Molecular Cell, Vol. 11, 69–78, January, 2003, Copyright 2003 by Cell Press
ATP Hydrolysis by the Proteasome
Regulatory Complex PAN Serves Multiple
Functions in Protein Degradation
Nadia Benaroudj,1,3 Peter Zwickl,2 Erika Seemüller,2
Wolfgang Baumeister,2 and Alfred L. Goldberg1,*
1
Harvard Medical School
Department of Cell Biology
240 Longwood Avenue
Boston, Massachusetts 02115
2
Max-Planck-Institute for Biochemistry
Department of Molecular Structural Biology
Am Klopferspitz 18a
D-82152 Martinsried
Germany
Summary
To clarify the role of ATP in proteolysis, we studied
archaeal 20S proteasomes and the PAN (proteasomeactivating nucleotidase) regulatory complex, a homolog of the eukaryotic 19S ATPases. PAN’s ATPase activity was stimulated similarly by globular (GFPssrA)
and unfolded (casein) substrates, and by the ssrA recognition peptide. Denaturation of GFPssrA did not accelerate its degradation or eliminate the requirement
for PAN and ATP. During degradation of one molecule
of globular or unfolded substrates, 300–400 ATP molecules were hydrolyzed. An N-terminal deletion in the
20S ␣ subunits caused opening of the substrate-entry
channel and rapid degradation of unfolded proteins
without PAN; however, degradation of globular GFPssrA
still required PAN’s ATPase activity, even after
PAN-catalyzed unfolding. Thus, substrate binding activates ATP hydrolysis, which promotes three processes: substrate unfolding, gate opening in the 20S,
and protein translocation.
Introduction
In all cells, degradation of intracellular proteins requires
ATP hydrolysis (Goldberg, 1992). In eukaryotes, ATP
hydrolysis is necessary for ubiquitination of protein substrates, as well as for the proteolytic activity of 26S
proteasomes, the principal site of proteolysis in the cytosol and nucleus (Coux et al., 1996). 26S proteasomes
contain six homologous ATPase subunits in their 19S
regulatory particles. The precise roles of those ATPases
in proteasome function, as well as the amount of ATP
consumed during proteolysis, are uncertain. It has been
proposed that the ATPases catalyze unfolding of protein
substrates and promote their translocation into the central chamber of 20S proteasomes where proteolysis occurs (Lupas et al., 1993). More recently, it has been
shown that bacterial ATP-dependent proteases (ClpAP
and ClpXP) and the PAN (proteasome-activating nucleotidase) complex (the archaeal 19S homolog) unfold globular proteins (Benaroudj and Goldberg, 2000; Hoskins
*Correspondence: [email protected]
3
Present address: Institut Pasteur, Unité de Repliement et Modélisation des Protéines, 25-28 rue du Dr Roux, 75015 Paris, France.
et al., 2000; Kim et al., 2000; Navon and Goldberg, 2001;
Singh et al., 2000; Weber-Ban et al., 1999). The amount
of ATP consumed and the specific functions of ATP
hydrolysis are particularly difficult to analyze with 26S
proteasomes due to the requirement for ubiquitin conjugation to most substrates, the low intrinsic stability of
the 19S and its complexity (it contains at least 17 distinct
subunits), and the presence of six different ATPases
that probably serve distinct functions (Glickman et al.,
1998; Holzl et al., 2000; Rubin et al., 1998).
Although prokaryotes lack ubiquitin and 26S proteasomes, archaebacteria and certain eubacteria contain
20S proteasomes whose structural organization and
catalytic mechanism are very similar to those of the
eukaryotic 20S particles (Baumeister et al., 1998; De Mot
et al., 1999; Zwickl et al., 2000). Consequently, studies of
archaeal proteasomes have provided important insights
into the structure and enzymatic mechanisms of their
eukaryotic homologs. 20S particles have a barrel-shaped
structure composed of two outer ␣ rings and two central
␤ rings, which contain the proteolytic active sites sequestered within a central chamber (Lowe et al., 1995;
Wenzel and Baumeister, 1995). Because of the tight
packing of subunits, substrates enter through a narrow
channel in the ␣ ring which only permits entrance of
unfolded polypeptide chains or short peptides (Lowe et
al., 1995). In the native form of yeast 20S particles, this
channel is blocked by the amino-terminal extremities of
the ␣ subunits (Groll et al., 1997). As a consequence,
eukaryotic 20S particles exhibit low peptidase activity
because substrates cannot readily enter the internal
chamber. Deletion of the N termini of the ␣3 subunit
(residues 2–10) in yeast 20S prevents closing of the
channel and greatly enhances peptide hydrolysis (Groll
et al., 2000). Recent studies have suggested that the
19S ATPase Rpt2 is particularly important in opening
the axial pore (Kohler et al., 2001). In contrast to the
eukaryotic particles, the X-ray diffraction analysis of the
20S particles from Thermoplasma acidophilum did not
reveal a closed channel, presumably because the aminoterminal extremities of the ␣ subunits are disordered
and therefore not visible (Lowe et al., 1995). Moreover,
these particles by themselves appear fully active in degradation of fluorogenic tetrapeptides, but not of proteins. Therefore, it has been generally assumed that
the channel formed by the ␣ rings of the archaeal 20S
particles is in an open state (Groll et al., 2000; Kohler et
al., 2001; Lowe et al., 1995).
