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Originally published In Press as doi:10.1074/jbc.M511951200 on January 30, 2006

J. Biol. Chem., Vol. 281, Issue 14, 9569-9575, April 7, 2006
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20S Proteasomes Have the Potential to Keep Substrates in Store for Continual Degradation*Formula

Michal Sharon{ddagger}1, Susanne Witt§, Karin Felderer§, Beate Rockel§, Wolfgang Baumeister§, and Carol V. Robinson{ddagger}2

From the {ddagger}Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom and Department of Molecular Structural Biology, §Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany

Received for publication, November 7, 2005 , and in revised form, January 3, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The 20S core of the proteasome, which together with the regulatory particle plays a major role in the degradation of proteins in eukaryotic cells, is traversed by an internal system of cavities, namely two antechambers and one central proteolytic chamber. Little is known about the mechanisms underlying substrate binding and translocation of polypeptide chains into the interior of 20S proteasomes. Specifically, the role of the antechambers is not fully understood, and the number of substrate molecules sequestered within the internal cavities at any one time is unknown. Here we have shown that by applying both electron microscopy and tandem mass spectrometry (MS) approaches to this multisubunit complex we obtain precise information regarding the stoichiometry and location of substrates within the three chambers. The dissociation pattern in tandem MS allows us to conclude that a maximum of three green fluorescent protein and four cytochrome c substrate molecules are bound within the cavities. Our results also show that >95% of the population of proteasome molecules contain the maximum number of partially folded substrates. Moreover, we deduce that one green fluorescent protein or two cytochrome c molecules must reside within the central proteolytic chamber while the remaining substrate molecules occupy, singly, both antechambers. The results imply therefore an additional role for 20S proteasomes in the storage of substrates prior to their degradation, specifically in cases where translocation rates are slower than proteolysis. More generally, the ability to locate relatively small protein ligands sequestered within the 28-subunit core particle highlights the tremendous potential of tandem MS for deciphering substrate binding within large macromolecular assemblies.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cells depend upon the regulated degradation of their various proteins to maintain homeostasis and change metabolic state. A key component of this process is the proteasome, a large multisubunit proteolytic machine (for reviews, see Refs. 1-3). The two major subcomplexes of the 26S proteasome are the ~700-kDa proteolytic core particle, known as the 20S proteasome, and the ATP-dependent regulatory particle known in eukaryotes as the ~900-kDa 19S complex. It is generally accepted that archaeal 20S proteasomes function in conjunction with a similar, albeit simpler, ATPase complex termed the proteasome-activating nucleotidase (4-6). In both kingdoms the regulatory "cap" selects, unfolds, and translocates substrates into the 20S core particle for proteolysis.

The structure of the 20S core particle of the proteasome from Thermoplasma acidophilum is comprised of only one type of {alpha}- and beta-subunit and has been studied extensively by electron microscopy and x-ray crystallography (7-10). The assembly is built from four stacked rings containing seven subunits in each ring: two internal beta-rings harboring catalytic beta-subunits and two outer {alpha}-rings that define a gated channel leading into the internal proteolytic chamber (11). The four rings form a hollow interior with three large chambers interconnected by a narrow channel with restricted orifices. The selectivity of the proteasome is achieved by this architecture that occludes its active sites within its central chamber, leading to a model by which unfolded substrates are fed into the central catalytic cavity as extended chains (12). The proteins are degraded in a processive manner without releasing the substrate before it is degraded to peptides (13-15). The rate of proteolysis in both eukaryotic and prokaryotic proteasomes is determined not only by the kinetics of degradation but also by the translocation of substrate molecules into and product molecules out of the proteasome (16, 17).

These steric constraints raise several questions about how substrate molecules are translocated through the internal cavities to the proteolytic core: specifically, whether more than one protein can bind simultaneously and the role of the two antechambers in the translocation process. These questions are challenging to address using established biochemical and structural biology approaches. Results from solution-based methods dependent upon molecular mass or changes in cross-sectional area are often ambiguous because the differences between free and ligand-bound forms of the complex are relatively small. Because the proteasome has broad substrate specificity and there are no constraints on the substrate conformation within the internal cavities, it is difficult to interpret additional density in terms of stoichiometry in both electron microscopy (EM)3 and x-ray crystallography analyses of the ligand-bound proteasome. Moreover, the D7 symmetry of the archaeal proteasome poses an additional difficulty in defining unambiguously the alignment of the substrate-bound proteasome particles in EM images. We therefore decided to apply mass spectrometry (MS) in conjunction with EM to resolve the various ligand-bound forms.

