Proteasomes are responsible for most intracellular protein degradation in eukaryotes. The 20S proteasome comprises a dyad-symmetric stack of four heptameric rings made from 14 distinct subunits. How it assembles is not understood. Most subunits in the central pair of b-subunit rings are synthesized in precursor form. Normally, the b5 (Doa3) propeptide is essential for yeast proteasome biogenesis, but overproduction of b7 (Pre4) bypasses this requirement. Bypass depends on a unique b7 extension, which contacts the opposing b ring. The resulting proteasomes appear normal but assemble inefficiently, facilitating identification of assembly intermediates. Assembly occurs stepwise into precursor dimers, and intermediates contain the Ump1 assembly factor and a novel complex, Pba1-Pba2. b7 incorporation occurs late and is closely linked to the association of two half-proteasomes. We propose that dimerization is normally driven by the b5 propeptide, an intramolecular chaperone, but b7 addition overcomes an Ump1-dependent assembly checkpoint and stabilizes the precursor dimer.

The proteasome is the central regulatory protease of eukaryotic cells. Heteroheptameric alpha-subunit and beta-subunit rings stack to form the 20S proteasome, which associates with a 19S regulatory particle (RP). Here we show that two yeast proteins, Pba3 and Pba4, form a previously unidentified 20S proteasome-assembly chaperone. Pba3-Pba4 interacts genetically and physically with specific proteasomal alpha subunits, and loss of Pba3-Pba4 causes both a reduction and a remodeling of cellular proteasomes. Notably, mutant cells accumulate proteasomes in which a second copy of the alpha4 subunit replaces alpha3. 20S proteasome-assembly defects also are associated with altered RP assembly; this unexpected result suggests that the 20S proteasome can function as an RP-assembly factor in vivo. Our data demonstrate that Pba3-Pba4 orchestrates formation of a specific type of proteasome, the first example of a trans-acting factor that controls assembly of alternative proteasomal complexes.

Dedicated chaperones facilitate eukaryotic proteasome assembly, yet how they function remains largely unknown. Here we demonstrate that a yeast 20S proteasome assembly factor, Pba1–Pba2, requires a previously overlooked C-terminal HbYX (hydrophobic-tyrosine-X) motif for function. HbYX motifs in proteasome activators open the 20S proteasome entry pore, but Pba1–Pba2 instead binds inactive proteasomal precursors. We discovered an archaeal ortholog of this factor, here named PbaA, that also binds preferentially to proteasomal precursors in a HbYX-dependent fashion using the same proteasomal α-ring surface pockets bound by activators. Remarkably, PbaA and the related PbaB protein can be induced to bind mature 20S proteasomes if the active sites in the central chamber are occupied by inhibitors. Our data suggest an allosteric mechanism in which proteasome active-site maturation determines assembly chaperone binding, potentially shielding assembly intermediates or misassembled complexes from non-productive associations until assembly is complete.

The properties of the ATPase activity of the type II chaperonin from Methanococcus maripaludis (Mm-cpn) were examined. Mm-cpn can hydrolyze not only ATP, but also CTP, UTP, and GTP, albeit with di¡erent e¡ectiveness. The ATPase activity is dependent on magnesium and potassium ions, and is e¡ectively inhibited by sodium ions. Maximal rates of ATP hydrolysis are achieved at 600 mM potassium. Initial rates of ATP hydrolysis by Mm-cpn were determined at various ATP concentrations, revealing for the ¢rst time the presence of both positive intra-ring and negative inter-ring cooperativity in the archaeal chaperonin.