A Gated Channel Into the Proteasome Core Particle (original) (raw)

Protein targeting to ATP-dependent proteases

Current Opinion in Structural Biology, 2008

ATP-dependent proteases control diverse cellular processes by degrading specific regulatory proteins. Understanding how these regulatory proteins are targeted to ATP-dependent proteases is of central importance to understanding their biological role as regulators. Recent work has shown that protein substrates are specifically transferred to ATP-dependent proteases through different routes. These routes can function in parallel or independently. In all of these targeting mechanisms it can be useful to separate two steps: substrate binding to the protease and initiation of degradation.

The central unit within the 19S regulatory particle of the proteasome

Nature Structural & Molecular Biology, 2008

The 26S proteasome is a multisubunit enzyme composed of a cylindrical catalytic core (20S) and a regulatory particle (19S) that together perform the essential degradation of cellular proteins tagged by ubiquitin. To date, however, substrate trajectory within the complex remains elusive. Here we describe a previously unknown functional unit within the 19S, comprising two subunits, Rpn1 and Rpn2. These toroids physically link the site of substrate recruitment with the site of proteolysis. Rpn2 interfaces with the 20S, whereas Rpn1 sits atop Rpn2, serving as a docking site for a substrate-recruitment factor. The 19S ATPases encircle the Rpn1-Rpn2 stack, covering the remainder of the 20S surface. Both Rpn1-Rpn2 and the ATPases are required for substrate translocation and gating of the proteolytic channel. Similar pairing of units is found in unfoldases and nuclear transporters, exposing common features of these protein nanomachines.

Assembly of the regulatory complex of the 26S proteasome

Molecular biology reports, 1999

The 19S regulatory complex (RC) of 26S proteasomes is a 900-1000 kDa particle composed of 18 distinct subunits (S1-S15) ranging in molecular mass from 25 to 110 kDa. This particle confers ATP-dependence and polyubiquitin (polyUb) recognition to the 26S proteasome. The symmetry and homogenous structure of the proteasome contrasts sharply with the remarkable complexity of the RC. Despite the fact that the primary sequences of all the subunits are now known, insight has been gained into the function of only eight subunits. The six ATPases within the RC constitute a subfamily (S4-like ATPases) within the AAA superfamily and we have shown that they form specific pairs in vitro. We have now determined that putative coiled-coils within the variable N-terminal regions of these proteins are likely to function as recognition elements that direct the proper placement of the ATPases within the RC. We have also begun mapping putative interactions between non-ATPase subunits and S4-like ATPases. ...

Hexameric assembly of the proteasomal ATPases is templated through their C termini

Nature, 2009

Substrates of the proteasome are recognized and unfolded by the regulatory particle (RP), then translocated into the core particle (CP) to be degraded1. A hetero-hexameric ATPase ring, containing subunits Rpt1-Rpt6, is situated within the base subassembly of the RP1. The ATPase ring sits atop the CP, with the Rpt C-termini inserted into pockets in the CP2-6. We have identified a novel function of the Rpt proteins in proteasome biogenesis through deleting the Cterminal residue from each Rpt. Our results indicate that assembly of the hexameric ATPase ring is templated on the CP. We have also identified an apparent intermediate in base assembly, BP1, which contains Rpn1, three Rpts, and Hsm3, a chaperone for base assembly. The Rpt proteins with the strongest assembly phenotypes, Rpt4 and Rpt6, were absent from BP1. We propose that Rpt4 and Rpt6 form a nucleating complex to initiate base assembly, and that this complex is subsequently joined by BP1 to complete the Rpt ring. Our studies show that assembly of the proteasome base is a rapid yet highly orchestrated process.

Mechanism of Gate Opening in the 20S Proteasome by the Proteasomal ATPases

Molecular Cell, 2008

Substrates enter the cylindrical 20S proteasome through a gated channel that is regulated by the ATPases in the 19S regulatory particle in eukaryotes or the homologous PAN ATPase complex in archaea. These ATPases contain a conserved C-terminal hydrophobic-tyrosine-X (HbYX) motif that triggers gate opening upon ATP binding. Using cryo-electron microscopy, we identified the sites in the archaeal 20S where PAN's C-terminal residues bind and determined the structures of the gate in its closed and open forms. Peptides containing the HbYX motif bind to 20S in the pockets between neighboring a subunits where they interact with conserved residues required for gate opening. This interaction induces a rotation in the a subunits and displacement of a reverse-turn loop that stabilizes the open-gate conformation. This mechanism differs from that of PA26/28, which lacks the HbYX motif and does not cause a subunit rotation. These findings demonstrated how the ATPases' C termini function to facilitate substrate entry.

Proteasomes and other self-compartmentalizing proteases in prokaryotes

Trends in Microbiology, 1999

I ntracellular proteases in prokaryotic cells perform many tasks, including cleavage of signal peptides during protein export, timely inactivation of regulatory proteins, and removal of aberrant nonfunctional proteins 1. Obviously, proteolysis inside prokaryotic cells needs to be controlled to avoid unwanted degradation. Spatial safeguarding can be provided by directing a protease to a specific compartment of the cell, such as the periplasmic space of Gram-negative bacteria. Autocompartmentalization, as a common strategy to curtail the potential hazard associated with intracellular protein breakdown, has recently emerged from the elucidation of the structure of a subset of cytoplasmic proteases in prokaryotes 2. Isolation of proteolytic activity is achieved by self-assembly of proteolytic subunits into a cylinder-shaped complex, in which the active sites are confined to nanocompart-ments in their interior (Fig. 1). Narrow entrances to the cylinder restrict access to unfolded proteins. The ATPase complexes that can bind to both ends of such barrels are thought to be involved in the initial binding, unfolding and translocation of substrates. Proteases creating nanocompartments The proteasome, which has become the paradigm of a selfcompartmentalizing protease 3 , was first discovered in eukaryotic cells, where it constitutes the major non-lysosomal proteolytic system 4. The proteolytic core (20S proteasome), capped with 19S regulatory complexes (including several ATPases), is designated the 26S proteasome and mediates the ATP-dependent degradation of ubiquitin-bound proteins 5. The archaeal counterpart of the 20S proteasome was identified in Thermoplasma acidophilum about a decade ago 6. The discovery of The proteasome represents the major nonlysosomal proteolytic system in eukaryotes. It confines proteolytic activity to an inner compartment that is accessible to unfolded proteins only. The strategy of controlling intracellular breakdown of proteins by self-compartmentalization is also used by different types of prokaryotic energy-dependent proteases. Genomic sequencing data reveal that various combinations of these energy-dependent proteases occur in prokaryotic cells from different lineages.