We have identified a 650 kDa ATPase complex from the
archaebacterium Methanococcus jannaschii named PAN,
which is probably the evolutionary precursor of the eukaryotic 19S (Zwickl et al., 1999). It is composed of identical
subunits closely homologous to the six ATPases of the
19S and stimulates markedly the degradation of proteins, but not of tetrapeptides, by archaeal 20S proteasomes. This process requires hydrolysis of ATP (Zwickl
et al., 1999), although the mechanisms linking ATP hydrolysis and protein degradation are unknown. Because
of its simple structural organization, lack of requirement
for substrate ubiquitination, and ease of expression in
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70
E. coli, PAN offers many advantages for mechanistic
studies. PAN exhibits certain activities characteristic of
molecular chaperones, e.g., it prevents aggregation and
promotes the refolding of denatured proteins (Benaroudj
and Goldberg, 2000). In addition, in the presence of
ATP, PAN catalyzes the unfolding of the globular green
fluorescent protein when its C terminus is fused to the
11 residue ssrA peptide (GFPssrA) (Benaroudj and Goldberg, 2000). This ATP-dependent “unfoldase” activity is
essential for the degradation of GFPssrA by archaeal
20S proteasomes, which otherwise do not degrade
GFPssrA. Like ATP-dependent proteases in E. coli and
other molecular chaperones, PAN’s ATPase activity is
stimulated by protein substrates (our unpublished data),
but the mechanism of this substrate-activated ATP hydrolysis and its role in translocation and degradation of
proteins remain unclear.
In the present studies, we have used the PAN complex
to analyze further the coupling between ATP hydrolysis,
substrate unfolding, and translocation. We have investigated how different types of protein substrates influence
ATP hydrolysis by PAN and the actual amount of ATP
consumed during hydrolysis of globular and denatured
proteins. In order to clarify the process of protein entry
into 20S particles, we have investigated whether archaeal 20S proteasomes, like eukaryotic 20S, have a functional gate that precludes protein entry. We have analyzed the degradation of different proteins by a 20S
proteasome deletion mutant ⌬␣(2-12)20S that lacks the
residues of ␣ subunits which in eukaryotic 20S function
as a gate for the channel. This study shows that one
function of PAN and ATP is to open the gate in the ␣
ring, and that ATP hydrolysis also serves an additional
role in facilitating the entry of substrates after their unfolding.
Results
Globular and Unfolded Substrates Stimulate PAN’s
ATPase Activity to Similar Extents
Like the ATPase component of bacterial ATP-dependent
proteases (lon, ClpA, and HslU) (Hwang et al., 1988;
Menon et al., 1987; Seol et al., 1997), PAN’s ATPase
activity is stimulated by protein substrates (our unpublished data). In order to understand the mechanism of
this activation, we compared the rates of ATP hydrolysis
by PAN in the presence of casein, which contains little
secondary or tertiary structures (Creamer et al., 1981),
and GFPssrA, a globular protein that is unfolded by PAN
(Benaroudj and Goldberg, 2000). These experiments
were performed with a 1000-fold molar excess of these
substrates, which causes maximal stimulation of PAN’s
ATPase activity (data not shown). Casein stimulated the
ATPase activity of PAN 5-fold. Surprisingly, PAN’s
ATPase activity was stimulated by GFPssrA to a similar
extent as casein (Figure 1A). Therefore, the maximal
stimulation of ATP hydrolysis is the same whether or not
the substrate is globular. Prior observations suggested
that only proteolytic substrates of the PAN-20S complex
enhance ATP hydrolysis (our unpublished data). Accordingly, native GFP (i.e., lacking the ssrA peptide), which
is not unfolded by PAN (Benaroudj and Goldberg, 2000),
did not stimulate PAN’s ATPase activity. When a 5-fold
molar excess of 20S proteasomes was added so that
all PAN should be bound to proteasomes, the basal and
the substrate-stimulated ATPase activity of PAN were
not significantly affected (Figure 1B). Thus, the proteinactivated PAN’s ATPase activity is not increased further
when PAN binds to 20S and when the substrate is translocated into the proteolytic chamber and degraded.
Peptide Binding to PAN Stimulates Its ATPase Activity
Native GFP lacking the ssrA peptide at its C terminus is
not unfolded or degraded by PAN and 20S proteasomes
(Benaroudj and Goldberg, 2000), and did not stimulate
ATP hydrolysis by PAN, presumably because it does not
interact with PAN. Thus, the binding of PAN to GFPssrA
seems to depend on the 11 residue ssrA extension. We
first tested whether this peptide could interact with PAN
and inhibit its ability to unfold GFPssrA. Upon transfer
of GFPssrA to 45⬚C, there was an immediate small drop
in its fluorescence signal (Figure 1C, closed circle),
which by 5 min reached a new steady state corresponding to GFPssrA fluorescence at 45⬚C. This decrease
reflected the effects of high temperature on the quantum
efficiency of fluorescence and does not indicate any
unfolding of GFP, which is a very thermostable protein
(Tm ⬎ 65⬚C) (Bokman and Ward, 1981). However, upon
addition of PAN (Figure 1C, closed triangle), there was
a large further decrease in fluorescence, indicating an
ATP-dependent unfolding of GFPssrA catalyzed by PAN
(Benaroudj and Goldberg, 2000). When the ssrA peptide
was added to the reaction containing GFPssrA-PAN and
ATP, the fall was prevented. Thus, the ssrA peptide
inhibited the unfolding of GFPssrA by PAN, presumably
because this peptide binds to the same site on PAN as
GFPssrA. Interestingly, the ssrA peptide also inhibited
the PAN-stimulated degradation of casein by 20S particles (Figure 1D), suggesting that these two very different
protein substrates bind to the same site on PAN.
We then tested whether the ssrA peptide could stimulate PAN’s ATPase activity. By itself, the ssrA peptide
stimulated PAN’s ATPase activity to a comparable extent as GFPssrA or casein when used at the same concentration (Figures 1E). Together, these findings indicate
that the binding of a peptide to PAN is sufficient to
stimulate its ATPase activity. It is very likely that substrate binding induces a change in the conformation
of PAN to a form that has a higher maximal ability to
hydrolyze ATP.
