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Structural Organization of the 19S Proteasome
Lid: Insights from MS of Intact Complexes
Michal Sharon
1
, Thomas Taverner
1
, Xavier I. Ambroggio
2
, Raymond J. Deshaies
2
, Carol V. Robinson
1*
1 Department of Chemistry, University of Cambridge, Cambridge, United Kingdom, 2 Division of Biology, California Institute of Techno logy, Pasadena, California, United
States of America
The 26S proteasome contains a 19S regulatory particle that selects and unfolds ubiquitinated substrates for
degradation in the 20S catalytic particle. To date there are no high-resolution structures of the 19S assembly, nor of
the lid or base subcomplexes that constitute the 19S. Mass spectra of the intact lid complex from Saccharomyces
cerevisiae show that eight of the nine subunits are present stoichiometrically and that a stable tetrameric subcomplex
forms in solution. Application of tandem mass spectrometry to the intact lid complex reveals the subunit architecture,
while the coupling of a cross-linking approach identifies further interaction partners. Taking together our results with
previous analyses we are able to construct a comprehensive interaction map. In summary, our findings allow us to
identify a scaffold for the assembly of the particle and to propose a regulatory mechanism that prevents exposure of
the active site until assembly is complete. More generally, the results highlight the potential of mass spectrometry to
add crucial insight into the structural organization of an endogenous, wild-type complex.
Citation: Sharon M, Taverner T, Ambroggio XI, Deshaies RJ, Robinson CV (2006) Structural organization of the 19S proteasom e lid: Insights from MS of intact complexes. PLoS
Biol 4(8): e267. DOI: 10.1371/journal.pbio.0040267
Introduction
In eukaryotes, the ubiquitin–proteasome pathway is essen-
tial for eliminating damaged or misfolded proteins and for the
degradation of short-lived regulatory proteins [1,2]. The two
major subcomplexes of the 26S proteasome are the 20S
proteolytic core particle (;700 kDa) and the 19S regulatory
particle (;900 kDa). The 19S is attached at either or both ends
of the 20S [3]. Selection of proteasomal substrates by the 19S
regulatory particle occurs via the recognition of polyubiquitin
chains bound to proteins that are destined to be degraded [4].
After selection of the labeled substrate, the polyubiquitin
chain is then removed by the deubiquitinating enzymes Rpn11
[5,6] and Ubp6 [7]. The substrate is then unfolded and
translocated into the 20S channel, where it is degraded.
Nineteen different subunits have been identified in the 19S
proteasome from yeast as well as mammals [8–10]. This
regulatory particle can be dissociated further into two
subcomplexes, the base that binds directly to the 20S and a
peripheral lid [11]. The base consists of six AAA-ATPase
subunits, Rpt1–Rpt6, and four non-ATPase subunits, Rpn1,
Rpn2, Rpn10, and Rpn13. The ATPases in the base are
required for unfolding of substrate proteins [12] and channel
opening [13] before translocation of substrate into the 20S
cavity. The lid is composed of nine non-ATPase subunits:
Rpn3, Rpn5–Rpn9, Rpn11, Rpn12, and Sem1. The major
activity of the lid is proposed to be deubiquitination [5–7].
Rpn10 and additional ubiquitin receptor proteins that
reversibly associate with the proteasome, such as Rad23 and
Dsk2, deliver the ubiquitinated protein to the proteasome [14].
The proteasome lid subunits exhibit high homology to the
COP9 signalosome complex (CSN) [11], an essential regulator
of diverse cellular and developmental processes, and has been
found in most eukaryotic organisms ranging from yeast to
human (for reviews see [15–17]). The CSN contains eight core
subunits that assemble into a 450-kDa particle. The subunits
show a remarkable one-to-one sequence correspondence with
those of the 19S lid, suggesting a common ancestry [11]. Of
the lid components, Rpn11 is the most highly conserved
subunit, with 65% identity between yeast and human proteins
[6]. Recently it was identified as a Zn
2þ
-dependent metal-
loprotease responsible for substrate deubiquitination during
proteasomal degradation [5,6,18,19].
Although in the last few years functional characterization of
the 19S lid has progressed considerably, the assembly poses a
considerable challenge to structural biologists. High-resolu-
tion structural analyses are difficult since the complex is
assembled in vivo and different subunits are expected to be
dynamic and heterogeneous [20]. Only a low-resolution 3-D
image of the 26S has been observed by electron microscopy
[3], and the structural features of the lid complex were
difficult to discern. Pairwise interactions among the lid
subunits have been studied using the yeast two-hybrid system
[20–26]. Insight into the organization of the lid has also been
gleaned from analysis of lid subcomplexes in cells that express
mutant forms of specific lid subunits [25,26]. However, a
comprehensive model of subunit interactions within the lid
has not b een proposed, and no distinct ive t opolo gical
features, such as rings or well-defined channels, have been
identified.
In this study, we aim to obtain novel information regarding
Academic Editor: Michael Glickman, Biology Department, Technion-Israel Institute
of Technology, Israel
Received March 31, 2006; Accepted June 9, 2006; Published August 1, 2006
DOI: 10.1371/jou rnal.pbio.0040267
Copyright: Ó 2006 Sharon et al. This is an open-access article distributed under the
terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author
and source are credited.