Non-covalent macromolecular complexes such as GroEL (18), proteasomes (19, 20), and ribosomes (21) can survive the phase transition from the electrospray process in solution to the gas phase of the mass spectrometer. This survival, together with an understanding of the factors that influence the transmission of macromolecular ions, prompted us to develop an instrument optimized for high mass tandem experiments (22). Because of the small difference in mass between ligand-bound and free forms of the proteasome it has not been possible to resolve the various substrate-bound forms of the core particle from mass spectra alone (20). However, using a tandem MS approach we have shown previously that it is possible to dissociate only a selected region of the mass spectrum, allowing us to resolve overlapping charge states that arise from polydispersity (23) and the presence of different substrate-bound forms of complexes (24). We reasoned that such an approach would also allow us to resolve the various ligand-bound forms of the proteasome.

To trap substrate molecules within the T. acidophilum proteasome we formed host-guest complexes between the 20S proteasome and either cytochrome c (Cyt c) (25) or green fluorescent protein (GFP). To prevent degradation of the two substrates the proteasome was covalently inhibited. In both host-guest complexes the EM data indicate additional density within the three proteasome cavities. However, because particle-averaging methods were employed to generate these images, it is not possible to determine unambiguously whether the three chambers are occupied simultaneously. Accelerating selected ions formed from the proteasome-substrate complex through a gas-filled collision cell induced dissociation of {alpha}-subunits from the outer rings with concomitant loss of substrate molecules. By contrast, under the same conditions, substrate molecules protected within the catalytic chamber are retained, enabling us to define the stoichiometry and location of the sequestered Cyt c and GFP molecules.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Substrates, Inhibitors, Antibodies, and Chemicals—Horse heart ferricytochrome c (Cyt c) was purchased from Sigma. His6-GFP was obtained by recombinant high level expression of an isopropyl-1-thio-beta-D-galactopyranoside-inducible plasmid (pTrc99A_His6-GFP) using the E. coli strain KY2266. His6-GFP was purified in a two-step purification scheme. A nickel-affinity chromatography step (His-Trap columns; GE Healthcare), followed by a size-exclusion chromatography step (HiLoad Superdex 200; GE Healthcare), yielded a homogeneous His6-GFP preparation. Purity of the proteins was confirmed by native and SDS-PAGE. The proteasome inhibitor clasto-lactacystin-beta-lactone was purchased from CalBiochem. The monoclonal antibodies anti-Cyt c and anti-GFP were used at dilutions of 1:500 and 1:200, respectively, and were purchased from Santa Cruz Biotechnology Inc.

Preparation of Proteasome-Substrate Complexes—The T. acidophilum proteasome was isolated as described previously (26). Formation of the host-guest proteasome complex was monitored using their spectroscopic signatures, 409 nm (Cyt c) and 395 nm (GFP) as previously described (25). Briefly, 20S proteasomes (1 µM) were inhibited with clasto-lactacystin-beta-lactone (70 µM) for 30 min at 22 °C prior to mixing with substrate. Substrates (100 µM) were unfolded in 2.3 M guanidine HCl at 60 °C and mixed with the inhibited proteasome to give a final concentration of 2 M guanidine HCl. The proteasome-substrate solution was incubated at 60 °C for 30 min and then cooled rapidly on ice for 1 h. For GFP the mixture was diluted 10-fold and both complexes separated from unbound substrate by size exclusion chromatography. Control samples for single particle EM and MS were prepared as described above without heating of the substrate molecule prior to incubation with 20S proteasomes. For the MS control experiment excess ligand was not removed prior to analysis, whereas for the EM control unbound substrate was removed as described above. To distinguish between sequestered and externally bound substrate, SDS-PAGE followed by Western immunoassay was carried out with either anti-Cyt c or anti-GFP antibodies on denatured host-guest complexes, control complexes, and Cyt c or GFP. The antibodies reacted only with the individual Cyt c and GFP proteins and the denatured host-guest complexes (data not shown); however, no interaction was obtained with the EM control complexes.