Substrate Denaturation Does Not Eliminate the
Requirement for PAN and ATP in Degradation
by 20S Proteasomes
Archaeal 20S proteasomes by themselves can degrade
certain unfolded proteins, e.g., the fully reduced ␣-lactalbumin (Wenzel and Baumeister, 1995). However, rapid
degradation of even the loosely folded casein, like the
breakdown of the globular GFPssrA, required PAN’s
ATPase activity (Benaroudj and Goldberg, 2000; Zwickl
et al., 1999). To further investigate the influence of the
substrate’s folding status, we tested whether denaturation of GFPssrA would make it competent for degradation by 20S independently of PAN and ATP. GFPssrA
was denatured by treatment with acid, which causes
a complete loss of fluorescence, modification of the
Role of ATP Hydrolysis in Proteasomal Degradation
71
Figure 1. Loosely Folded Substrates, Globular Substrates, and Peptides Activate ATP
Hydrolysis by PAN to the Same Extent and
Irrespective of the Presence of 20S Proteasomes
(A) ATPase activity of PAN in the presence of
loosely folded and globular substrates. 2.8
nM of PAN complex were incubated with 4
␮M of casein, GFP, or GFPssrA as indicated
at 45⬚C, and ATP hydrolysis was measured
in 50 mM Tris (pH 7.5), 1 mM DTT, 1 mM ATP,
and 10 mM MgCl2. Values are means plus
standard deviations of three independent experiments.
(B) Effect of 20S proteasomes on the basal
and substrate-activated ATPase activity of
PAN. 2.8 nM of PAN complex were incubated
as in (A) in the presence of 14 nM of 20S
proteasomes.
(C) SsrA peptide inhibits unfolding of GFPssrA
by PAN. The time course of fluorescence
change of 500 nM of GFPssrA alone (䊉) or in
the presence of 250 nM of PAN complex (䉱)
or 250 nM of PAN complex and 100 ␮M of
ssrA peptide (䊊) was followed at 45⬚C in 50
mM Tris (pH 7.5), 1 mM DTT, 2 mM ATP, and
10 mM MgCl2. The initial decrease in GFPssrA
fluorescence reflects the effects of 45⬚C on
GFP fluorescence and does not indicate unfolding, which occurred only after addition of
PAN and ATP.
(D) SsrA peptide inhibits casein degradation
by the PAN-20S complex. Degradation of 500
nM 14C-casein was followed with 2.1 nM of
20S proteasomes (open bars) or 2.1 nm 20S
proteasomes and 9.3 nM of PAN complex
(filled bars) in the presence of 550 ␮M of ssrA
peptide as indicated at 45⬚C in 50 mM Tris
(pH 7.5), 1 mM DTT, 1 mM ATP, and 10 mM
MgCl2. Values are means plus standard deviations of three independent experiments.
(E) SsrA peptide activates PAN’s ATPase activity. ATP hydrolysis by PAN was measured
without or with 4 ␮M of ssrA peptides as described in (A). Values are means plus standard deviations of three independent experiments.
absorption spectrum, and a disruption of the secondary
structure of GFP, as measured by circular dichroism
(Bokman and Ward, 1981; Ward and Bokman, 1982)
(data not shown). Surprisingly, the acid-denatured
GFPssrA was not degraded faster than globular
GFPssrA by 20S proteasomes (Figure 2). The addition
of PAN and ATP led to a similar stimulation of degradation of denatured GFPssrA as was found for globular
GFPssrA. Therefore, unfolding of this substrate does
not appear to be the rate-limiting step in its degradation
by 20S. It is noteworthy that the rate of casein degradation was three to four times higher than those of globular
and denatured GFPssrA, although casein and GFPssrA
activated PAN’s ATPase activity to a similar extent and
have comparable molecular weights (24 and 30 kDa,
respectively). The reason for the faster degradation of
casein is still unclear.
ATP Consumption during Degradation of Globular
and Unfolded Proteins
In order to determine how many ATP molecules are
consumed when a protein is degraded by the PAN-20S
complex, the rates of degradation of different proteins
and of ATP hydrolysis (in the presence of protein substrates) were measured simultaneously with different
concentrations of the PAN-20S complex. In initial experiments, conditions were established where hydrolysis of
both ATP and protein were linear with time. In order to
ensure that each PAN complex interacted with a 20S
particle (i.e., that PAN’s activated ATPase activity was
linked to proteolysis), these experiments were performed with a 5-fold molar excess of 20S proteasomes
over PAN. The rates of ATP hydrolysis were then plotted
against those of protein degradation (Figure 3). During
degradation of 1 molecule of casein, 312 molecules of
ATP were hydrolyzed (upper panel).
In similar experiments, when a molecule of globular
GFPssrA was degraded, 331 molecules of ATP were
hydrolyzed (Figure 3, middle panel). Thus, surprisingly,
ATP consumption during degradation by the PAN-20S
complex appears to be similar for a loosely folded and
a tight globular substrate. To test further if ATP consumption is in fact independent of the folding state of
the substrate, GFPssrA was fully denatured by acid
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72
Figure 2. PAN Stimulates the Degradation of Globular and Unfolded
Proteins by 20S Proteasomes
The degradation of 1 ␮M of 14C-GFPssrA, acid-denatured 14C-GFPssrA,
or 14C-casein was followed with 2.1 nM of 20S proteasomes alone
(open bars), with 9.3 nM of PAN complex added (striped bars), or
with 9.3 nM of PAN complex and 1 mM ATP added (filled bars) at
45⬚C in 50 mM Tris (pH 7.5), 1 mM DTT, and 10 mM MgCl2. Values are
means plus standard deviations of three independent experiments.
treatment just before incubation with PAN and 20S proteasomes. Three hundred and seventy three molecules
of ATP were hydrolyzed during the degradation of a
molecule of acid-denatured GFPssrA (Figure 3, lower
panel). Together, these findings indicate that a large
number of ATP molecules (at least 300) are hydrolyzed
during degradation of a protein substrate, regardless of
its folding status. These findings are consistent with our
observation that folded and unfolded protein substrates
stimulate PAN’s ATPase activity to a similar extent.
