insomecasesalsomakeinteractionsthatstabi-
lize the closed conformation shown here.
(B) Same as (A) for the PA26 complex, with Tyr8, Asp9, Pro17, and Tyr26 colored yellow. The C-terminal three residues of PA26 (blue) are shown in the four S.
cerevisiae proteasome pockets where they are visible in the crystal structure (Forster et al., 2005). The pocket between a5 and a6 is boxed to encompass the
PA26 C-terminal residues and also the Glu102 activation loop residue, which lies closer to the pseudo seven-fold axis and is shown for all seven PA26 subunits.
The same open conformation is induced in an archaeal proteasome, when PA26 C-terminal residues bind equivalently to all seven pockets.
(C) Same as(B) for the Blm10 complex.Blm10 makes extensive contacts that completely surround theproteasome entrance pore (Figure 2G), and theC-terminal
residues (red) bind in the a5/a6 pocket (boxed). In contrast to the PA26-bound structures, many N-terminal residues become disordered upon binding Blm10
(white, in the position of the unliganded structure).
(D)SuperpositionoftheTyr8, Asp9, Pro17, andTyr26residuesof theopen(yellow)andclosed (pink)gate conformationsfollowing an alignment onthebsubunits.
This movement destabilizes packing in the closed conformation and widens the pore to allow a belt of Tyr8, Asp9 residues to assemble. The a2/a3 cluster, which
undergoes the largest (3.5 A˚) displacement of a Pro17 residue, is boxed, and the outward direction of displacement upon opening is indicated with an arrow.
(E) Close-up of the a2/a3 cluster boxed in (D).
(F) Close-up showing a subset of interactions that occur in the pocket boxed in (B) and (C) with PA26 and Blm10 C termini superimposed. Main-chain groups of
PA26 and Blm10 C-terminal residues make equivalent hydrogen bonding interactions (dashed lines).
(G) Superposition of the PA26 and Blm10 complexes in the a5/a6 pocket illustrating the different mechanisms of displacing the Pro17 reverse turn. PA26
displaces Pro17(inallseven subunits)by contactingadjacent residueswithactivation loopresidue Glu102(blue). Blm10(red) stabilizesthesamePro17displace-
ment(justof a5)by forming ahydrogen bond (dashed lines)between itsTyrside chainand the main-chain oxygenof Gly19. ATPase activators likelyuse an equiv-
alent penultimate Try interaction to induce gate opening.
Molecular Cell 41, January 7, 2011 ª2011 Elsevier Inc.
also induced by the ATP-dependent activators, which are found
in essentially all species that express proteasomes. This idea
was validated by biochemical analysis of mutant archaeal pro-
teasomes and the ATPase activator PAN, which demonstrated
that degradation of a model substrate protein is severely attenu-
ated by mutation of Tyr8, Asp9, Pro17, or Tyr26 (Forster et al.,
2003). Moreover, a similar argument that ATP-dependent activa-
tors use equivalent C-terminal interactions to those seen for
PA26 was supported by the observation that the conserved
lysine residue at the bottom of the pocket and the C-terminal
residue of PAN subunits are both important for biochemical
activity (Forster et al., 2005) and by electron microscopic recon-
struction of proteasome complexes with peptides correspond-
ing to the C terminus of PAN (Rabl et al., 2008).
While structural and biochemical studies on 11S have shed
light on proteasome gate opening, an explanation for their phys-
iological functions is less apparent. The central channel of PA28
might accommodate diffusion of small substrates and possibly
even natively unstructured polypeptides. PA26, however,
displays an insertion in helix 3 that projects into the central
channel to form a diaphragm-like structure that is expected to
impede passage of even peptide substrates (Figures 2B and
2C). The extent to which peptide diffusion is impeded is difficult
to model, because the degree of flexibility is unclear. However,
this structure prompts consideration of other explanations for
the activation of proteasomes by 11S in biochemical assays,
including the following: (1) 11S activators might leave the
unliganded proteasome temporarily in an open conformation
that can accept substrates after they dissociate, and (2)
substrates might bind coincidently with the activator, perhaps
located in the activator’s central opening. The relevance of the
biochemical activity for physiological function is also unclear,
but one possibility is that they function in the context of hybrid
proteasomes, in which they bind to the opposite end of the
20S proteasome from an 19S activator, and through as-yet-un-
characterized interactions localize the degradative capacity of
the 19S activator complex to a specific cellular environment
(Rechsteiner and Hill, 2005).