Abbreviations: CSN, COP9 signalosome; MALDI, matrix-assisted laser desorption/
ionization; MS, mass spectrometry; MS/MS, tandem mass spectrometry; TOF, time
of flight
* To whom correspondence should be addressed. E-mail: cvr24@cam.ac.uk
PLoS Biology | www.plosbiology.org August 2006 | Volume 4 | Issue 8 | e2671314
P
L
o
S
BIOLOGY
the structural organization of the 19S lid from Saccharomyces
cerevisiae using both tandem mass spectrometry (MS/MS) of
the intact nine-component complex together with chemical
cross-linking and MS analysis. It is established that MS and
MS/MS can be applied to the study of intact noncovalent
complexes, but to date, the majority of examples were of
various complexes reconstituted in vitro [27–29]. While some
studies have been carried out on complexes isolated directly
from cells, including bacterial RNA polymerase [30], ribo-
somes [31,32], and RNA-processing complexes from yeast
[33], existing structural information aided the interpretation
of the resulting MS and MS/MS spectra. Here we show that the
end ogenous wild-type complex can be studied directly,
without the need to generate mutants, and the results
incorporated with existing yeast two-hybrid data to yield a
comprehensive model. This represents a significant advance
in MS methodology given the absence of existing high-
resolution structural data. Moreover, the fact that all directly
or indirectly interacting subunits can be observed within a
single spectrum highlights one of the fundamental advantages
of our methodology.
Using this approach the mass spectrum revealed not only
the intact complex but also a substoichiometric complex. In
addition, we identified the presence of an independent
subcomplex containing Rpn5, Rpn6, Rpn8, and Rpn9, where-
in Rpn8 occupies a central p osition. We could also
demonstrate that Rpn6, Rpn9, and Rpn12 are found on the
periphery of the complex. Complementary information from
cross-linking approaches enabled us to show that Rpn5 binds
to Rpn3. In addition, we could determine that the recently
identified lid subunit Sem1 [9] binds to both Rpn7 and Rpn3.
Taken together, our data allow us to propose a comprehen-
sive model for the subunit organization of the 19S lid that
incorporates our findings from MS with those of prior
analyses of pairwise interactions.
Results
Electrospray Ionizatio n MS and MS/MS of the Intact Lid
The electrospray mass spectrum of the intact 19S lid
isolated from S. cerevisiae is shown in Figure 1. Despite the fact
that the sample analyzed was the endogenous complex and
was not overexpressed, the spectrum is well resolved and has
one major series of peaks centered at 8,000 m/z. This major
charge state series corresponds to the nine protein compo-
nents of the lid, demonstrating clearly that all subunits must
be interacting, either directly or indirectly, and present at
unit stoichiometry. The measured mass of the intact complex
is 376,151 6 369 Da (Table 1). Interestingly, an overlapping
less-intense charge state series discerned at 7,750 m/z,
indicates the existence of a substoichiometric complex that
results from the absence of a subunit from the complex.
Calculation of the mass difference of these series indicates a
mass that is 50,270 Da less than that of the intact lid. This
corresponds to the absence of Rpn6 from the intact lid. The
similarity in the number of positive charges between the two
complexes suggests that both complexes are present in
solution (see below). An additional minor series of peaks is
centered at 5,500 m/z. The calculated mass for this lower m/z
charge series is 185,556 6 137 Da, indicating that a smaller
subcomplex of the lid is also present in solution.
To probe the composition and subunit organization of the
lid we used an MS/MS approach [31,34]. In such experiments,
a specific well-defined m/z range that encompasses the parent
ion is selected and accelerated through a collision cell at
increased argon pressures. This process gives rise to multiple
collisions in which the internal energy of the ions is
accumulated. When this energy reaches a threshold value,
dissociation occurs, yielding product ions. It is established
that for noncovalent assemblies, individual highly charged
subunits are dissociated from the parent ion, producing
‘‘ stripped’’ complexes with lower charge states than the
original ion [35–38]. As a consequence, we can distinguish
complexes formed in solution from those generated in the
gas phase since the former will have charge states commen-
surate with their mass [39], while those formed in the gas
phase will have anomalously low charge states. If we consider
the propensity for dissociation of different protein subunits
in the gas phase, it is known that under vacuum ionic
interactions are enhanced, while hydrophobic interactions
are weakened [40,41]. However, we cannot rule out the effect
of other factors that govern protein–protein interactions
such as contact area, planarity, shape complementarity, etc.
There is, however, a growing number of studies demonstrat-
ing that individual proteins that are expelled during tandem
MS are those that are known from high-resolution structures
to be more exposed and peripheral, while those at the core of
the assembly are retained in the ‘‘ stripped’’ complex
[30,32,42,43].
Applying this tandem MS strategy to the 376-kDa lid
complex, using a collision cell acceleration voltage of 100 V,
five series of ions were formed (Figure 2A). At m/z values
below 2,400, two highly charged series are observed. Their
measured masses correspond to 45,782 6 4 and 31,796 6 10
Da, in very close agreement with the mass calculated for Rpn9
and Rpn12, respectively. At high m/z values between 10,000
and 18,500 three further charge series are observed. Their
masses are consistent with the lid stripped of Rpn9 and/or
Rpn12. These results indicate that both Rpn9 and Rpn12 have
Figure 1. Electrospray Mass Spectrum of the 19S Lid Complex Isolated
from S. cerevisiae
The intact lid complex is observed between 7,250 and 9,000 m/z, with
the most intense peak at ’8,000 m/z. Well-resolved charge states are
observed, and the measured mass confirms the presence of all nine
subunits. An overlapping charge series is detected and corresponds to a
substoichiometric complex in which the Rpn6 subunit is absent (labeled
with asterisks). A subcomplex of the lid gives rise to a signal in the range
of 5,000–6,250 m/z.
DOI: 10.1371/journal.pbio.0040267.g001
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MS of the Proteasome Lid
weaker interactions with other subunits and are peripheral,
and hence are first to dissociate. Increasing the collision cell
voltage, and hence the internal energy of the lid complex,
induces the dissociation of an additional subunit (Figure 2B).