This calculation of energy cost for protein breakdown
includes the basal and the protein-activated ATP hydrolysis. This analysis assumed that all the ATP consumption in the presence of substrate is linked to proteolysis.
An alternative assumption would be that only the increase in ATP hydrolysis upon addition of substrate is
linked to proteolysis. Therefore, the basal ATP hydrolysis (35% of the total ATP hydrolysis on the average)
should be subtracted. On this basis, the actual ATP
cost during degradation of a molecule of those three
substrates is then about 220 molecules.
Entry of Unfolded Proteins Is Gated by the Amino
Termini of the 20S ␣ Subunits and Regulated by PAN
One apparent difference between the X-ray structure of
the prokaryotic and eukaryotic 20S particles lies in the
␣ rings. In the X-ray structure of 20S from T. acidophilum,
an axial channel with a diameter of 1.3 nm appeared
to connect the surrounding medium with the internal
chamber of the particle (Lowe et al., 1995). Such a channel through the ␣ rings was not observed in the X-ray
structure of yeast 20S particles (Groll et al., 1997). Indeed, entry of small peptides into the eukaryotic particle
appeared to be blocked by the N-terminal extremities
of the ␣ subunits that act as a gate for the channel
(Groll et al., 2000). However, these differences between
archaeal and yeast 20S might only reflect differences
between the forms of the particles crystallized in these
studies.
Figure 3. ATP Consumption by PAN during Degradation of Globular
and Unfolded Proteins by 20S Proteasomes
1 ␮M of 14C-casein (upper panel), 14C-GFPssrA (middle panel), or
acid-denatured 14C-GFPssrA (lower panel) were incubated with different concentrations of PAN (0, 1.54, 3.08, 4.62, 6.16, and 7.7 nM)
and of 20S proteasomes (0, 7.14, 14,28, 21.42, 28.56, and 35.7 nM)
at 45⬚C in 50 mM Tris (pH 7.5), 1 mM DTT, 1 mM ATP, and 10 mM
MgCl2 in a 100 ␮l vol reaction. 50 ␮l were used to measure ATP
hydrolysis, and 50 ␮l were used to measure protein degradation as
described in Experimental Procedures. The rates of ATP hydrolysis
were plotted against those of protein degradation. The data were
fitted to a linear function, and the slope (shown in the lower right
corner of each panel) was used to determined the number of moles
of ATP hydrolyzed during degradation of one mole of protein. Values
are means plus or minus standard deviations of three independent
experiments.
To investigate whether protein entry in the archaeal
20S is limited by a similar gate that was missed in prior
studies, we have deleted the residues 2–12 of the ␣
subunits in archaeal 20S particles. These residues correspond to those found to block the axial pore of the yeast
20S and whose deletion led to rapid substrate entry and
hydrolysis (Groll et al., 2000). The ␣ subunits of the wildtype and ⌬␣(2-12)20S proteasomes were expressed in
E. coli, and the resulting particles were analyzed by
electron microscopy. Previous studies showed that expression of the archaeal ␣ subunits alone leads to formation of seven membered rings which can assemble further into double rings (Zwickl et al., 1994). Accordingly,
both wild-type and ⌬␣(2-12) proteasome ␣ subunits form
Role of ATP Hydrolysis in Proteasomal Degradation
73
Figure 4. Deletion of Amino-Terminal Residues of 20S ␣ Subunits
Opens the Axial Channel of ␣ Rings
Electron microscopy top views of the ␣ subunits of wild-type (A)
and ⌬␣(2-12) 20S proteasomes (B) were performed as described in
Experimental Procedures. (C) represents the difference mapping of
the averaged pictures. The diameter of both complexes is approximately 11 nm.
particles with 7-fold symmetry (Figures 4A and 4B). Difference maps of averaged images of the wild-type and
the ⌬(2-12) ␣ ring showed the presence of mass in the
center of the ␣ rings formed by the wild-type 20S ␣
subunits, which was lacking in the center of the ⌬(2-12)
␣ ring (Figure 4C). Therefore, the wild-type archaeal proteasomes normally have their axial channel in a closed
form, and deletion of residues 2–12 in the ␣ subunits
led to the opening of this channel.
The ⌬␣(2-12) 20S variant enabled us to examine the
effect of gate opening as an isolated process, and thus
the ability of the ⌬␣(2-12) 20S variant to degrade peptides, unfolded proteins, or globular proteins was tested.
In contrast to eukaryotic 20S particles, which degrade
small peptides at a slow rate when their axial channel
is closed, the wild-type and the ⌬␣(2-12) archaeal 20S
particles did not differ in degradating tetrapeptides (Figure 5A). However, the ⌬␣(2-12) 20S degraded casein
29 times and acid-denatured GFPssrA 46 times faster
than wild-type 20S proteasomes (Figure 5B). In fact, the
⌬␣(2-12) 20S degraded protein substrates even more
efficiently than the wild-type 20S in the presence of
PAN and ATP (three and five times more for casein and
denatured GFPssrA, respectively). Moreover, degradation of casein and acid-denatured GFPssrA by the
⌬␣(2-12) 20S variant was not stimulated further by the
addition of PAN and ATP. However, the ⌬␣(2-12) 20S,
like the wild-type 20S, was not able to degrade the
globular GFPssrA, in the absence of PAN or ATP (Figures
5B and 5C). Presumably, the folded GFPssrA molecule,
unlike the unfolded one, is incapable of entering the
20S particles even when the channel is open. Therefore,
although the amino-terminal extremities of the ␣ subunits are not visible in the X-ray structure of archaeal
proteasomes, they function in gating, as they do in
eukaryotic 20S. The axial channel of archaeal 20S proteasomes in its native state is still permeable to tetrapeptides, but is not large enough to accommodate polypeptides, which require the presence of PAN and ATP
for efficient degradation. Once the channel of the 20S
proteasome is opened by deleting the N-terminal extremities of the ␣ subunits, the dependence on PAN and
ATP is abrogated for unfolded proteins. Therefore, we
conclude that PAN, in the presence of ATP, facilitates
the opening of the gate in the 20S proteasome, enabling
the translocation of unfolded polypeptides.
