The proteasome, a protease of over 2.5 MegaDaltons, functions primarily to degrade proteins that have been modified by the attachment of the small protein ubiquitin (1
) (). A cascade of enzymes, known as E1, E2, and E3, conjugate ubiquitin via its C-terminus to lysine residues in target proteins. The ubiquitin-proteasome system (UPS) is the major cytosolic proteolytic system in eukaryotes, with critical functions in cell cycle control, apoptosis, inflammation, transcription, signal transduction, protein quality control, and many other biological processes. The number of genes involved in the UPS varies significantly among eukaryotes, occupying, at the high end, over 6% of the genome of A. thaliana
). The breadth of the UPS owes mainly to the multiplicity of ubiquitin ligase (E3) enzymes, the specificity factors for ubiquitination, but the proteasome is the single most complex enzyme known in the system.
Figure 1 The ubiquitin-proteasome pathway for protein breakdown. Ubiquitin in green, substrate in yellow. A ubiquitin chain, synthesized via a cascade of E1, E2, and E3 enzymes, is thought to be the predominant signal for substrate recognition by the proteasome. (more ...)
The proteasome works via a multistep mechanism, with peptide bond cleavage being only the last step in a complex program of substrate manipulation. Critical upstream processes–recognition, unfolding, translocation, and deubiquitination of the substrate–take place distantly from the site of proteolysis, and are mediated by a large separable subassembly, the regulatory particle, largely under the direction of six distinct ATPases. Structurally, the proteasome is a hybrid between a proteolytic machinery and an ATP-dependent regulatory machinery, functionally linked by a gated protein translocation channel. But more interestingly it is also a hybrid between an ancient core biochemistry consisting of mechanisms for protein unfolding and degradation, which was developed in the early precursors of all cells, and the more recent eukaryotic inventions associated with ubiquitination. These two periods of evolutionary innovation, separated by billions of years, produced an unusually complex molecular machine. The most basic questions about the proteasome are those that apply generally to molecular machines: how is the energy of nucleotide hydrolysis converted into useful work, how is the fidelity of the process ensured, how are the core mechanisms of the machine regulated to provide for robust function and adaptation to changing cellular conditions. The answers are only beginning to emerge, but have already proven novel because of the unique biochemical features of the proteasome.
The proteasome exists in multiple forms, but contains two major assemblies, the 28-subunit core particle (CP, also known as the 20S particle) and a regulatory particle of 19–20 subunits, depending on the species (RP, also known as the 19S particle, and PA700) (). The CP is a barrel-like structure whose subunits are arranged in four stacked seven-membered rings (4
) (). The proteasome’s proteolytic active sites are sequestered within the large internal space of the CP (). Thus, for the free CP, substrate access into the proteolytic chamber is blocked through an essentially topological mechanism. In the RP-CP holoenzyme, substrate entry is controlled by the RP: the RP both opens a substrate translocation channel into the CP (6
) and guides substrates into this channel. The RP binds to the cylinder end of the CP (13
), and opens a channel located centrally within the cylinder end. Thus, the axial channel opens directly onto the RP. The RP is in turn assumed to possess its own substrate translocation channel, the outlet of which is apposed to that of the CP, although the RP channel is essentially uncharacterized.
Figure 2 Structure of the proteasome holoenzyme. The lid is highlighted in green, the base in purple, and the CP in orange. The position of the joint between the lid and base is only an estimate. The image was generated by averaging of electron micrographs of (more ...)
Figure 3 Gallery of proteasome core particle images. Upper left, Surface representation of the CP, showing its organization into 4 heptameric rings of subunits. The CP is shown along its 2-fold symmetry axis. Each subunit and its symmetry mate were painted in (more ...)
The substrate translocation channel, even when open, is sufficiently narrow (6
) that it prevents the bulk of cytoplasmic proteins from being degraded spuriously. The channel also imposes a constraint on true substrates, that they must be unfolded by the RP prior to translocation into the CP. Substrate entry in an unfolded state presumably also facilitates efficient hydrolysis of substrates within the CP. Finally, in contrast to the distributive mechanism typical of proteases, the proteasome degrades substrates processively, which reflects its compartmentalized structure and substrate translocation mechanism. Processive protein degradation avoids the generation of truncated reaction products, which could compromise cell function. However, in rare instances, regulatory proteins are subjected to partial proteolysis by the proteasome, which activates them through the removal of inhibitory domains. Partial proteolysis requires specialized features in the substrate, as discussed below.
The protein-unfoldase activity of the RP is thought to be mediated by its six ATPase subunits, which are members of the AAA protein family (15
). These ATPases, known as the Rpt proteins in yeast, are thought to form a pseudo-symmetrical ring structure, which is embedded within the highly asymmetric structure of the RP (10
). Docking of the RP to the CP is stabilized by the alignment of two ring assemblies–the Rpts and the outer ring of the CP (the α ring). Because the Rpt ring is thought to have six members and the α ring seven, RP-CP docking might induce a major break in the symmetry of either the Rpt or α ring.
