Architecture and assembly of the archaeal Cdc48⋅20S proteasome

Abstract

ATP-dependent proteases maintain protein quality control and regulate diverse intracellular functions. Proteasomes are primarily responsible for these tasks in the archaeal and eukaryotic domains of life. Even the simplest of these proteases function as large complexes, consisting of the 20S peptidase, a barrel-like structure composed of four heptameric rings, and one or two AAA+ (ATPase associated with a variety of cellular activities) ring hexamers, which use cycles of ATP binding and hydrolysis to unfold and translocate substrates into the 20S proteolytic chamber. Understanding how the AAA+ and 20S components of these enzymes interact and collaborate to execute protein degradation is important, but the highly dynamic nature of prokaryotic proteasomes has hampered structural characterization. Here, we use electron microscopy to determine the architecture of an archaeal Cdc48·20S proteasome, which we stabilized by site-specific cross-linking. This complex displays coaxial alignment of Cdc48 and 20S and is enzymatically active, demonstrating that AAA+ unfoldase wobbling with respect to 20S is not required for function. In the complex, the N-terminal domain of Cdc48, which regulates ATP hydrolysis and degradation, packs against the D1 ring of Cdc48 in a coplanar fashion, constraining mechanisms by which the N-terminal domain alters 20S affinity and degradation activity.AAA+ protease | dynamic wobbling model | p97/VCP P roteasomes are large macromolecular complexes that degrade misfolded or damaged proteins to maintain cellular homeostasis and quality control in all domains of life. In addition, selective proteasomal turnover of regulatory proteins is often a critical element of signaling cascades that allow cells to respond to changing conditions and environmental stress (1, 2). Proteasomes consist of the self-compartmentalized 20S peptidase and one or two AAA+ (ATPase associated with a variety of cellular activities) family ring hexamers. The α 7 β 7 β 7 α 7 ring topology of the 20S enzyme ensures that the proteolytic active sites, which reside in the β-subunits, are sequestered and can only cleave substrates that enter the chamber through narrow axial pores formed by the α-subunits (3). As a consequence, ATP-dependent unfolding of natively folded protein substrates by the AAA+ ring and subsequent polypeptide translocation through a narrow axial channel and into the 20S chamber are required for degradation (4).Although the 20S peptidase is the degradation module in all proteasomes, the associated AAA+ unfolding machines differ. For example, homohexamers of Mpa/Arc in actinobacteria and PAN in archaea serve as proteasomal motors, whereas a heterohexameric Rpt 1-6 ring in the 19S particle is the motor of the eukaryotic 26S proteasome (2, 5). Characteristic of type-I AAA+ enzymes, Mpa/Arc, PAN, and Rpt 1-6 subunits contain a single AAA+ module for ATP binding and hydrolysis (6). By contrast, as found in type-II AAA+ enzymes, the homohexameric Cdc48 motor in the recently discovered archaeal Cdc48·20S proteasome cont...

Unfolding the mechanism of the AAA+ unfoldase VAT by a combined cryo-EM, solution NMR study

“…Copper-1,10-phenanthroline was used as an oxidizing catalyst to promote disulfide bond formation (44). The solvent accessibility of the added cysteines can result in intermolecular crosslinking, leading to dodecamers or higher order oligomers of ADP-VAT, as depicted in Fig.…”

Regulation of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) Activase

“…Although several Rca-Rubisco binding geometries have been proposed (9,32,48), the actual mechanisms of recognition, interaction, and activation of Rubisco remain unknown. It is tempting to speculate that the sequestration of less ordered Rubisco segments by the central pore of Rca toroids plays a role, as demonstrated for a number of other AAA ϩ assemblies, such as FtsH (42), PAN (49), ClpX (50), Cdc48 (51), and ClpA/B (18). A mutational analysis of tobacco ␤-Rca pore loop residues has provided evidence consistent with a peptide binding or threading mechanism (32).…”

Proteasome assembly

“…In the case of the RP base, the chaperones assist in the incorporation of the six different Rpt ATPase subunits and two non-ATPase subunits, such that each subunit finds its position in the complex with high fidelity and efficiency [100]. No chaperones are known to be required for the assembly of eukaryotic and prokaryotic ATPase ring complexes consisting of a single ATPase subunit, like archaeal PAN and VAT, eubacterial ARC and Mpa, yeast Cdc48 and mammalian p97, suggesting that the hexagonal symmetry of the complex and the demand of space for each subunit does not allow alternative configurations [22,101].…”