Figure 5. Deletion of Amino-Terminal Residues of 20S ␣ Subunits
Increases Rate of Degradation of Unfolded Proteins and Eliminates
Stimulation by PAN
(A) Wild-type and ⌬␣(2-12) 20S proteasomes degrade a tetrapeptide
at similar rates. The degradation of 100 ␮M of Suc-LLVY-amc was
carried out as described in Experimental Procedures with 420 nM
of wild-type (solid line) or ⌬␣(2-12) 20S (dashed line) at 45⬚C in 50
mM Tris (pH 7.5), 1 mM DTT, and 10 mM MgCl2.
(B) Degradation of GFPssrA, acid-denatured GFPssrA, and casein
by ⌬␣(2-12) 20S. Degradation of 1 ␮M of 14C-radiolabeled protein
was followed as in Figure 2. Values are means plus standard deviations of three independent experiments.
(C) PAN promotes degradation of GFPssrA by the ⌬␣(2-12) 20S. The
time course of fluorescence change of 250 nM of GFPssrA was
followed at 45⬚C in 50 mM Tris (pH 7.5), 1 mM DTT, and 10 mM
MgCl2 in the presence of 20 nM of ⌬␣(2-12)20S and 2 mM of ATP
(䊉); or of 20 nM of ⌬␣(2-12)20S, 10 nM of PAN complex, and 2 mM
of AMPPNP (䊊); or of 20 nM of ⌬␣(2-12)20S, 10 nM of PAN complex,
and 2 mM of ATP (䉱).
After Unfolding by PAN, GFPssrA Degradation
by the ⌬␣(2-12)20S Proteasome Still Requires
ATP Hydrolysis by PAN
It is noteworthy that the ⌬␣(2-12)20S by itself could
degrade the acid-denatured GFPssrA, but needed PAN
and ATP to hydrolyze the globular GFPssrA. We therefore tested whether this 20S variant could by itself degrade GFPssrA after this protein had first been unfolded
by PAN. We first incubated GFPssrA with PAN and ATP,
and when the loss of fluorescence had reached its maximal extent, further ATP hydrolysis was prevented by
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74
Figure 6. After Unfolding of GFPssrA by PAN,
Hydrolysis of ATP by PAN Is Still Essential
for Its Degradation by the ⌬␣(2-12)20S Proteasome Variant
(A) The time course of fluorescence change
of 250 nM of GFPssrA was followed in 50 mM
Tris (pH 7.5), 1 mM DTT, 1 mM ATP, and 10
mM MgCl2 at 45⬚C in the presence of 50 nM
of PAN (䉭, 䊉, 䉱). After 15 min of incubation
to allow unfolding of GFPssrA, 0 nM (䉭) or 20
nM of ⌬␣(2-12)20S was added to the samples
together with 2 mM of ATP␥S (䊉) or the equivalent volume of buffer (䉭, 䉱). If ⌬␣(2-12)20S
were present together with PAN and ATP
from the onset (䊊), only buffer was added.
(B) GFPssrA was incubated as in (A), and at
the indicated times, aliquots were withdrawn,
loaded on a 12% SDS-PAGE, and analyzed
by Western blot using an anti-histidine antibody. The antibody also recognizes the ␤
subunit of 20S proteasomes which carries a
(His)6 tag.
(C) GFPssrA amount on (B) was quantified
and relative amounts were determined using
the 0 time point as 100%. Relative amounts
of GFPssrA in lanes 1–3 (䊊), 4–6 (䉭), 7–9 (䉱),
and 10–12 (䊉) are plotted as indicated.
addition of an excess of ATP␥S that totally inhibited
PAN’s ATPase activity, as well as its ability to unfold
GFPssrA (data not shown). Then, the ⌬␣(2-12)20S was
added, and degradation of GFPssrA was assayed by
monitoring its fluorescence (Figure 6A) and the amount
of the protein on a SDS-PAGE gel (Figures 6B and 6C).
After incubating GFPssrA with PAN and ATP, when
the 20S variant was added to the preincubated mixture
of GFPssrA, PAN, and ATP (Figure, 6, closed triangles,
lanes 7–9) degradation of GFPssrA occurred to a similar
extent as when the ⌬␣(2-12)20S, PAN, and ATP were all
present from the onset (open circles, lanes 1–3). However, when the ⌬␣(2-12)20S was added in the presence
of ATP␥S to prevent further ATP hydrolysis, GFPssrA
was not degraded (closed circles, lanes 10–12), even
though most of it had been unfolded by PAN during
the preincubation (open triangles). Similar results were
obtained with wild-type 20S proteasomes (data not
shown). Some disappearance of GFPssrA was observed
on the electrophoretic gel at 15 min just after the addition
the ⌬␣(2-12)20S (lanes 7–9). This decrease corresponds
probably to inevitable degradation that occurs between
the withdrawing of the sample from the fluorescence
cuvette just after addition of 20S and the loading on the
electrophoretic gel due to the very high ability of this
variant to degrade unfolded substrates as noted in Figure 5B. Such degradation was minimized in experiments
using the wild-type 20S and when aliquots were withdrawn before addition of proteasomes at 15 min (data
not shown). Thus, although GFPssrA is unfolded by PAN,
its degradation by 20S still requires ATP hydrolysis, even
when the axial channel in the proteasome ␣ ring is in
an open state.