Like 11S activators, Blm10/PA200 (Saccharomyces cerevisiae/
human) does not utilize ATP and is generally believed to stimu-
late the hydrolysis of peptides but not proteins. Blm10/PA200
has been proposed to function in a surprisingly broad variety
of processes, including 20S proteasome assembly (Fehlker
et al., 2003), DNA repair (Schmidt et al., 2005; Ustrell et al.,
2002), genomic stability (Blickwedehl et al., 2008), proteasome
inhibition (Lehmann et al., 2008), spermatogenesis (Khor et al.,
2006), and mitochondrial checkpoint regulation (Sadre-Bazzaz
et al., 2010). This remarkable variety of functions may reflect
the difficulty of identifying proximal action from a sea of indirect
effects. Electron microscopic reconstructions of the PA200 and
Blm10 proteasome complexes revealed similar dome-like archi-
tectures (Iwanczyk et al., 2006; Ortega et al., 2005; Schmidt
et al., 2005). A recently reported yeast Blm10 20S crystal struc-
ture (Sadre-Bazzaz et al., 2010) revealed that Blm10 forms
a HEAT repeat-like solenoid that makes 1.5 superhelical turns
to form the dome that encloses a large (110,000 A˚3) volume
and offers only a small (18 A˚3 9 A˚) opening through which
substrates might pass (Figures 2D–2G). This closed architecture
degradation of proteins, and is consistent with the suggestion
that Blm10/PA200 and 11S activators might serve as adaptors
that function in the context of hybrid proteasomes. Further sup-
porting the proposal that Blm10 is not a direct activator of prote-
olysis in vivo, it induces a proteasome gate structure that is
disordered rather than fully open (Figure 3C). This structure is
expected to allow passage of small peptides that can enter the
opening through the dome, but, as with the disordered pore of
the unliganded archaeal proteasome, it is not expected to allow
passage of protein substrates. Thus, it is unclear how the gate
conformation relates to Blm10/PA200 physiology beyond the
requirement that it is an energetically accessible conformation
that is compatible with binding.
Binding of Blm10 reveals an intriguing parallel with PA26 and,
as discussed more fully in the following section, may provide
insight into gate opening by the ATP-dependent activators. Of
the many interactions in the large 10,000 A˚2interface between
Blm10 and the proteasome, contacts made by the Blm10 C
terminus are especially notable. The three C-terminal residues
insert into the pocket between a5 and a6 in a conformation
that is superimposable with the PA26 C-terminal residues,
including maintenance of the same hydrogen bonds between
main-chain groups and the salt bridge between the activator
C-terminal carboxylate and the conserved proteasome lysine
side chain (Figure 3F). Unlike 11S activators such as PA26, but
like ATPase subunits that function in gate opening (below), the
penultimate residue of Blm10 is a tyrosine, or phenylalanine in
some homologs. This tyrosine side chain contacts a5 Gly19 O
to stabilize the a5 Pro17 reverse turn in the same position as
seen in the open conformation with PA26 (Figure 3G). Thus,
11S conformation to repositioning of a single subunit into the
open conformation. This has the effect of moving the entire
proteasome gate partially toward the fully open conformation
seen with PA26, although the gate remains disordered rather
than fully open because repositioning of a5 alone is insufficient
additional Blm10 contacts prevent the fully open conformation
from forming (Sadre-Bazzaz et al., 2010).
Gate Opening by ATP-Dependent Activators
As noted above, evolutionary and biochemical evidence indicate
that PAN, and presumably other ATP-dependent activators,
utilize their C-terminal residues to bind 20S proteasomes in the
same manner as PA26/11S and induce the same seven-fold
symmetric open conformation. An important difference is that
the penultimate tyrosine residue and a preceding hydrophobic
residue found in the C termini of PAN subunits and in some of
the 19S ATPase subunits can stimulate peptide degradation
without employing an activation loop mechanism as observed
for PA26/11S (Gillette et al., 2008; Smith et al., 2007).