The new charge series observed at 2,000–2,400 m/z is assigned
to Rpn6, and the corresponding stripped complex appears
between 13,000 to 17,000 m/z. These results enable us to
deduce therefore that Rpn6, Rpn9, and Rpn12 interact
weakly and are presumably at the periphery of the lid
subcomplex.
Tandem Mass Spectrum of the Lid Subcomplex
In order to examine the composition of the 185-kDa
subcomplex observed in Figure 1 we again applied MS/MS. A
single charge state isolated from this complex and subjected
to collision with argon at an accelerating potential of 130 V
yielded a well-resolved tandem mass spectrum, despite the
low intensity of the subcomplex relative to the intact complex
(Figure 3A). Three series of ions are observed at the low m/z
region (Figure 3B). On the basis of their measured masses we
assign these species to Rpn5, Rpn6, and Rpn9. Between 8,000
to 16,000 m/z, ions arising from the removal of a single
subunit are observed (Figure 3C). Although Rpn8 and the
modified Rpn11 have similar masses, by simulating the charge
states based on the known sequence of the subunits we can
clearly distinguish between the two possibilities. We could
then conclude that Rpn8 and not Rpn11 is a component of
the subcomplex. These ions are therefore attributed to three
different heterotrimers; Rpn5:6:8, Rpn5:8:9, and Rpn6:8:9.
Interestingly, while Rpn8 is not detected as an individual
subunit, it is present in all three heterotrimers. By extrap-
olation, therefore, we can conclude that prior to the
collisional activation, the subcomplex is a tetramer composed
of Rpn5, Rpn6, Rpn8, and Rpn9. From the dissociation
pattern generated by the MS/MS experiment we can
determine that Rpn8 occupies a central position within this
subcomplex (Figure 3D). It is noteworthy that the two charge
series assigned to monomeric Rpn9 and its stripped hetero-
trimer, namely Rpn5:Rpn6:Rpn8, are the most intense series
in the spectra. This is further evidence for the ease of
dissociation of Rpn9, implying a relatively weak interaction
with neighboring subunits.
Chemical Cross-Linking Analysis
The cross-linking agent BS
3
is a bifunctional reagent
linking amines, and is selective for the N-terminal amino
group and lysine side chains, with an eight-carbon spacer arm
of 11.4 A
˚
. Denaturing gel electrophoresis before and after
chemical cross-linking [44] shows that the reaction resulted in
the production of covalently linked species labeled A to F
according to their electrophoretic mobility (Figure 4A). The
bands were assigned as described in Materials and Methods
(Table 2). As an example, Figure 4B shows part of the matrix-
assisted laser desorption/ionization (MALDI) mass spectrum
of the peptide mixture from band D containing Rpn3, Rpn5,
and Rpn8. The inset within the figure illustrates the MS/MS
analysis of one of the peptides corresponding to Rpn5.
Several conclusions can be drawn from the information
extracted from the six cross-linked bands. As Sem1 is present
in two bands cross-linked with Rpn7 and with Rpn3, we can
assume that Sem1 interacts with both subunits. Moreover,
due to the small molecular mass of Sem1, it is reasonable to
assume that Rpn3 and Rpn7 are in close proximity to one
another. In band C, Rpn3 is observed accompanied by Rpn5,
and we can therefore conclude that Rpn3 binds to Rpn5. In
addition, the subunits identified in bands D and E further
confirm the formation of the subcomplex comprising Rpn5,
Rpn8, and Rpn9 identified from our intact complex data. For
Table 1. Theoretical and Measured Masses of Proteins and Complexes
Protein/Complex Theoretical Mass (Da) Experimental Mass (Da) Figure Number
Individual components Rpn3
a,b
60,303 58,815 6 14 —
Rpn5
a,b
51,679 51,695 6 8—
Rpn6
a,b
49,685 49,688 6 11 —
Rpn7
a
48,827 48,829 6 11 —
Rpn8
a,b
38,223 38,224 6 8—
Rpn9 45,782 45,782 6 10 —
Rpn11
b,c
37,903 37,921 6 14 —
Rpn12
a
31,788 31,789 6 9—
Sem1
d
10,386 10,298 6 1—
Complex
e
Intact lid 373,040 376,151 6 369 1
Lid-Rpn6 323,352 325,881 6 160 1
Lid-Rpn6 323,352 323,836 6 294 2B
Lid-Rpn9 327,258 327,175 6 118 2A
Lid-Rpn12 341,252 341,082 6 98 2A
Lid-Rpn9:12 295,470 295,347 6 255 2A
Rpn5:6:8:9 185,389 185,556 6 137 1
Rpn5:6:8 139,607 139,574 6 42 3
Rpn5:8:9 135,701 135,575 6 261 3
Rpn6:8:9 133,694 133,435 6 146 3
a
The initiator Met is removed [54].
b
The N-terminal amino group is acetylated [54].
c
The sequence of Rpn11 was modified for purification purposes.
d
According to the experimental mass the initiator Met is removed and the N-terminal amino group is acetylated.
e
The masses were calculated from experimental masses.
DOI: 10.1371/journal.pbio.0040267.t001
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MS of the Proteasome Lid
Rpn11 (band F) we can deduce that it binds either directly to
Rpn5:9 or is anchored by Rpn3:7 to Rpn5:9. In summary,
cross-linking analysis allows determination of four additional
interactions within the lid.
