The Mammalian Proteasome Activator PA28 Forms an Asymmetric α4β3 Complex

The heptameric proteasome activator (PA) 28 alpha beta is known to modulate class I antigen processing by docking onto 20S proteasome core particles (CPs). The exact stoichiometry and arrangement of its alpha and beta subunits, however, is still controversial. Here we analyzed murine PA28 complexes regarding structure and assembly. Strikingly, PA28 alpha, PA28 beta, and PA28 alpha beta preparations form heptamers, but solely PA28 alpha and PA28 alpha beta associate with CPs. Co-expression of a and b yields one unique PA28ab species with an unchangeable subunit composition. Structural data on PA28 alpha, PA28 beta, and PA28 alpha beta up to 2.9 angstrom resolution reveal a PA28 alpha(4)beta(3) complex with an alternating subunit arrangement and a single alpha-alpha interface. Differential scanning fluorimetry experiments and activity assays classify PA28 alpha(4)beta(3) as most stable and most active, indicating that this assembly might represent the physiologically relevant species. Together, our data resolve subunit composition and arrangement of PA28 alpha beta and clarify how an asymmetric heptamer can be assembled from two highly homologous subunits.