How the PAN/19S C-terminal residues induce gate opening is
somewhat controversial. One model, based on low-resolution
structural studies, holds that binding induces closing of the
binding pocket and a 4?rotation of the a subunits around the
Molecular Cell 41, January 7, 2011 ª2011 Elsevier Inc.
a-annulus that subsequently induces gate opening (Rabl et al.,
2008; Yu et al., 2010). A strength of this model is that it is
based on electron microscopic observations of free complexes
that are not restricted to a crystal lattice, although it does not
provide a clear mechanism for how the conformational changes
would be propagated. We favor an alternative model based on
the crystal structure of the Blm10 complex (Sadre-Bazzaz
et al., 2010) and on a series of chimeric PA26-proteasome
complex crystal structures and binding studies (Stadtmueller
et al., 2010). These structures verify that sequences correspond-
and Blm10 while using their penultimate tyrosine residue to
contact and reposition the proteasome Pro17 reverse turn,
thereby inducing the same open gate conformation as seen in
the PA26 complex structures. These structures also reveal that
a penultimate phenylalanine, which is conserved in PAN from
many archaeal species, can stabilize the same repositioning of
the Pro17 reverse turn, and surface plasmon resonance data
indicate that a phenylalanine can contribute to activator binding
affinity at a level similar to that of a penultimate tyrosine residue
(Stadtmueller et al., 2010). This model relies heavily upon crystal
structures, but its strengths include high-resolution structural
information and a clear mechanism for how binding is coupled
to gate opening. Another appealing aspect of this model is that
it suggests a unified mechanism for gate opening in which all
three types of activator make superimposable contacts through
their C-terminal residues (Figure 3F) that provide binding affinity
and dictate additional interactions that open the proteasome
gate (Figure 3G). In the case of 11S activators, the activation
loop provides gate-opening interactions, whereas the other acti-
vators use penultimate tyrosine or phenylalanine residues.
Blm10 provides only one penultimate tyrosine interaction, which
is not sufficient to fully open the gate, while the two or more
penultimate tyrosine interactions ofPAN/19S and thesevenacti-
vation loop contacts of 11S result in complete gate opening.
Additional factors may contribute to mechanisms of gate
opening by ATP-dependent activators. For example, ATP
binding seems to be important (Liu et al., 2006; Smith et al.,
2005), probably because ATP-induced conformational changes
increase accessibility of C-terminal residues and hence binding.
Binding of associated proteins or polyubiquitin to the 19S
activator isalsoreportedtopromotegateopening (Bech-Otschir
et al., 2009; Li and Demartino, 2009; Peth et al., 2009). These
interactions could propagate conformational changes through
the ATPase subunits to the 20S proteasome entrance pore,
and/or they could influence substrate access through the pore
in the center of the ring of ATPase subunits. Untangling these
two possibilities remains a challenge.
A further consideration for gate opening is that a population of
free 20S proteasome has been reported to exist in eukaryotic
cells (Shibatani et al., 2006), and a substantial number of
substrates are reportedly degraded by these proteasomes
withoutthe assistance ofanactivator (Baughetal.,2009). Mech-
anisms associated with activator-independent degradation are
poorly understood. One possibility is that thermal fluctuations
induce spontaneous gate opening with some frequency, thereby
allowing unstructured proteins to gain entry. Other possibilities
are that specific peptides may stimulate gate opening (Kisselev
et al., 2003) or that some substrates may contain binding deter-
minants that induce formation of the open conformation, and
thereby serve as their own activator for proteasome entry.