Figure 1
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  		FIGURE 1.
Cryo-electron microscopy of host-guest complexes of the proteasome. Left, image of control 20S complexes taken at 160 kV and a defocus of -3.5 µm showing that most particles appear as side view projections. Average of controls (i) and host-guest complexes (ii) as well as difference images host-guest complex minus control (iii). Upper row, Cyt c; lower row, GFP. Protein is shown in black. Scale bar, 10 nm.

 
Cryo-electron Microscopy—For cryo-electron microscopy (cryo-EM) 3 µl of protein solution containing host-guest complexes consisting of inhibited 20S proteasomes with either Cyt c or GFP were applied to glow-discharged lacy carbon grids, incubated for 1 min, washed twice with 3 µl of deionized water, and plunged into liquid ethane. Focal pairs (defocus values -2.5 and -3.5 µm, respectively) of ice-embedded 20S particles were recorded under low dose conditions on a Philips CM20 microscope operated at 160 kV and at a nominal magnification of x46750 using a CCD camera (pixel size 14 µm, pixel size on the specimen level 0.3 nm). The protocols used for image processing are described in the supplemental information.

Mass Spectrometry—Electrospray ionization-MS and tandem MS (MS/MS) experiments were conducted on a high mass Q-TOF-type instrument (22) adapted for a QSTAR XL platform (19). Immediately prior to MS, aliquots were buffer exchanged using Bio-Rad Biospin columns into 1 M ammonium acetate solution and stored on ice. Typically, 2 µl of solution was electrosprayed from gold-coated borosilicate capillaries prepared in-house as described (27). The following experimental parameters were used: capillary voltage up to 1.2 kV, declustering potential 150 V, focusing potential 250 V, declustering potential-2 55 V, and collision energy up to 130 V, MCP 2350. In tandem MS argon was used as a collision gas. All spectra were calibrated externally by using a solution of cesium iodide (100 mg/ml). The assignment approach is described in detail in the supplemental information.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
To visualize bound substrate within 20S proteasomes, we imaged host-guest complexes using cryo-EM. 20S proteasomes appear well separated when negatively stained on continuous carbon but aggregate when embedded in vitreous ice, resulting in high particle density (Fig. 1). Therefore a defocus -2.5 µm was used for the first image of a focal pair in order to have enough contrast to align individual images. The side view averages obtained for the Cyt c and GFP host-guest complexes together with the two controls clearly display the catalytic chamber and the two antechambers. In the control samples all three chambers appear to be unoccupied. For both host-guest complexes, the contrast inside the particles is diminished, which we attribute to bound substrate. The difference images show density for substrates in all three cavities. The Cyt c difference image appears narrower than the corresponding GFP complex. Furthermore, although the densities in all three cavities are similar for GFP, for Cyt c the density in the catalytic chamber appears more elongated than the respective densities at the positions of the antechambers. These observations might reflect the presence of more than one Cyt c molecule in the central chamber. Because we cannot group the particle projections unambiguously, according to their different rotational states around the cylinder axis of the 20S particle, we cannot conclude whether the two antechambers and the catalytic chamber of the proteasome are de facto occupied simultaneously.

To determine the number of bound substrate molecules we first recorded MS spectra of the bound proteasome complexes (Fig. 2). However, because of heterogeneity of binding and overlap of charge states we could not determine unambiguously the number of bound substrates using this method. We therefore decided to apply a tandem MS approach because the asymmetric dissociation that is a characteristic of this approach leads to highly charged monomer ions and relatively low charged "stripped" complexes (28-31). Consequently the peaks of the stripped complexes have a greater separation between the charge states than traditional MS experiments (23), and such an approach has been used previously to separate overlapping charge states that arise from polydispersity or other heterogeneous complex systems (23).