This finding strongly suggests that ATP hydrolysis
by PAN serves an additional function after substrate
unfolding and gate opening. This additional function
very likely is translocation of unfolded substrates. One
alternative possible explanation of this lack of degradation of unfolded GFPssrA by the open channel 20S variant in the presence of ATP␥S would be that, in the
absence of ATP hydrolysis, the unfolded substrate
forms a stable association with PAN, which precludes
entry of GFPssrA into the proteolytic chamber. However,
prior work has established that the complex between
GFPssrA and PAN is quite short lived, since PAN and
GFPssrA, even when it is acid denatured, could not be
Role of ATP Hydrolysis in Proteasomal Degradation
75
coimmunoprecipitated, even in the presence of ATP␥S
(Navon and Goldberg, 2001) (our unpublished data). Accordingly, PAN could not be retained by a GFPssrAcontaining resin (our unpublished data). In addition,
when PAN was added without ATP, it did not affect the
degradation of acid-denatured GFPssrA or of casein
(Figure 5B), as would be expected if these substrates
remained bound to PAN in the absence of ATP hydrolysis. This observation also allows us to rule out a model
whereby PAN hinders the entry of unfolded substrates
into the open channel 20S particle in the presence of
ATP␥S.
Thus, once a globular protein has been unfolded by
PAN, it is not simply released into the medium and does
not reach the 20S by passive diffusion. Instead, the
unfolded polypeptide has to be actively translocated by
PAN into 20S proteasomes, by a process that requires
ATP hydrolysis, even if 20S proteasomes have their axial
channel sufficiently open to digest the denatured protein.
Discussion
ATP Consumption during Protein Degradation
Although it has long been appreciated that intracellular
proteolysis requires ATP hydrolysis, the actual amount
of ATP required for the breakdown of a protein by proteasomes is not known. The present studies show that
when the protein-activated ATP hydrolysis and proteolysis proceed at linear rates, there is an apparent stoichiometry between the amount of ATP molecules hydrolyzed and protein molecules degraded that appears
independent of the nature of the protein substrate. At
least 300 ATP molecules are hydrolyzed by PAN when
proteasomes degrade 1 molecule of the protein substrate. This surprisingly high number indicates that protein unfolding and translocation into the 20S particle
requires many cycles of ATP hydrolysis. Even more surprising was the finding that the amount of ATP hydrolysis
per protein degraded is similar with the globular and
unfolded substrates. Thus, the rates of ATP hydrolysis
by PAN are independent of whether or not PAN has to
catalyze substrate unfolding. This conclusion is also
supported by our finding that globular and unfolded
substrates stimulate ATP hydrolysis to similar extents.
ATP-driven changes in the conformation of PAN must
underlie both the unfolding and translocation through
the ATPase ring. With PAN alone, the protein is probably
released into the solution. With 20S particles present,
these events are linked to substrate injection into the
proteolytic chamber. ATP consumption was identical
whether or not proteasomes were present. In contrast,
with the ATP-dependent proteases from E. coli, the
ATPase activity is altered by the proteolytic component;
e.g., ATP consumption by ClpA and ClpX decreases
upon addition of the ClpP protease (Burton et al., 2001;
Hwang et al., 1988) while ATP hydrolysis by HslU increases in the presence of HslV (Seol et al., 1997).
Because the 11 residue recognition sequence in
GFPssrA (ssrA peptide) by itself stimulated ATP hydrolysis to a similar extent as GFPssrA or casein, the activation of ATP consumption must be triggered by the interaction between the ATPase and the substrate. Although
no prior studies have addressed the energy costs for
proteasome function, the energy costs for protein breakdown by two E. coli ATP-dependent proteases have
been investigated. It has been shown that during degradation of Arc-ssrA, 150 mol of ATP were hydrolyzed by
ClpXP, and this number was independent of the substrate’s thermodynamic stability (Burton et al., 2001).
Thus, in degrading this small globular protein (about 75
residues), ClpXP consumed almost as much ATP as
found here for PAN-20S proteasome degrading the
much larger polypeptides casein and GFPssrA (220 and
268 residues). Menon et al. (1987) found that ATP hydrolysis by the lon protease is proportional to the number
of cleavages made in the substrate, but with this type of
enzyme (which contains ATPase and proteolytic active
sites in the same polypeptide chain and where these
processes may be more tighly coupled), significantly
less ATP is consumed than by PAN-20S complex.
The Rate-Limiting Step in Protein Degradation
by Proteasomes
Because acid treatment of GFPssrA destroyed its globular conformation and its ability to fluoresce, but did not
accelerate its rate of degradation, gross unfolding of
this protein can not be the rate-limiting step in its degradation. One alternative explanation of this result might
be that once the acid-denatured GFPssrA was diluted
into the reaction buffer (pH 7.5) at 45⬚C, it refolded rapidly and therefore showed similar rates of proteolysis
and ATP dependence as globular GFPssrA. However,
several findings make this trivial explanation very unlikely. First, after removing the acid, the denatured
GFPssrA did not regain its fluorescence, and after 2 hr,
its absorption spectrum was still indistinguishable from
that of denatured GFPssrA maintained in acid (data not
shown). Second, the yield of GFP refolding at 50⬚C is
only 19% of that at 37⬚C (Makino, 1997). Perhaps the
strongest evidence against refolding was that the aciddenatured GFPssrA, unlike globular GFPssrA, was rapidly degraded by the ⌬␣(2-12)20S proteasome. Therefore, its structure must differ from that of the globular
GFPssrA at 45⬚C. Although gross unfolding of globular
substrates does not appear to be the rate-limiting step in
degradation, translocation of the unfolded polypeptide
through the ATPase ring into the 20S particle could be
rate limiting. Consistent with this conclusion was the
finding that deletion of the N-terminal residues of the ␣
subunit that obstruct entry into the 20S led to much
faster degradation of unfolded proteins, even faster than
with PAN and ATP.
It has been generally assumed that protein unfolding
is the rate-limiting step in the functioning of ATP-dependent proteases. For example, it has been shown that
when isolated ClpX ATPase complex catalyzes the unfolding of GFPssrA, fluorescence of GFPssrA decreases
at a similar rate as GFPssrA is degraded by the holoenzyme ClpXP (Kim et al., 2000). The authors concluded
that unfolding is the rate-limiting step during ClpXPmediated proteolysis. On the other hand, Singh et al.