Discussion
We acquired remarkably well-resolved mass spectra of the
intact lid from S. cerevisiae despite the fact that we anticipated
that the nine-component endogenous complex would be
heterogeneous. From our data we were able to determine the
subunit composition of the intact complex, the substoichio-
metric complexes present in solution, and those generated as
a result of our MS/MS approach. Together with chemical
cross-linking, our MS approach allowed us to obtain precise
additional information regarding the subunit organization of
the lid complex. Based on our data we constructed an
interaction map of the lid subunits (Figure 5). We identified a
heterotetrameric core structure formed by Rpn5, Rpn6,
Rpn8, and Rpn9, in which Rpn8 is centered. The absence of
Rpn11 from this complex shows that it forms after
purification and indicates that it is a stable subcomplex in
solution. In addition, we revealed that Sem1 binds to both
Rpn7 and Rpn3. Considering the small size of Sem1, we
assume that Rpn3 and Rpn7 are also interacting. We could
also determine that Rpn3 binds Rpn5. Interestingly, we
determined a substoichiometric complex of the lid in which
Rpn6 is absent. Close inspection of the spectrum suggests that
20%–30% of the purified lid complexes lack Rpn6. The MS/
MS results clearly show that both Rpn9 and Rpn12 and, to a
lesser extent, Rpn6, readily dissociate from the lid complex. It
is established that the extent of interaction between protein
subunits as well as their exposure underlies the release of
subunits in tandem MS of heteroligomeric complexes [45].
Figure 2. MS/MS of the 47þ Charge State of the Intact 19S Lid
(A) Applying a voltage of 100 V to the collision cell generates product ions from the loss of only Rpn9 and/or Rpn12. Peaks between 12,500–17,000 m/z
correspond to the loss of Rpn9 (blue squares), while the series at 10,000–12,000 m/z correspond to the loss of Rpn12 (red squares). The low intensity
charge series at 17,000–18,000 m/z indicate the loss of both Rpn9 and Rpn12 (magenta squares). At low m/z 1,000–2,400 series of peaks are assigned to
individual Rpn9 (blue circles) and Rpn12 (red circles). The data indicate the Rpn9 and Rpn12 have a relatively small contact surface with the other
subunits, which implies that they are exposed.
(B) Increasing the voltage to 120 V induces the dissociation of Rpn6 in addition to Rpn9 and Rpn12. The highly charged Rpn6 series is centered at ;
2,200 m/z (green circles), while the remaining stripped complex is observed between 13,000 to 18,000 m/z (green squares). In summary, Rpn9 and
Rpn12 are easily dissociated from the lid complex (blue). An increase in the collision cell voltage also induces dissociation of Rpn6 (purple).
DOI: 10.1371/journal.pbio.0040267.g002
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MS of the Proteasome Lid
This indicates that these subunits are therefore located at the
periphery of the complex. Consistent with this, Rpn9 is
known to be a nonessential subunit of the lid [10].
Interestingly, it has been shown previously that Rpn10, which
stabilizes the association of the lid and base, interacts with
both Rpn9 and Rpn12 [20]. This suggests that Rpn9 and
Rpn12 are located near the contact surface between the base
and the lid.
A very recent study has shown that an rpn6 temperature-
sensitive mutant grown at restrictive temperatures contained
a subcomplex comprising four out of the nine lid components,
Rpn5, Rpn8, Rpn9, and Rpn11 [26]. A similar study with an
rpn7 temperature-sensitive mutant demonstrated the pres-
ence of an Rpn5:6:8:9:11 subcomplex [25]. From our cross-
linking data, we could not conclude whether Rpn3:7 or Rpn5:9
were anchored to Rpn11. However the results from the rpn6
Figure 3. MS/MS of the Lid Subcomplex
(A) Spectrum showing the dissociation of the 34þ charge state.
(B) Expansion of the spectrum over the m/z range of 1,600–2,800. Three individual subunits are assigned Rpn5, Rpn6, and Rpn9 (orange, green, and blue
circles, respectively).
(C) Expansion of the 8,000–16,000 m/z region. Three different hetrotrimers are assigned: Rpn5:Rpn6:Rpn8, Rpn5:Rpn8:Rpn9, and Rpn6:Rpn8:Rpn9 (blue,
green, and orange stars, respectively). By extrapolation we can conclude that the subcomplex is composed of four subunits, Rpn5, Rpn6, Rpn8, and
Rpn9. Rpn8 is not observed in its monomeric form; however, because it exists in all three different hetrotrimers, we can conclude it adopts a central
position within the subcomplex.
(D) A schematic summarizes the structural data obtained from MS and MS/MS results. Red indicates core subunits, while purple and to a greater extent
blue specifies peripheral subunits.
DOI: 10.1371/journal.pbio.0040267.g003
Figure 4. Chemical Cross-Linking of the Lid Complex
(A) Bis-Tris 4%–12% denaturing gel, stained with Colloidal Blue, enables separation of proteins before () and after (þ) cross-linking revealed six new
bands, A-F. The absence of Sem1 in this analysis is attributed to its low molecular mass and is consistent with other analyses [10,25,56].
(B) A MALDI-TOF/TOF spectrum of trypsin digestion of band D. Three proteins were identified within this band: Rpn3 (yellow), Rpn5 (cyan), and Rpn8
(pink). Peaks labeled by asterisks were sequenced in order to identify the peptide unambiguously. The inset shows a low-energy collision-induced
dissociation of an Rpn5 peptide producing mainly C-terminal (y) sequence ions.
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MS of the Proteasome Lid
and rpn7 mutants support the scenario in which Rpn5:9
recruits Rpn11 into the Rpn5:6:8:9:11 subcomplex. In addi-
tion, the rpn7 mutation prevented the incorporation of Rpn3,
Rpn7, and Rpn12 into the lid. These results, together with the
evidence that Rpn5:6:8:9 exists as a stable independent
subcomplex in solution (Figure 1), are consistent with the
Rpn5:6:8:9 subcomplex forming the scaffolding core of the lid.