Although models for gate opening of eukaryotic and archaeal
proteasomes are advanced, it is not clear how to think about
eubacterial proteasomes in this regard. The lysine that is func-
tionally important for binding 11S, Blm10, and PAN/19S appears
to be conserved in the presumed activator binding pocket of
eubacterial proteasomes, and eubacterial ARC/Mpa ATPases
conserve the pentultimate tyrosine found in PAN and 19S
subunits. These observations suggest that eubacterial protea-
somes will bind their ATP-dependent ARC/Mpa activator in the
same manner as their archaeal and eukaryotic cousins. On the
other hand, eubacteria do not conserve the a subunit Tyr8,
Asp9, Pro17, or Tyr26 residues that stabilize the open conforma-
tion in archaeal and eukaryotic 20S proteasomes. Thus, it will be
of interest to determine the extent to which eubacterial protea-
some gating parallels or diverges from the eukaryotic and
Substrate Unfolding and Translocation
by ATP-Dependent Activators
The ATP-dependent proteasome activators are classical AAA
ATPase family members (Erzberger and Berger, 2006) that
actively unfold and translocate substrates. The archaeal/eubac-
terial PAN/ARC/Mpa activators are homohexameric ATPase
rings, and the core of the eukaryotic 19S activator is a hetero-
hexameric ATPase ring that is comprised of six different but
related ATPase subunits (Rpt1-6) that each occupy a unique
position within the ring (Tomko et al., 2010). The N-terminal
and C-terminal regions of the six ATPases each interact to
form two hexameric rings in the fully assembled complex.
Binding studies and electron microscopy (Bohn et al., 2010; da
Fonseca and Morris, 2008; Forster et al., 2010; Smith et al.,
2005) have demonstrated that the C-terminal region, which
includes the ATPase catalytic sites, is adjacent to the protea-
some a ring, and the central pore of the ATPase subunit rings
is roughly alignedwiththe
Crystal structures have been reported for the N-terminal
region of the Rhodococcus erythropolis ARC, Archaeoglobus
fulgidus PAN (Djuranovic et al., 2009), Methanocaldococcus
jannaschii PAN (Zhang et al., 2009a), and Mycobacterium tuber-
coiled coils, formed by N-terminal residues from adjacent pairs
of subunits,that arefollowed byaringof OB domains (or tandem
OB domains in eubacterial variants) to form a chalice-like
hexamer with a central pore (Figures 4B and 4E). The striking
mismatch between the trimeric arrangement of coiled-coil
dimers and six-fold rotational symmetry of the OB regions (and
presumably the ATPase domains) is accommodated by a
conserved proline residue, Pro91 (M. jannaschii numbering), in
the linker between the OB and coiled-coil domains. The confor-
mation of the peptide bond preceding this residue alternates
between cis and trans in subunits around the ring, with each
coiled coil being formed by a Pro91 cis-trans pair of ATPase
subunits. This model has been extended to the heteromeric
ATPase hexamer at the heart of the 19S activator, where
Molecular Cell 41, January 7, 2011 ª2011 Elsevier Inc.
Pro91 is conserved in Rpt2, Rpt3, and Rpt5, which appear to be
the three subunits that require a cis proline configuration to
accommodate the asymmetric structure (Zhang et al., 2009a).
This finding is consistent with studies indicating that 19S assem-
bles from precursor complexes containing the Rpt pairs Rpt1-
Rpt2, Rpt6-Rpt3, and Rpt4-Rpt5 (Park et al., 2010), and targeted
disulfide crosslinking studies of S. cerevisiae proteins demon-
strate that Rpt subunits are ordered Rpt1-Rpt2-Rpt6-Rpt3-
Rpt4-Rpt5 around the ring (Tomko et al., 2010).
A crystal structure has also been reported for the C-terminal
ATPase domain of PAN from Methanocaldococcus jannaschii
(Zhang et al., 2009a). Although this structure represents the
monomeric, unassembled state, modeling based on the bacte-
rial ATPase HslU (Bochtler et al., 2000; Sousa et al., 2000) indi-
cates that the ATPase domain forms a ring with a central pore
that displays an Ar-F loop (aka pore loop 1) from each of the
six subunits (Figure 4C) (Zhang et al., 2009a). AR-F loops are
conserved among ATPase domains of AAA ATPase such as
HslU and are thought to move upon ATP hydrolysis to drive
substrate translocation (Park et al., 2005). Thus, the PAN Ar-F
loop (Phe244-Ile245-Gly246) likely paddles substrates through
the pore, with a leading role being played by the aromatic
Phe244 side chain and the Gly246 being required to allow the
conformational changes to occur (Zhang et al., 2009b). Genetic
analysis in S. cerevisiae demonstrated that mutation of the
equivalent residues in 19S Rpt subunits led to proteolysis
Figure 4. ATP-Dependent Activators
activator are indicated. The volume assigned to the ATPase subunits is colored orange.