(2000) observed a difference in the rates of GFPssrA
unfolding and degradation. Nevertheless, both studies
suggested that the rate-limiting step precedes peptide
bond cleavage, in agreement with our conclusion that
the rate-limiting step occurs after protein unfolding, and
Molecular Cell
76
Figure 7. Model for the Energy-Dependent Steps of PAN and Proteasomes-Mediated Protein Degradation
Cut side views of 20S particles are schematized with the active sites represented as blue dots in the internal chamber. PAN is schematized
as a hexameric ring. The circle and pentagonal shape of the subunits represent the substrate-free and substrate-bound forms of PAN,
respectively. The gate which precludes entry of protein into 20S particles is represented in red.
thus probably is the translocation into the 20S particle.
For the unfolded proteins, translocation into the 20S is
clearly rate limiting, since their degradation was accelerated dramatically by deletion that enlarges the gate and
facilitates entry into the 20S (see below).
Gating of the Archaeal 20S Proteasomes
These studies have demonstrated that archaeal 20S proteasomes contain a functional gate in their outer ring
whose opening appears to be regulated by PAN. Indeed,
substrate entry into the archaeal 20S is normally blocked
by the N termini of the ␣ subunits, since deletion of
residues 2–12 of those subunits dramatically increased
the degradation of unfolded proteins and led to an opening in the ␣ ring visible by electron microscopy analysis.
Those residues are highly conserved among prokaryotic
and eukaryotic proteasomes, and the present findings
are similar to those obtained with analogous deletions
in yeast 20S particles (Groll et al., 2000). With eukaryotic
20S proteasomes in their native state, entry and exit of
small peptides requires gate opening by the 19S regulatory
particle (Kohler et al., 2001) or by PA28-related activators (Whitby et al., 2000). By contrast, entry of tetrapeptide into archaeal 20S particles occurs readily since the
⌬␣(2-12) 20S variant and the wild-type 20S-PAN complex did not degrade these small peptides faster than
the wild-type 20S proteasome (this work; Zwickl et al.,
1999). Thus, although archaeal proteasomes show ATPregulated gating that prevents entry of polypeptides, in
the basal state these particles do appear open to small
peptides, unlike the eukaryotic 20S.
ATP-Dependent Gate Opening
and Substrate Translocation
The most fundamental function of the PAN ATPase is
to facilitate entry of protein substrates into proteasomes
for degradation. Since the axial channel in the ␣ ring
has a gate that normally prevents protein entry, it is
not surprising that PAN and ATP trigger gate opening.
However, such a function was unexpected because the
X-ray structure of archaeal proteasomes showed an
open axial channel (Lowe et al., 1995). By interacting
with 20S particles, PAN probably induces an ATPdependent movement of the N termini of the ␣ subunits
that displaces them from the surface of the axial pore.
A similar mechanism has been suggested for the activation of 20S proteasomes by PA28 (Whitby et al., 2000).
When the channel into the 20S was opened by the
␣(2-12) deletion, degradation of casein and acid-denatured GFPssrA were rapid and independent of ATP and
PAN, while in the wild-type, where the channel was in its
closed state, degradation of these unfolded substrates
required PAN’s ATPase activity. Therefore, PAN appears
to open the gate in the ␣ ring. Interestingly, the rates of
degradation of unfolded proteins by the PAN-wild-type
20S complex was lower than those by the ⌬␣(2-12) 20S.
Therefore, protein binding to PAN and ATP-dependent
translocation through the ATPase ring into the 20S particle appear to be slower than diffusion directly through
the open axial channel, perhaps because the PANinduced opening in the ␣ ring may not be as large as in
the deletion mutant.
In eukaryotes, a homologous ATPase in the 19S particle activates proteolysis apparently by promoting the
opening of the gate into the 20S particles. Mutation in
one of the six ATPases (rpt2RF) strongly reduced the
ability of 20S particles to hydrolyze peptides (Rubin et
al., 1998). This defect could be suppressed by a mutation
in the 20S particle that leads to the opening of the channel in the ␣ ring (Kohler et al., 2001). Therefore, Rpt2
appears to be particularly important in regulating opening of the axial channel and peptide entry, although
analogous mutations in the other five ATPase subunits
were not analyzed.
Even after its unfolding by PAN, and even when the
axial channel of the 20S particles was open, hydrolysis
of ATP by PAN was still required for degradation of
globular GFPssrA. In contrast, when the GFPssrA was
denatured with acid, PAN was not required for its translocation into the ⌬␣(2-12)20S. This difference in the requirement for PAN between GFPssrA unfolded by PAN
Role of ATP Hydrolysis in Proteasomal Degradation
77
and GFPssrA denatured by acid is intriguing. Perhaps,
when PAN unfolds this substrate, it does not adopt a
stable extended conformation that can diffuse readily
into the 20S particles. The gross unfolding of GFPssrA
occurs on the surface of PAN’s ring, and probably precedes its PAN-mediated substrate threading through
PAN’s central opening into the 20S (Navon and Goldberg, 2001). Perhaps, this ATP-dependent threading
through PAN might also eliminate residual secondary
structural elements of GFPssrA that prevent its diffusion
into ⌬␣(2-12)20S particles.
In summary, these findings allow us to dissect several
distinct steps where PAN is required during protein degradation (Figure 7). The substrate binds to PAN, which
triggers a change in its conformation leading to an increased capacity to hydrolyze ATP. Substrate binding
to PAN can occur on the isolated PAN or on the PAN-20S
complex. This substrate-activated ATPase (i) facilitates
unfolding of the substrate, probably at the surface of
the ATPase ring, (ii) favors an open form of the 20S axial
channel, and (iii) promotes substrate translocation into
the internal chamber of the 20S particles where peptide
bond cleavage occurs. (Because ATP hydrolysis by PAN
can also occur in the absence of a protein substrate,
although at a lower rate, it seems likely that gate opening
in the 20S by PAN occurs also in the absence of a
substrate.) These distinct functions of PAN’s ATPase
activity are very likely to apply to the closely homologous
19S ATPases, although in addition one ATPase subunit
in the 19S is involved in interacting with ubiquitinated
proteins (Lam et al., 2002).