Figure 2
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  		FIGURE 2.
Nanoflow mass spectrum of Cyt c host-guest complex. The vertical lines represent calculated m/z values for different charge states of the following complexes (with their charge state at ~10,800 indicated in parentheses): free proteasome in blue (64+), proteasome bound to one Cyt c molecule in orange (65+), proteasome with two Cyt c molecules in green (66+), and proteasome bound to three Cyt c molecules in pink (67+). These isotope models illustrate the difficulty in defining the number of bound substrates using MS. Similar results were obtained for GFP.

 
We first established optimal conditions for collisional activation and tandem MS of the free 20S proteasome. A single charge state (69+)was isolated from the complex using the quadrupole mass filter and subjected to collisions with argon to induce dissociation (Fig. 3A). Two series of charge states are observed at higher m/z values than 69+, assigned to stripped complexes having lost one and two {alpha}-subunits (Table 1). At low m/z values only {alpha}-subunits are dissociated from the proteasome, consistent with the architecture in which the two {alpha}7 ring structures are exposed and in accord with previous analysis of free 20S proteasomes (19, 20). Comparison of these tandem MS with those recorded for the host-guest complex formed with Cyt c reveals additional peaks, presumably due to the presence of substrate molecules within the proteasome assembly and also to formation of a dimer (65+) (Fig. 3B). To ensure that we could distinguish nonspecific binding from occupancy within cavities we mixed Cyt c with the 20S proteasome at a ratio of 10:1 without applying heat or denaturant to unfold substrate molecules and without removing excess ligand bound to the proteasome. The MS/MS spectrum recorded for this control solution when compared with that of the Cyt c host-guest complex shows a clear difference in peak width (Fig. 3, B and C). Moreover, at low m/z, the dissociation of both Cyt c (with an average charge state of 9+) and {alpha}-subunit ions is clearly observed for the mixed solution, whereas only {alpha}-subunits are observed for the corresponding host-guest complex. Charge states in electrospray mass spectra are dictated by the surface area of the protein or complex (32). For the proteasome this gives rise to a series of charges from 65+ to 75+. Binding of substrate proteins within the proteasome would not be expected to change the overall charge state of the complex because substrate molecules will be buried within the proteasome. This broadening of peaks and release of highly charged Cyt c is attributed to multiple copies of the substrate protein adhering to the outer surface of the proteasome. Conversely Cyt c molecules sequestered within the proteasome have no overall charge and are not therefore detected in tandem mass spectra. Because analogous results were obtained for the GFP host-guest complexes we conclude that there is no evidence for Cyt c or GFP adhering to the outer surfaces in agreement with results from antibody binding experiments.


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  		TABLE 1
Theoretical and measured masses of proteins and complexes

 


Figure 3
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  		FIGURE 3.
Tandem MS of the free, Cyt c host-guest complex and Cyt c mixed with 20S proteasome from T. acidophilum. Free proteasome (A), proteasome/Cyt c host-guest complex (1:100 ratio) (B), and intact proteasome mixed with Cyt c (1:10 ratio) (C). Dissociation of the complex generates product ions from the loss of only the exposed {alpha}-subunits, consistent with the known architecture of the complex. Peaks between 12,000 and 16,000 m/z correspond to the loss of one {alpha}-subunit, and the series at 17,000-25,000 correspond to the loss of two {alpha}-subunits. At low m/z 1,000-2,000 series of peaks are assigned to individual {alpha}-subunits. The schematic shows the sequential dissociation pathway. The charge states that were isolated for tandem MS are labeled. The tandem MS of the mixed proteasome Cyt c sample (C) gives rise to stripping of Cyt c molecules, labeled by asterisk, while the broadening of the charge distributions is due to the unspecific binding of Cyt c molecules. The region between 12,000 and 15,000 m/z in panel C was magnified three times. Fourteen molecules of a covalent bound inhibitor are attached to all proteasome species.