(B) Top view of the N-terminal domains of PAN. The six subunits have identical sequences, but adopt alternating cis/trans conformations of Pro91 that allow
formation of a trimer of coiled coils above a hexamer of OB domains. Inset shows a superposition of yellow (trans) and blue (cis) subunits on their OB domains
to illustrate how the different conformations result in very different orientations for the N-terminal helix.
(C) Top view of the C-terminal ATPase domain of PAN. The monomer crystal structure was determined and is modeled here on the hexameric structure of HslU,
following the approach of Zhang et al. (2009a). ATP/ADP sites are indicated in orange. Ar-F pore loop residues are colored green.
(D) Cutaway side view of a composite model of the PAN hexamer based on the available domain structures shown in (B) and (C). A substrate (red) is shown
interacting with the N-terminal coiled coils, which promote protein unfolding, and with a flexible segment extending through the conduit of OB domains to reach
the ATPase pore loops that drive translocation. PAN C termini (dashed lines) bind directly to 20S to induce gate opening.
(E) Model of Mycobacterium tuberculosis Pup-Mpa interactions based on crystal structures of Mpa N-terminal domains (Wang et al., 2010). Mpa displays
a tandem OB domain, rather than the single OB domain of other ATPase activators. OB domains from a single Mpa subunit have been removed for clarity.
Pup (purple) binds the Mpa coiled coil (teal) to present a disordered N-terminal segment (dashed line) that can traverse the OB domain conduit, with both
Pup and conjugated substrate beingsubsequently dragged intotheproteasomeby the ATPase translocationactivity.Binding stoichiometryand packing consid-
erations suggest the arrangement shown here, with just one Pup-substrate associated with a Mpa hexamer.
Molecular Cell 41, January 7, 2011 ª2011 Elsevier Inc.
defects (Zhang et al., 2009b), which further supports the model
that PAN and 19S ATPases adopt equivalent structures and
mechanisms. The paddling model accounts for how ‘‘simple’’
sequences (Tian et al., 2005), that are thought to interact weakly
with the pore loops, allow adjacent, stable domains to escape
degradation, as reported for a number of transcription factors
that use this mechanism to release active signaling domains
that relocate to the nucleus (Rape and Jentsch, 2004). An impor-
tant, outstanding mechanistic question is how movements of the
pore loops in a hexamer are coordinated to promote substrate
translocation. Indeed, extensive studies on other AAA ATPases
have provided support for a variety of models, including sequen-
tial action of each subunit (Enemark and Joshua-Tor, 2006;
Thomsen and Berger, 2009), stochastic/probabilistic firing of
individual subunits (Martin et al., 2005), and concerted move-
ment of all pore loops (Gai et al., 2004).
A composite PAN model proposes that the coiled coils sit
above a conduit of OB domains through which substrates pass
before engaging the translocating pore loops of the ATPase
domains (Zhang et al., 2009a) (Figure 4D). The corresponding
Rpt assembly has been localized in the 19S activator (Bohn
et al., 2010) (Figure 4A), and the structure of an N-terminal region
of Mycobacterium Mpa supports ananalogous model for eubac-
terial activators (Wang et al., 2010) (Figure 4E). This model
explains why proteasome substrates must include a flexible
segment, which can be an internal loop, in order to be pro-
cessed, because only an unstructured sequence could reach
from the top surface to engage the ATPase pore loops and
initiate translocation (Prakash et al., 2004, 2009). Although
substrate translocation promotes unfolding by forcing the
substrate through a narrow channel, ATP-independent mecha-
nisms also contribute to the functions of ATP-dependent activa-
tors. The N-terminal coiled coils structurally resemble the chap-
erone prefoldin and, by virtue of their overall structure, can
promote protein unfolding (Djuranovic et al., 2009). It also
appears that unfolding on the ATPase surface can be promoted
by nucleotide binding and hydrolysis, although the mechanism
of coupling between the N- and C-terminal regions of the
ATPase subunits is currently unclear (Zhang et al., 2009b).
