Insights into the molecular architecture of the 26S proteasome

Abstract

Cryo-electron microscopy in conjunction with advanced image analysis was used to analyze the structure of the 26S proteasome and to elucidate its variable features. We have been able to outline the boundaries of the ATPase module in the ''base'' part of the regulatory complex that can vary in its position and orientation relative to the 20S core particle. This variation is consistent with the ''wobbling'' model that was previously proposed to explain the role of the regulatory complex in opening the gate in the ␣-rings of the core particle. In addition, a variable mass near the mouth of the ATPase ring has been identified as Rpn10, a multiubiquitin receptor, by correlating the electron microscopy data with quantitative mass spectrometry.ATPase ͉ cryo-electron microscopy ͉ mass spectrometry ͉ protein degradation ͉ AAA-ATPase C ellular protein levels are regulated through protein synthesis and degradation. Given its destructive potential, intracellular protein degradation must be subject to rigorous spatial and temporal control. In eukaryotic cells, most proteins in the cytosol and the nucleus are degraded by the ubiquitinproteasome system, and malfunctions of this system have been implicated in a wide variety of diseases (1-4). Unlike constitutively active proteases, the proteasome has the capacity to degrade almost any protein, yet it acts with exquisite specificity. The key stratagem is self-compartmentalization: the active sites of the proteolytic machine are sequestered from the cellular environment in the interior of the barrel-shaped 20S proteasome (5). Proteins destined for degradation are marked by ubiquitin, a degradation signal that is recognized by the regulatory 19S complexes (RPs) that associate with the core 20S proteasome or core particle (CP) to form the holoenzyme called the 26S proteasome. The 26S complex is a multimeric assembly with a mass of approximately 2.5 MDa (6).The 20S core complex, which is highly conserved from archaea to higher eukaryotes, was amenable to structure determination by X-ray crystallography, and the resulting structures have revealed the salient features of this prototypical selfcompartmentalizing protease (7-9). In contrast, the 26S holocomplex with 1 or 2 RPs attached to the barrel-shaped core has so far resisted all crystallization attempts. For protein complexes refractory to crystallization, cryo-electron microscopy (cryo-EM) of ''single particles'' is an alternative approach. The amounts of material required for cryo-EM are minute and, moreover, some degree of heterogeneity is tolerable. Although high resolution is often elusive, the medium resolution (1-2 nm) structures afforded by cryo-EM provide a platform for hybrid approaches which, in turn, can provide useful insights into the structure and mechanism of macromolecular assemblies (10). Results and Discussion MS Analysis of Purified 26S Proteasomes.In the case of the 26S proteasome, progress in elucidating its structure has been hampered by the complexity of the system, its variability, and its fragility. 26S p...

Inherent Asymmetry in the 26S Proteasome Is Defined by the Ubiquitin Receptor RPN13

“…1). These results further confirm a recent conclusion regarding S5a stoichiometry (33), whereas data raised the possibility of a non-stoichiometric amount of S5a in the proteasome (24,25). Using the same methodology, we were unable to detect the presence of two Rpn13 copies in a DC proteasome, both in yeast and in mammals (Fig.…”

“…Proteasomes-Previous studies raised the possibility that in each DC 26S complex, only one of the 19S regulatory particles has an integral Rpn10 subunit (24,25). This is an attractive possibility, as it may provide the basis for preventing concomitant processing of substrate by two 19S particles in the DC 26S proteasome.…”

Molecular architecture of the 26S proteasome holocomplex determined by an integrative approach

“…The density corresponding to Rpn1 is highly variable (Fig. 1B), as already observed (49), which makes it impossible to discern secondary structure elements. This variability may be due to Rpn1's function as a recruitment factor for shuttling Ub receptors and the DUB Ubp6, which dock to the LRRs of Rpn1 (50,51).…”

Structural characterization of the interaction of Ubp6 with the 26S proteasome

“…At least three distinct states, which we refer to as "s1-s3," can be distinguished (13). In ATP-containing buffer, purified 26S proteasomes primarily adopt the s1 state, which is characterized by pronounced off-axis positioning of the AAAATPase hexamer with respect to the CP and a staircase arrangement of the Rpts with Rpt3 in the most elevated position (6)(7)(8)(9). Under the same conditions a minority of particles (∼20%) adopts the s2 state, in which the axis of the AAA-ATPase is positioned closer to that of the CP and the Rpns concomitantly rotate largely en bloc by ∼25° (13).…”

“…Although the structure of the CP has been known for more than two decades (4,5), the molecular architecture of the RP was unraveled by cryo-electron microscope (EM)-based approaches only recently (6)(7)(8)(9). It comprises six RP triple A (AAA) ATPases (Rpt), 1-6, and 13 RP non-ATPases (Rpn), 1-3, 5-13, and 15.…”