Taking together our data with existing data from analysis
of mutants [25,26] and results of previous two-hybrid analysis
[20,22,23,46] (Table 3), we propose a detailed interaction map
(Figure 6). The fact that all the data could be integrated into a
single model increases our confidence in the MS analysis. In
the model, two clusters become apparent. The first structural
cluster includes Rpn5, Rpn6, Rpn8, Rpn9, and Rpn11, in
agreement with the cluster suggested previously [20]. Within
the second cluster Rpn3, Rpn7, Rpn12, and Sem1 are
included, in accord with recent observations [25,26]. The
link between the two subcomplexes is between Rpn5:Rpn3,
and not Rpn7 as suggested by the analysis of an rpn7 mutant
[25]. These results explain previous observations in Schizo-
saccharomyces pombe that in cells in which rpn5 is deleted the
26S proteasomes misassemble [47]. Moreover, our structural
organization indicates that Rpn11 forms extensive interac-
tions with all three subunits, Rpn5, Rpn8, and Rpn9.
Furthermore, in our model Rpn6 is opposite Rpn9 and
Rpn12. As these may interact with Rpn10 at the lid–base
junction, it follows that Rpn6 is exposed to the cellular
environment.
Given the significant genetic similarity shared between the
lid and the COP9 signalosome, we investigated their
structural homology. Table 4 summarizes the protein–protein
interactions identified within the CSN subunits. Out of the 21
interactions detected, nine were observed among the 19S lid
complex (Figure 6), and eight of the interactions, although
not physically recognized, are feasible with our current
model, while 4 interactions are not in correspondence with
the lid structure. We speculate that the dense web of protein–
protein interactions detected within the CSN complex could
also be due to the tendency of the two-hybrid system to
produce false positives [22]. However, it is interesting to note
that six of the nine similar interactions and two of the eight
feasible interactions are found within the Rpn5, Rpn6, Rpn8,
Rpn9, and Rpn11 cluster. It is also worth noting that a 2-D
electron microscopy study [48] did not deduce a common
architecture for the two complexes. However, since both
particles have similar sizes, show asymmetric arrangements of
their subunits, possess a central channel, and share a large
number of similar interactions, the two particles are likely to
possess some common structural features.
The major function of the lid is determined by Rpn11, a
specialized isopeptidase that tightly couples the deubiquiti-
nation and degradation of substrates [5–7]. So far, no
catalytic activity has been described for any other subunit.
A recent study suggests that the role of these subunits is of a
scaffold for the other binding partners [49], namely Rpn11,
and the base subunits as well as the proteolyic substrates and
soluble cofactors. Interestingly, Rpn11 is unable to fold as an
Figure 5. An Interaction Map Summarizing the Data Obtained from Both
Native MS and Cross-Linking MS Analysis
Rpn5, Rpn6, Rpn8, and Rpn9 form a tetrameric structure, in which Rpn8
occupies a central position. The dashed line circling Rpn6 indicates that it
is present in substoichiometric amounts. Both Rpn7 and Rpn3 bind to
Sem1. Considering the small size of Sem1, it is reasonable to suggest that
Rpn3 and Rpn7 are in close proximity. In addition, we could determine that
Rpn3 binds to Rpn5. Rpn11 interacts with Rpn3:7 or Rpn5:9; these possible
interactions are labeled with a dashed line. No Interactions were
determined for Rpn12. Subunits are colored either red, purple, or blue,
indicating their increasing exposure within the complex.
DOI: 10.1371/journal.pbio.0040267.g005
Table 3. Subunit–Subunit Interactions within the 19S Lid from
Saccharomyces cerevisiae
Pair Species Method References
Rpn3-Sem1 Sc, MSc This study
Rpn7-Sem1 Sc, MSc This study
Rpn3-Rpn7 Sc 2h, MSc [20,23,25,46], and this study
Rpn3-Rpn5 Sc MSc This study
Rpn3-Rpn12 Sc 2h [20]
Rpn5-Rpn6 Sc 2h [20,26]
Rpn5-Rpn8 Sc 2h, MSn [20], this study
Rpn5-Rpn9 Sc 2h [20]
Rpn5-Rpn11 Sc 2h [20]
Rpn6-Rpn8 Sc MSn This study
Rpn8-Rpn9 Sc, Ce 2h, MSn [20,46], this study
Rpn8-Rpn11 Sc, Ce 2h [20,22,46]
Rpn9-Rpn11 Sc, Ce 2h [20,46]
Species: Ce, C. elegans; Sc, S. cerevisiae; methods: 2h, yeast two-hybrid; MSn, native mass
spectrometry; MSc, cross-linking mass spectrometry.
DOI: 10.1371/journal.pbio.0040267.t003
Table 2. Cross-Linked Subunits Obtained from Bands A–F
Band Cross-Linked Proteins
A Rpn7-Sem1
B Rpn3-Sem1
C Rpn3-Rpn5
D Rpn3-Rpn5-Rpn8
E Rpn3-Rpn5-Rpn8-Rpn9
F Rpn3-Rpn5-Rpn7-Rpn9-Rpn11
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MS of the Proteasome Lid
isolated subunit into a native, active conformation [6].
Neighboring subunit interactions are probably required to
position conjugates for cleavage by Rpn11. The multiple
protein–protein interactions observed in this study for S.
cerevisiae Rpn11 are in accordance with this assumption.