Substrate Targeting to ATP-Dependent Activators
Proteasome ATP-dependent activator function is coupled to
cellular substrate targeting strategies. Substrates must include
both a flexible sequence that can reach the ATPase pore loops
and affinity for the ATPase that may be either inherent or
provided by posttranslational modification. The eubacterium
Mycobacterium tuberculosis targets substrates to Mpa using
Pup (prokaryotic ubiquitin-like protein), which is a natively
unstructured protein that when conjugated to a substrate
provides both affinity for the ATPases and a means to engage
the pore loops. In contrast to ubiquitin, which is typically
removed prior to degradation of the substrate, Pup is translo-
cated and degraded together with the substrate (Burns et al.,
2010; Pearce et al., 2008; Striebel et al., 2010), a targeting
strategy that may explain why the eubacterial a-annulus is wider
than that of eukaryotic (or archaeal) proteasomes. An attractive
model for how Pup delivers substrates to the Mpa activator
and on to the eubacterial 20S proteasome is provided by crystal
structures of Mpa and Pup complexes (Wang et al., 2010), which
indicate that Pup binding to Mpa coiled coils induces the forma-
tion of a Pup helix that positions the flexible Pup N terminus for
engagement with the pore loops (Figure 4E). The archaeal
SAMP1/2 proteins appear to play proteasome-targeting roles
analogous to those of Pup and ubiquitin, although mechanistic
details are less clear (Humbard et al., 2010).
Whereas the archaeal and eubacterial ATPase activators
appear to be autonomous complexes that can process
substrates without the assistance of additional factors, the
eukaryotic 19S activator is generally estimated to comprise 19
different stoichiometric subunits including the six ATPases.
This considerably expanded complexity appears to provide an
interface withtheeukaryotictargetingpathways, themostprom-
inent of which involve ubiquitylation (Finley, 2009). Hundreds of
human proteins are thought to function in ubiquitylation and
deubiquitylation pathways, some of which are 19S subunits.
Several of the 19S non-ATPase subunits can recognize ubiquitin
conjugated proteins, and some are enzymes that can edit the
chain (extend/trim) to alter substrate affinity for 19S or remove
polyubiquitin chains as the ATPases translocate the substrate
into the proteasome. This process can be further regulated by
many tens of additional proteins that have been characterized
as substoichiometric 19S subunits that presumably associate
with the core machinery and tailor its activity to specific physio-
logical contexts (Finley, 2009).
19S Structure and Dynamics
The challenging task of obtaining structural information on the
19S activator is a topic of intense effort and debate. An attractive
composite model, provided by electron microscopic and
informatics analysis (Bohn et al., 2010; Forster et al., 2010), pla-
ces the ATPases adjacent to the 20S proteasome (Figure 4A). In
this model, 19S subunits form two major subassemblies, the
base and the lid (Glickman et al., 1998). The base includes the
six Rpt ATPases, two scaffolding proteins (Rpn1-2) and several
proteins (Rpn10, Rpn13, and Uch37) involved in ubiquitin recog-
nition and processing, whereas the lid contains at least one deu-
biquitylating enzyme, Rpn11, and eight other proteins (Rpn3,5-
9,12,15) whose individual functions are uncharacterized. An
alternative model suggests that the Rpn1 and Rpn2 scaffolding
proteins form a tower above the 20S proteasome entrance
pore (Rosenzweig et al., 2008), although, as discussed above,
the model in which the ATPase ring occupies this position
is generally preferred (Forster et al., 2010; Tomko et al.,
2010). Another controversial proposal is that 26S proteasome
assembly and disassembly are integral to the mechanism of
protein degradation (Babbitt et al., 2005), although the generally
preferred view is that 26S proteasomes catalyze multiple rounds
of degradation without disassembly (Kriegenburg et al., 2008).
In addition to a need for high-resolution structural data, under-
standing 19S mechanisms will require unraveling the functional
consequences of subunit conformational changes beyond those
already inferred for the ATPase pore loops. For example, shuttle
receptors such as Rad23, which can bind both polyubiquitin and
associated (UBA) domains. These domains associate with each
other in the absence of substrate, but upon binding of
Molecular Cell 41, January 7, 2011 ª2011 Elsevier Inc.
polyubiquitin to the UBA domains the Ubl domain is liberated to
associate with its receptor on 19S (Walters et al., 2003). The
Ubp6/USP14 and Uch37 deubiquitylating enzymes are also
activated upon binding to 19S, although the mechanisms and
associated conformational changes are not currently well under-
stood (Lam et al., 1997; Leggett et al., 2002). Moreover, Uch37’s
binding partner, the ubiquitin receptor Rpn13 (Yao et al., 2006),
undergoes a functionally important conformational change
upon binding the 19S Rpn2 subunit, whereupon its ubiquitin-
binding activity is increased 26-fold (Chen et al., 2010).