 
The peaks assigned to the free proteasome are broader than corresponding peaks in the MS/MS spectra of proteasome-Cyt c complexes (Fig. 3, A and B). This broadness of peaks is an indication of the extent to which water/buffer molecules are trapped within gas phase assemblies (27) and allows us to conclude that a greater number are trapped within the internal cavities of the free 20S proteasome than in the Cyt c host-guest complex. Further comparison of the free and substrate-bound forms of the stripped proteasomes was complicated by the changing distribution of water/buffer molecules. The narrow peaks of the bound proteasome species indicate that a discrete number of substrates had replaced the trapped water/buffer molecules. We therefore compared the {alpha}-subunit region of the spectra recorded for the two host-guest complexes (Fig. 4). From this comparison we were able to identify peaks common to both spectra that arise from proteasomes without substrate molecules (A) -apo {alpha}13beta14(inhibitor)14 and peaks corresponding to binding of three (C) one and two (B) Cyt c molecules. Similarly for the same region of spectra recorded for the GFP host-guest complex, the dominant charge state series corresponds to two GFP molecules (see supplemental material). None of the charge states corresponds to binding of three GFP molecules. From the {alpha}-subunit region of the spectra recorded for the two host-guest complexes we can conclude therefore that up to three Cyt c or up to two GFP molecules can bind within the proteasome.


Figure 4
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  		FIGURE 4.
Analysis of the Cyt c and GFP proteasome complexes. Tandem mass spectra of the Cyt c (A) and GFP (B) host-guest complexes. The dotted line indicates coincidence with the onset of the peak assigned to apo-proteasomes. The bars correspond to simulation of the increase in m/z upon binding to I, II, or III substrate molecules. The peaks assigned to the apo-proteasome {alpha}13beta14(inhibitor)14 are labeled A. Series labeled B and C correspond in mass to binding of two or three Cyt c molecules, respectively (bold bars). The peaks labeled G correspond in mass to two bound GFP molecules (bold bar). No binding of three GFP molecules could be observed.

 
A key question arises as to whether or not this represents the maximum number of substrate molecules that are accommodated in solution or whether the collisional activation process, which removes {alpha}-subunits, also releases substrate ligands. To investigate this possibility we varied the acceleration voltage in the collision cell, and consequently the internal energy of the ions, and examined the resulting mass spectra of the host-guest complexes (Fig. 5). In the case of the Cyt c host-guest complex at the lowest collision energy employed (100 V) in the {alpha}-subunit region of the spectrum, peaks assigned to binding of three Cyt c molecules predominate at >95% of the intensity in this region of the spectrum. Interestingly, as the collision energy is increased to 120 and 130 V the intensity of the apo form is found to increase; at 130 V the apo and ligand-bound forms of the complexes are of approximately equal intensity. This implies that a greater proportion of Cyt c molecules have been expelled from the complex as the internal energy of the ions increased.

If we compare the 2{alpha}-subunit region of the spectrum at the lowest energy (100 V), we observe predominantly apo forms of the proteasome {alpha}12beta14(inhibitor)14, M-monomer (Table 1) as well low intensity peaks assigned to binding of two and three Cyt c molecules, confirming our assignment above (Fig. 5, 100 V). Interspersed between peaks assigned to monomers, a second series of peaks appears equidistant from the monomeric charge states, consistent with their interpretation as dimers (D [{alpha}12beta14(inhibitor)14]2) (33). Because this dimer formation was not observed for the free form of the proteasome, we conclude that gas phase dissociation of two {alpha}-subunits from the substrate-bound 20S proteasome promotes dimer formation. At higher energies (120 and 130V) the intensity of the substrate-bound forms decreased relative to apo forms, whereas peaks assigned to dimeric forms have increased. However, the fact that a population of substrate molecules is retained within the complex, even after two {alpha}-subunits have been stripped, implies that at least a fraction of the 20S proteasome structure survives the collisional activation process. Moreover at low collision cell voltages, when only one {alpha}-subunit has been lost, substrate binding is observed for >95% of proteasome molecules.


Figure 5
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  		FIGURE 5.
Investigation of the effect of collisional activation on the 71+ charge state of the Cyt c host-guest complex. Acceleration voltages of 100, 120, and 130V were employed. At the lowest voltages low intensity peaks can be assigned to two (*) or three Cyt c (+) molecules within proteasomes. Contrary to the free proteasome, upon the loss of the second {alpha}-subunit the proteasome with Cyt c coexists as a monomer (labeled M) and dimer (labeled D).