Given that the only interaction observed linking the two
protein clusters in the lid is between Rpn3 and Rpn5, this
implies that there is a flexible hinge region which allows
movement necessary for productive interaction of substrates
with Rpn11. Taking into consideration the fact that Rpn6 is
present at substoichiometric amounts, and that Rpn11 forms
extensive protein interactions, it is interesting to speculate
that after substrate binding, Rpn6 dissociates to expose active
Rpn11. Support for our hypothesis comes from the finding
that Rpn6 is essential for assembly of the lid; however, rpn6
mutants do not affect the activity of 26S proteasome after
assembly [26]. This may imply that Rpn6 acts as a fail-safe
mechanism, at least in S. cerevisiae, ensuring that active sites
are exposed only after assembly is completed. Given the high
natural abundance of proteasome particles in the cell and the
need to prevent indiscriminate proteolysis of proteins, the
masking of active sites before full assembly could be an
essential regulatory mechanism. We anticipate that further
examination of the levels of Rpn6 within the 19S lid complex
will validate this scenario.
In summary, the results presented here extend significantly
previous models based on pairwise interactions and allow
interesting comparison with the structure of the signalosome.
Our model is consistent with a core assembly of four subunits
that provides a scaffold for recruitment of additional
subunits. Also apparent from our analysis is the fact that
subunits are assembled into two protein clusters, possibly
providing a cleft in which a polyubiquitinated substrate can
be accommodated. Based on our finding of substoichiometric
quantities of Rnp6, and its position within the complex, we
speculate that a regulatory mechanism involving dissociation
of Rpn6 is responsible for the exposure of the active site of
Rpn11. Overall, therefore, the subunit architecture that we
have described will not only benefit further structural analysis
but has also provided insight into the function and regulation
of the 19S lid.
More generally, the results of this study highlight major
advantages over existing approaches for generating interac-
tion maps. For example, it is established that the yeast two-
hybrid approach is complicated by the fact that protein
interactions are queried pairwise. Interactions of subunits
within protein complexes, such as the anaphase-promoting
complex, can be underrepresented in two-hybrid datasets
because assembly of such complexes may require higher-order
interactions between multiple subunits. A second potential
problem is that endogenous proteins may facilitate two-
hybrid interactions that are not direct, and the presence of
endogenous copies of proteins may confuse the analysis [22].
The other existing approach that has been widely applied is to
generate mutants for functional analysis of this complex
[25,26]. This method can also be limited by the availability of
appropriate mutants, lackofknowledgeabouthowto
generate appropriate mutants, and interpreting the effects
of the mutations. The fundamental advantage of our approach
is that it is carried out with the wild-type endoge nous
complex, and heterogeneity and subcomplex formation are
immediately apparent from the spectra. Moreover, when
combined with gas-phase dissociation we are able to probe the
composition of protein subcomplexes and reveal peripheral
subunits leading to definition of the structural organization of
Table 4. Subunit–Subunit Interactions within the COP9
Pair Species Interaction
Type
References Similar Interaction
Observed in Lid
Csn1-Csn2 Sp, Hs 2h [24,48] (Rpn7-Rpn6)
Csn1-Csn3 Hs 2h, fb [24,48] Rpn7-Rpn3
Csn1-Csn4 At, Hs 2h [24] (Rpn7-Rpn5)
Csn1-Csn5 At, Hs 2h, fb [24,48] Rpn7-Rpn11
Csn1-Csn7 At 2h [20,24] Rpn7-Rpn9
Csn2-Csn3 Hs fb [48] Rpn6-Rpn3
Csn2-Csn5 Dm, Hs 2h, fb [24,48] (Rpn6-Rpn11)
Csn2-Csn6 Hs fb [48] Rpn6-Rpn8
Csn2-Csn7 Dm, Hs 2h, fb [24,48] (Rpn6-Rpn9)
Csn3-Csn4 At 2h [20] Rpn3-Rpn5
Csn3-Csn5 At 2h [20] Rpn3-Rpn11
Csn3-Csn7 At 2h [20] (Rpn3-Rpn9)
Csn3-Csn8 At 2h [20] Rpn3-Rpn12
Csn4-Csn5 At 2h [20,24] Rpn5-Rpn11
Csn4-Csn7 At, Dm 2h [20,24] Rpn5-Rpn9
Csn4-Csn8 At 2h [24] (Rpn5-Rpn12)
Csn5-Csn6 At 2h [20] Rpn11-Rpn8
Csn5-Csn7 Hs 2h, fb [20,48] Rpn11-Rpn9
Csn5-Csn8 At 2h [20] (Rpn11-Rpn12)
Csn6-Csn7 Hs fb [20,48] Rpn8-Rpn9
Csn7-Csn8 At, Hs 2h, fb [20,24,48] (Rpn9-Rpn12)
A bold font indicates that a similar interaction was identified in the 19S lid (Fig 6B); an
italic font corresponds to an interaction that was not found in the 19S lid complex;
parenthesis designates a possible interaction that could exist in the lid complex.
At, Arabidopsis thaliana; Dm, Drosophila melanogaster; Hm, Homo sapiens; Sp, S. pombe;
2h, yeast two-hybrid; fb, filter binding.
DOI: 10.1371/journal.pbio.0040267.t004
Figure 6. Structural Organization of the 19S Lid
A plot summarizing the interactions within the lid, based on interactions
identified here in combination with those determined previously. The
dashed line circling Rpn6 indicates that it is present in substoiciometric
amounts. Rpn10 (in gray) is added to the model to emphasize the
orientation of the complex relative to the base and the cellular
environment. The two apparent clusters are labeled. Subunits are
colored either red, purple, or blue, indicating their increasing exposure.
DOI: 10.1371/journal.pbio.0040267.g006
PLoS Biology | www.plosbiology.org August 2006 | Volume 4 | Issue 8 | e2671320
MS of the Proteasome Lid
the complex. The fa ct th at the assembly state of this
asymmetric complex, with multiple distinct subunits, can be
determined and results incorporated into a comprehensive
model highlights the tremend ous potential of MS. We
anticipate that this approach will be used for determining
the organization of many other important cellular complexes
for which very little structural data exists.