A dramatic example illustrating how conformational changes
contribute to 19S function is provided by the 19S lid subunit
Rpn11/POH1 (S. cerevisiae/human), which is a deubiquitylating
enzyme that removes ubiquitin chains en bloc in order to facili-
tate entry of substrate to the proteasome and to avoid depleting
pools offree ubiquitin (Vermaetal.,2002;Yaoand Cohen,2002).
Ubiquitin has seven lysine residues, each of which can be used
to build a polyubiquitin chain, with all types of chain linkage
except Lys63 serving as efficient tags for proteasomal degrada-
tion (Xu et al., 2009). Interestingly, POH1 preferentially cleaves
Lys63 isopeptide bonds (Cooper et al., 2009), which suggests
that it rapidly disassembles Lys63chains in anATP-independent
manner in order to spare mistargeted substrates from degrada-
tion. Paradoxically, authentic proteasome substrates are poor
Rpn11/POH substrates, and their processing requires ATP
hydrolysis by the Rpt subunits. This indicates that the ATPases
drive the substrate into a productive conformation in the
Rpn11/POH1 active site, and ensures that substrates engage
the ATPases before the chain is removed. This is an effective
strategy to ensure that substrates are translocated and are not
released prior to degradation (Cooper et al., 2009). The coupling
between ATPases in the 19S base and a deubiquitylating
enzyme in the 19S lid indicates that conformational changes
coordinated throughout the 900kDa complex are functionally
Currently we understand many fundamental aspects of protea-
some function and mechanism. We know the structure of the
20S proteasome and of many inhibitor complexes at high
We also understand that substrates enter through an axial pore
that is gated by activators. Detailed structural models are
available for proteasome complexes with the 11S and Blm10
activators, and emerging models describe how ATP-dependent
activators induce gate opening. We also have an approximate
model for how the ATP-dependent activators promote substrate
unfolding and translocation, and we are beginning to understand
other details of 19S mechanism, including high-resolution struc-
tures of some subunits and models describing functionally
important conformational changes.
Despite these advances, many unknowns cloud our under-
standing of proteasome function. We do not have a firm grasp
on the biological roles played by 11S and Blm10/PA200 activa-
tors, or the relevance of hybrid proteasomes. We lack atomic
structures of most 19S components and, more importantly, we
do not fully understand how these components interact with
each other and move during their functional cycle. Uncertainty
even exists regarding 19S composition and the designation of
some subunits as being stoichiometric or substoichimetric,
and considerable uncertainty exists regarding the functional
relevance of the many substoichiometric subunits that have
use their C-terminal residues to bind 20S in the same manner,
and the finding that some C-terminal tripeptide sequences
have higher affinity and can trigger gate opening, suggests that
currently unrecognized proteins may use the same principles
to bind, and possibly activate, the proteasome. While it is clear
that coordinated conformational changes are an important
between 19S and the 20S proteolytic sites, which are at least
60 A˚distant, is unclear. Although some reports indicate that
such an allosteric relationship exists (Kisselev et al., 2003;
Kleijnen et al., 2007; Li et al., 2001; Osmulski et al., 2009) as
yet no direct visualization of relevant conformational changes
has been described.
regulation of proteasome activity, as indicated by the recent
finding that the 19S subunit Rpn10 is itself ubiquitylated in order
to inhibit its ubiquitin-binding activity and to target it for protea-
somal degradation (Isasa et al., 2010), and the finding that an
inhibitor of the proteasome-associated USP14 deubiquitylating
enzyme can enhance 26S proteolytic function (Lee et al.,
2010). Finally, an important area of research that we have not
discussed is the assembly pathways of 20S and 26S protea-
such as a possible role of the 20S proteasome in promoting 19S
assembly, are still outstanding (Murata et al., 2009; Park et al.,
2010). Overall, these challenges invite future efforts to better
define functions of protein complexes that stably interact with
the proteasome, and the mechanisms by which they regulate
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