 
The fact that protein substrates can be retained within the proteasome, even during collisional activation, can be rationalized because the constrictions that prevent folded proteins from entering the cavities may also be expected to prevent substrates from exiting. Because Cyt c and GFP are approximately half the size and a similar volume to {alpha}-subunits, respectively, we propose that dissociation of an {alpha}-subunit in the gas phase opens a cavity along the proteasome entrance channel, facilitating release of substrate. Removal of a second {alpha}-subunit can take place either from the same ring as the first or from the opposing one. In the case of proteasomes in the absence of substrate we anticipate that loss of the second {alpha}-subunit occurs mainly from the same ring as the first, because a higher energy is required to disrupt protein interactions in fully formed rings.4 If this lowest energy scenario were the case for the host-guest complexes we would expect the number of substrate molecules to remain constant, trapped within the opposing ring and the catalytic chamber. We find, from analysis of the -2{alpha}-subunit region of the spectrum, dissociation products containing both two and three Cyt c molecules (Fig. 5) and one and two GFP molecules (data not shown). As the number of substrate molecules is not constant and two populations are observed for both complexes, we conclude that loss of {alpha}-subunits can occur simultaneously from both rings (see schematic, Fig. 6). One plausible explanation for loss of {alpha}-subunits from both rings is that ligand binding destabilizes the antechamber, promoting dissociation from the opposing ring and concomitant loss of an additional substrate molecule.

In summary, we can conclude that loss of an {alpha}-subunit occurs concomitantly with loss of substrate in cases in which the antechamber is occupied initially. Therefore the minus {alpha}-subunit regions of the spectra, assigned to assemblies containing two or three GFP or Cyt c molecules, respectively, correspond to a proportion of assemblies that have lost one substrate molecule. By extrapolation therefore we conclude that prior to MS a maximum of four Cyt c molecules or three GFP molecules are bound within host-guest complexes (Fig. 7). Given the dissociation pattern together with the additional density observed in host-guest complexes by cryo-EM, we propose that Cyt c and GFP molecules are located singly in each of the antechambers but that one GFP and two Cyt c molecules occupy the central cavity.


Figure 6
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  		FIGURE 6.
Schematic representation of the dissociation pattern of the host-guest complexes during tandem mass spectrometry. The MS/MS spectra of the proteasome Cyt c host-guest complex is shown in red (A) and with GFP in green (B). Initially four Cyt c or three GFP molecules bind within the proteasome. During the first dissociation step, the stripping of one {alpha}-subunit exposes the substrate molecule within the antechamber and triggers its dissociation. In the second dissociation step an additional {alpha}-subunit is lost, either from the same ring or from the opposing one. In the case that the {alpha}-subunit dissociates from the opposing ring, another substrate molecule is exposed and released. The stripping of an additional {alpha}-subunit from the same ring will not trigger further dissociation of substrate molecules. The region between 12,000 and 15,000 m/z in panel A was magnified 3-fold.

 


Figure 7
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  		FIGURE 7.
Space-filling model of the proteasome host-guest complexes. The substrates Cyt c (A) and GFP (B) are placed within the two antechambers and the catalytic cavity to illustrate their fitting. For clarity 50% of the proteasome complex was removed to show the inner cavities. The substrates are shown in their folded conformation, although based on our results we conclude that the substrates are less compact than the native state. The figure was prepared using Rasmol (34), and coordinates are from the Protein Data Bank. For the proteasome, Cyt c, and GFP structures coordinates 1PMA, 1HRC, and 1GFL were used, respectively.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we have employed both cryo-EM and tandem MS to examine the stoichiometry of substrate binding within the intact proteasome, a complex with a mass greater than 736 kDa. The fundamental advantage of using tandem MS to study complexes such as this lies in its ability to probe heterogeneous and large macromolecular complexes. Using this approach we have not only been able to determine the precise number and location of substrate molecules bound within proteasome host-guest complexes but also to confirm that ligand binding occurs in >95% of 20S particles. In conjunction with the cryo-EM data that indicated substrate binding in all three cavities, we can conclude that the electron density observed in the two antechambers and central cavity reflects simultaneous substrate binding. However, it is noteworthy that we cannot directly exclude contributions from a second population in which one of the antechambers remains empty, although we anticipate that any contribution is likely to be small given the predominance in MS of the fully bound forms. EM difference images also indicate that the amount of substrate present in the catalytic chamber of the Cyt c-bound complexes is greater than in the two antechambers, consistent with data from tandem MS.