Materials and Methods
Materials. Bis(sulfosuccinimidyl) (BS
3
) suberimidate was purchased
from Pierce Biotechnolo gy (Rock ford, Illinois, United Stat es).
Modified trypsin and RNasin were obtained from Promega (Madison,
Wisconsin, United States). Centricon YM10 centrifugal filters were
purchased from Millipore (Billerica, Massachusetts, United States)
and Nanosep centrifugal devices were purchased from Pall (East Hills,
New York, United States). Other reagents and solvents used were
analytical reagents or high-performance liquid chromatography
grade where appropriate, and were purchased from Sigma-Aldrich
(Saint Louis, Missouri, United States).
Protein expression and purification. Plasmid pJS-TM53H [50] was
modified by overlap PCR to replace the Myc epitopes with 12
histidine codons, resulting in plasmid pT2H12. The S. cerevisiae
RJD415 strain (MATa can1–100 leu2–3,112 his3 trp1–1 ura3–1 ade2–1
pep4::TRP1 bar1::LEU2) was transformed with a PCR product from a
reaction with pT2H12 as the template using a forward primer
homologous to the 42 nucleotides preceding the stop codon of
RPN11, and a reverse primer with 41 nucleotides that are
homologous to the region following the stop codon. The last 21
nucleotides of the forward and reverse primers are respectively
homologous to the first and last regions of the tagging cassette of
pT2H12. Transformants were selected on synthetic dropout–HIS
agar plates. Integrants were screened for by nickel precipitation using
Ni-NTA Agarose (Qiagen, Valencia, California, United States)
followed by Western blot analysis of the eluates with anti-His and
anti-Pad1 (S. pombe Rpn11) antibodies. A positive transformant was
selected and designated as RJD2909.
The RJD2909 strain was grown in yeast/peptone/dextrose (YPD)
medium, and levels of Rpn11
H12
were monitored through late
stationary phase by nickel precipitation and detection by anti-His
antibodies. Expression levels of Rpn11
H12
remained constant through
all phases of growth. A large (170 l) growth of RJD2909 in YPD was
performed at the UCLA Fermentor Facility. The cells were harvested at
an OD
600
of 18.4 yielding a 1.7 kg cell pellet and were frozen at 80 8C.
A cell pellet of 70 g was thawed in 140 ml of chilled lysis buffer (25
mM Tris [pH 7.5], 200 mM NaCl, 20 mM imidazole, 10 mM b-
mercaptoethanol, 0.3% Triton X-100, 50 mM NaF, 1 mM PMSF) and 3
complete EDTA-free protease inhibitor cocktail tab lets (Roche,
Indianapolis, Indiana, United States), and added to 200 ml of chilled
500-lm glass beads inside a stainless steel chamber of a BeadBeater
(BioSpec Products, Bartlesville, O klahoma, United States). The
chamber jacket was filled with ice water, and the BeadBeater was
run for 1 min and allowed to cool for 1 min for six cycles at 4 8C. The
contents of the chamber were applied with vacuum to a filter unit
(Nalgene, Rochester, New York, United States) equipped with a 180-
lm nylon net filter (Millipore) to separate the glass beads from the
supernatant. The filtrate was subsequently spun at 5,000 rpm in an
Eppendorf 5804 centrifuge (Eppendorf, Westbury, New York, United
States) for 20 min to remove intact cells and precipitated material.
The cell lysis solution was applied to a column of Ni-NTA
Superflow resin (Qiagen) at a flow rate of 2 ml/min. The resin was
subsequently washed in wash buffer (25 mM Tris [pH 7.5], 500 mM
NaCl, 0.3% Triton X-100, 50 mM imidazole, 10 mM b-mercaptoe-
thanol) and starting buffer (25 mM Tris [pH 7.5], 200 mM NaCl, 20
mM imidazole, 10 mM b-mercaptoethanol), and the bound protein
was eluted with elution buffer (25 mM Tris [pH 7.5], 100 mM NaCl,
300 mM imidazole, 10 mM b-mercaptoethanol). Peak fractions were
collected and concentrated to a final volume of 2 ml by ultra-
centrifu gation in a Centric on concentrator (Millipore), with a
molecular weight cutoff of 100 kDa.
The sample was loaded and run on a 26:60 Sephacryl S-400 gel
filtration column (Amersham Biosciences, Little Chalfont, United
Kingdom) at a flow rate of 0.5 ml/min with gel filtration buffer (50
mM Tris [pH 7.5], 200 mM NaCl, 5% glycerol, 1 mM DTT). The lid
particle elutes in three peaks at 156 ml (peak 1), 164 ml (peak 2), and
196 ml (peak 3). Based on calculations derived from calibration
standards, the theoretical molecular masses of the three peaks are
1,800 kDa for peak 1, 200 kDa for peak 2, and 500 kDa for peak 3. In
peaks 1 and 2, the lid particle elutes as part of the 19S proteasome
regulatory particle. In peak 3, the lid particle elutes in a relatively
pure form. Fractions corresponding to peaks 2 and 3 were
concentrated to ;200 ll by ultracentrifugation in an Amicon
Ultra-15 concentrator (Millipore) with a molecular weight cutoff of
100 kDa. The protein concentrations of peak 2 and 3 were 4 mg/ml
and 10 mg/ml, respectively, as determined by a DC protein assay (Bio-
Rad, Hercules, California, United States).