It is generally assumed that the initial recognition of substrates by the proteasome is mediated by the substrate’s ubiquitin tag (1
). This is a function of the RP; the CP itself cannot specifically degrade ubiquitin-protein conjugates. There is no indication that ubiquitin performs roles at the proteasome other than substrate tethering, though a signaling function for ubiquitin within the proteasome remains a distinct possibility. A surprising number of proteins can mediate ubiquitin recognition at the proteasome: two subunits of the RP, Rpn10 and Rpn13 (22
); and apparently three proteasome-associated proteins, Rad23, Dsk2, and Ddi1 (26
). The latter proteins will be referred to collectively as the UBL/UBA proteins. Each contacts the proteasome through its UBL (ubiquitin-like) domain and ubiquitin-conjugates through one or more UBA (ubiquitin-associated) domains. The UBL/UBA proteins are not integral proteasome subunits; they bind proteasomes only weakly and are usually substoichiometric components of purified proteasomes. Many other ubiquitin receptors exist; most are not associated with proteasome function, but instead mediate nonproteolytic functions of ubiquitination (34
The proteasome does not degrade proteins to amino acids (35
) but instead produces a highly heterogeneous mixture of peptides from a given protein (36
). These peptides serve as raw material for adaptive cell-mediated immunity. Product peptides of 8–10 residues dock onto the major histocompatibility (MHC) class I molecule in the endoplasmic reticulum after their export from the cytoplasm via the peptide-specific TAP transporter. Once routed to the cell surface, the peptide-MHC complex may be recognized by epitope-specific T cell receptors carried on cytotoxic T lympohocytes. If the presented epitope is derived from viruses, tumors, or other “foreign” sources, the presenting cell is induced to undergo apoptosis.
Eukaryotic cells contain a second proteolytic pathway that is responsible for the breakdown of less rapidly degraded proteins. The lysosome, a membrane-enclosed organelle, contains numerous proteases of low specificity, ensuring that almost any cytoplasmic protein that reaches its interior will be degraded efficiently. The uptake of intracellular proteins into the lysosome, known as autophagy, is a predominantly nonspecific process in which portions of cytoplasm or whole organelles are subsumed en mass into an autophagic vesicle, whose entire contents are then delivered to the lysosome upon membrane fusion. This contrasts strongly with the mechanism of substrate selection by the proteasome, which recognizes proteins via a specific tag and sorts proteins individually. It should be noted, however, that some autophagy pathways are selective (37
), and some evidence for ubiquitin-mediated targeting in this pathway has been presented (38
). The autophagic pathway might serve as a back up mechanism for the proteasome for some substrates. The regulatory mechanisms associated with autophagy are complex, fascinating, and highly relevant to human health (see recent reviews [39
Because proteins that are rapidly turned over are also typically present at low levels in cells, mass-spectrometry-based searches for ubiquitinated proteins and proteasome substrates have so far provided only incomplete surveys (28
). However, the number of proteins that are degraded via the proteasome is undoubtedly very high. The existence of hundreds of physiological substrates (43
), and potentially more, implies that the proteasome’s capacity to process protein substrates is robust and specifically that it can unfold a remarkable variety of proteins. However, in most cases the proteasome can only act on proteins if they are ubiquitinated.
Regulatory proteins such as cyclins, CDK inhibitors, IκB, and p53 are key substrates for the proteasome, and their ubiquitin-dependent degradation is under tight control. Other regulators are activated through partial proteolysis as described above. Finally, misfolded and aberrant proteins are substrates, and recent work has shown that numerous diseases are associated with poor clearance of these often deleterious proteins. Thus, the proteasome is important to human health in a variety of ways.
The proteasome literature is vast, and cannot be fully reviewed here. The reader is referred to excellent summaries of the earlier literature (1
). Space limitations and the availability of current reviews preclude discussion of either proteasome inhibitors (5
), which are anti-cancer agents in clinical use, subcellular localization of the proteasome (44
), nonproteolytic roles of the proteasome (46
), or the remarkable maneuvers of proteasome assembly (48
). An emerging area that will also not be covered is the regulation of proteasome levels, in which the transcription factors Rpn4 (in yeast) and Nrf2 (in mammals) play important roles (51
A nomenclatural note: unfortunately, most proteasome subunits have many different names, among species and within a species. The systematic nomenclature for RP and CP subunits is most easily apprehended, and will be used here (4
). For example, we will refer specifically to budding yeast Rpn10 as scRpn10, murine Rpn10 as mRpn10, and human as hRpn10. provides, for the RP subunits, both systematic names and other names found in the literature.
Subunits of the Proteasome Regulatory Particle