PAN belongs to the large family of AAA ATPases
(Ogura and Wilkinson, 2001), many of which have been
shown to catalyze protein remodeling. The present findings on substrate-activated ATPase activity, the energy
costs of unfolding and translocation, and the multiple
roles of ATP hydrolysis are likely to apply also to these
related multimeric ATPases. Analogous studies of these
AAA family members may also help clarify their molecular mechanisms.
Experimental Procedures
Proteins and Peptides
PAN and archaeal 20S proteasomes were purified as previously
described (Benaroudj and Goldberg, 2000; Zwickl et al., 1999). Molar
concentrations of PAN were expressed on the basis of a 650 kDa
complex (Zwickl et al., 1999). (His)6-GFP and (His)10-GFPssrA were
purified as described earlier (Benaroudj and Goldberg, 2000) with
the following modifications. After purification on an Ni-NTA agarose
column, the fractions containing GFP proteins were pooled and
subjected to a MonoQ HR5/5 (Amersham Pharmacia Biotech) anion
exchange column equilibrated with 50 mM Tris-HCl (pH 7.5) and
1 mM DTT. A linear gradient of NaCl from 0 to 500 mM was applied,
and the GFP proteins eluted at approximately 150–200 mM. Fractions containing (His)6-GFP or (His10)-GFPssrA were pooled and
dialyzed against 50 mM Tris-HCl (pH 7.5). The ssrA peptide (NH2CAANDENYALAA-CO2H) was a generous gift from Y.I. Kim (Kim et
al., 2000). Casein was purchased from Sigma.
Construction of ⌬␣(2-12)20S Proteasome Variant
Deletion of residues 2–12 in the ␣ subunits of archaeal 20S proteasomes was performed by reverse PCR mutagenesis using the
pRSET5a-␤(His)6-␣ described earlier (Seemuller et al., 1995; Zwickl
et al., 1992). The resulting pRSET5a-␤(His)6-⌬␣(2-12) plasmid was
then introduced in E. coli BL21(DE3) cells, and the ⌬␣(2-12)20S
proteasome variant was purified as the wild-type 20S proteasomes.
Expression and Purification of 20S ␣ Rings
The ␣ gene was elongated at the 3⬘ end by an affinity tag that codes
for 6 histidine residues and cloned into pRSET5a expression vector.
Then, the tagged ␣ gene was deleted for 33 nucleotides at the 5⬘
end, which codes for residues 2–12, thus generating the ⌬(2-12)␣
gene. Wild-type and mutant ␣ proteins were expressed in BL21(DE3)
and purified as 20S proteasomes.
Hydrolysis of ATP
Hydrolysis of ATP was assayed by following production of inorganic
phosphate at 45⬚C as described elsewhere (Ames, 1966; Zwickl et
al., 1999).
Degradation of 14C-Radiolabeled Proteins
14
C-radiolabeled casein and GFPssrA were prepared by reductive
alkylation, a gentle procedure that does not produce dramatic
changes in protein structure (Rice and Means, 1971) and stored in
50 mM Tris-HCl (pH 7.5). The radiolabeling of GFPssrA did not alter
its fluorescence properties. Breakdown of these 14C-radiolabeled
proteins was conducted in the presence of 20S proteasomes, PAN,
and ATP as indicated in 50 mM Tris (pH 7.5), 1 mM DTT, and 10
mM MgCl2 at 45⬚C. After 40–60 min incubation, trichloroacetic acid
at a final concentration of 12% was added together with 100 ␮g of
BSA as a carrier. Acid-soluble radioactive peptides products were
quantify by scintillation counting.
Peptides Hydrolysis
100 ␮M of fluorogenic Suc-LLVY-amc peptide diluted in 100%
DMSO was incubated with 420 nM 20S proteasomes at 45⬚C in 50
mM Tris (pH 7.5), 1 mM DTT, and 10 mM MgCl2. Hydrolysis of the
peptide was followed by measuring the relase of amc (7-amido-4methylcoumarin) in a spectrofluorometer (␭ex 380 nm; ␭em 440 nm).
GFPssrA Fluorescence
Measurements of GFPssrA fluorescence were obtained at 45⬚C with
excitation at 400 nm and emission at 510 nm. GFPssrA was diluted
in 50 mM Tris (pH 7.5), 1 mM DTT, and 10 mM MgCl2, in the presence
or the absence of PAN, 20S proteasomes, and the indicated nucleotide.
Denaturation of GFPssrA
GFPssrA or [14C]-GFPssrA were incubated with 50 mM HCl (pH 2.0)
for 10 min at room temperature. Denatured GFPssrA was diluted
directly into reaction mixture at 45⬚C and used immediately.
Electron Microscopy and Image Analysis
Purified proteins were negatively stained with 2% uranyl acetate.
600 end-on views of wild-type ␣ rings and of mutant ␣ rings were
extracted and aligned by conventional cross-correlation methods.
After performing correspondence analysis, the projections were interpreted according to their coordinates along the dominating first
factor.
Acknowledgments
This work was supported by research grants from NIH to A.L.G.
(GM46147 and GM 51923), postdoctoral fellowships from Association pour la Recherche contre le Cancer and Human Frontier Science
Program Organization to N.B., and by a postdoctoral fellowship
from the Deutsche Forschungsgemeinschaft to P.Z. Authors are
very grateful to Günter Pfeifer for her excellent technical assistance
in electron microscopy and image analysis. We thank Prasanna
Variath, Marion Schmidt, and Tom Rapoport for critically reading
this manuscript, and Sarah Trombley for assistance in preparation
of the manuscript.
Received: June 23, 2002
Revised: October 31, 2002
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