MS conditions. To confirm the protein composition of the lid
complex the sample was analysed by established proteomics methods
using a high-resolution MALDI time of flight (TOF)/TOF (Applied
Biosystems, Foster City, California, United States), and a C18 75 lm 3
15 cm column on an UltiMate capillary system (Dionex, Sunnyvale,
California, United States) connected to a Probot fraction collector
(Dionex). All 9 subunits were identified by automated MALDI-TOF/
TOF peptide mapping and sequence database searching of S. cerevisiae
proteins. Electrospray ionization MS and MS/MS experiments were
conducted on a high mass quadrupole TOF–type instrument [42,51]
adapted for a QSTAR XL platform, [42,51]. In this instrument a flow-
restricting sleeve is installed in the front part of the Q
0
to increase
the pressure locally and allow collisional cooling of heavy ions. In
addition, the low-frequency extended mass range of Q
1
permits the
isolation of ions up to 35,000 m/z.
For analysis of the intact complex, prior to mass spectrometry 25
ll of the 25 lM sample of the lid complex was buffer exchanged using
a Nanosep centrifugal device with a molecular weight cutoff of 10
kDa into 1 M ammonium acetate. For accurate mass determination of
the individual protein subunits, the sample was washed and eluted
from a C
4
ZipTip column (Millipore) under denaturing conditions
(50% acetonitrle and 0.1% formic acid). All other mass spectra were
recorded from aqueous solution conditions in 1 M ammonium
acetate. Typically, 1.5 ll of solution was electrosprayed from gold-
coated borosilicate capillaries prepared in-house as described [52].
The following experimental parameters were used: capillary voltage
up to 1.2 kV; declustering potential, 150 V; focusing potential, 250V;
second declustering potential, 55V; and collision energy up to 200 V,
microchannel plate 2350 V. In MS/MS the relevant m/z value was
selected in the quadrupole and argon was used as a collision gas at
maximum pressure. All spectra were calibrated externally by using a
solution of cesium iodide (100 mg/ml). Spectra are shown here with
minimal smoothing and without background subtraction.
Assignment of peaks in mass spectra. The masses of the individual
subunits and the complexes were calculated from the spectra
according to a method in which the charge is iterated over the
measured mass value to determine the best fit to the experimental
data [53]. The accurate masses of the individual subunits were
determined from a denatured solution of the complex. The molecular
masses of Rpn5, Rpn6, Rpn7, Rpn8, Rpn9, Rpn11, and Rpn12 (Table
1) are consistent with those reported in the database and consistent
with the N-terminal modifications previously reported [54]. We could
also clearly identify that the initiator methionine is removed and the
N-terminal amino group is acetylated in the Sem1 subunit. In
addition, from the mass measured for Rpn3 we could determine that
the first 16 residues of this subunit are truncated.
Cross-linking reactions and tryptic digestion. Solutions of the
cross-linking reagent BS
3
were prepared immediately before use at a
concentration of 10 mM in buffer (200 mM NaCl, HEPES 50 mM [pH
7.5]). The cross-linking reaction was performed with a lid concen-
tration of 4.8 lM and BS
3
ratio of 1:50 for 2 h on ice. The reaction was
terminated by addition of 2 M Tris-HCl (pH 7.4) to give a final
concentration of 50 mM Tris-HCl. SDS-PAGE gel analysis was
performed on Bis-Tris 4%–12% gels using the Novex Colloidal Blue
kit (Invitrogen, Carlsbad, California, United States). For analysis of
gel bands by in-gel digest, proteins were digested with 2% trypsin wt/
wt at 37 8C for 12 h in 50 mM ammonium bicarbonate [55].
MS/MS analysis of cross-linked peptides. The digestion mixture was
subjected to a Nano LC column coupled to a Probot MALDI spotter
followed by MS and MS/MS analysis using air as collision gas on a
MALDI-TOF/TOF instrument (Applied Biosystems). Resolution on
this instrument was within 30 ppm and MS data were acquired using
software from Applied Biosystems. Typically, 5,000 spectra were
acquired for each sample in MS or each MS/MS experiment.
Throughout the assignment a set of four conditions were applied:
(1) the peak intensity had to be 10%; (2) only peptides identical to
those observed in the absences of cross-linking reagent were
considered; (3) each protein had to be represented by at least four
independent peptides; and (4) peptides for which there was more
than one amino acid composition possible had to be sequenced. The
assignment was validated further by analysis of the MALDI-MS/MS
PLoS Biology | www.plosbiology.org August 2006 | Volume 4 | Issue 8 | e2671321
MS of the Proteasome Lid
peptide sequencing spectra. Each protein had at least one peptide
confirmed by the MALDI-TOF/TOF sequence analysis. It was not
practical to apply these conditions to Sem1 due to its low molecular
weight (10 kDa). The presence of Sem1 was confirmed by identifying
two Sem1 peptides and sequencing by MALDI-MS/MS spectra.
Using our set of criteria for assignment, proteins identified in bands
A–F were considered cross-linked. In the case of band A three proteins,
Rpn3, Rpn7, and Sem1, were identified. Considering the apparent
migration mass for this band, the only combination of proteins
consistent with this mass was Rpn7-Sem1. Therefore, the presence of
Rpn3 is likely to arise from the two neighboring bands of native Rpn3
and band B. In all the other 5 bands the apparent masses deduced from
migration on the gel were within error, taking into account changes in
migration anticipated from cross-linking [44], and were therefore
entirely consistent with the cross-linked protein components.
Acknowledgments
We thank Rati Verma and Sarah Maslen for valuable assistance in the
preparation of this manuscript. CVR acknowledges the Walters-
Kundert trust.
Author contributions. MS, RJD, and CVR conceived and designed
the experiments. MS and TT performed the experiments. MS and TT
analyzed the data. XIA and RJD contributed reagents/materials/
analysis tools. MS, RJD, and CVR wrote the paper.
Funding. M.S. is grateful for funding from the European Molecular
Biology Organization and from the Wingate Scholarship.
Competing interests. The authors have declared that no competing
interests exist.
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