Several aspects of this study reveal interesting structural differences between the free and bound proteasome complexes. For the free proteasome broad peaks imply that the free channels are occupied with a distribution of buffer molecules. We also noted the formation of dimeric forms of the proteasome after loss of {alpha}-subunits and substrate, a process not observed for the free proteasome. This implies that a conformational change is induced upon substrate binding and translocation, as suggested by other studies (35-37), and that this allosteric transition persists even after loss of {alpha}-subunit and substrate. The finding that the loss of two {alpha}-subunits occurs from both antechambers of the host-guest complexes implies that substrate binding destabilizes the {alpha}-subunit rings. This could represent two different mechanistic scenarios: in the first, substrate binding and translocation would generate, transiently, vacancies with a role in releasing degradation products, or in the second it could affect the interaction of the {alpha}-rings with the regulatory complexes, bringing about their disassembly as recently reported (38).

The location and number of Cyt c and GFP molecules emphasize the dependence on the size of the protein and the various cavities within the core particle (Fig. 7). The dimensions of the catalytic chamber are such that a maximum of three Cyt c and two GFP molecules could be accommodated within the catalytic chamber (39). However, our findings indicate that only one GFP and two Cyt c molecules are contained within this chamber. As the proteasome-GFP host-guest complex exhibits the specific absorption and emission spectrum characteristic of native GFP, it is possible that substrate has refolded within the internal cavities of the 20S proteasome. However, the observation that only one GFP and two Cyt c molecules are contained within this chamber suggests that the substrates occupy a slightly larger volume than would be predicted from their tightly packed native conformation.

The fact that partial occupancy of substrate within the proteasome is not observed in spectra of either host-guest complex strongly implies that binding of substrate molecules within the proteasome is a highly cooperative process. Binding of the first would therefore trigger binding of the second, third, and fourth substrate molecules. This is in accord with a previous proposal for cooperative binding of a much smaller protein (insulin); however, only binding in both antechambers, and not the central cavity, was considered (35). Additionally, atomic force microscope measurements have indicated a two-state model of allosteric transition that is dependent on substrate binding (40). Further support for our proposal comes from the observation that even at lower proteasome:substrate ratios (1:10) (data not shown) the dominant species corresponds to binding of the maximum number of ligands.

Our observation of substrate binding simultaneously in all three chambers of the inhibited proteasome has implications for the mechanism of substrate translocation. Specifically, it implies that in cases where substrate unfolding or translocation is not the rate-limiting step, as in this model system, storage must take place concurrently in both antechambers while the processive degradation is carried out within the catalytic chamber. Interestingly, accumulation of substrate does not appear to affect the proteolytic activity of the proteasome, because when a reversible inhibitor was employed Cyt c was digested as normal (25). It is possible therefore that the antechambers retain proteins in a partially folded state, allowing them to enter the catalytic chamber as soon as space allows (41). In summary, it is interesting to speculate that this mechanism of storage may have evolved to enhance protein degradation, providing a continuous stream of substrates to prevent effectively the accumulation of toxic, misfolded proteins within cells.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental text and supplemental Fig. S1. Back

1 Supported by the European Molecular Biology Organization and a Wingate scholarship. Back

2 Funded by the Walters Kundert Trust. To whom correspondence should be addressed. Tel.: 44-1223-763864; E-mail: cvr24{at}cam.ac.uk.

3 The abbreviations used are: EM, electron microscopy; MS, mass spectrometry; Cyt c, cytochrome c; GFP, green fluorescent protein. Back

4 J. L. Benesch, J. A. Aquilina, B. T. Ruotolo, F. Sobott, and C. V. Robinson, manuscript in preparation. Back


   ACKNOWLEDGMENTS
 
We thank Oana Mihalache (Max Planck Institute of Biochemistry, Martinsried, Germany) for recording the EM data and Dr. Reiner Hegerl (Max Planck Institute of Biochemistry, Martinsried, Germany) for helpful discussions.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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M. Sharon, S. Witt, E. Glasmacher, W. Baumeister, and C. V. Robinson
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