Intermediates in the formation of mouse 20S proteasomes: implications for the assembly of precursor beta subunits

Abstract

The assembly of individual proteasome subunits into catalytically active mammalian 20S proteasomes is not well understood. Using subunit‐specific antibodies, we characterized both precursor and mature proteasome complexes. Antibodies to PSMA4 (C9) immunoprecipitated complexes composed of α, precursor β and processed β subunits. However, antibodies to PSMA3 (C8) and PSMB9 (LMP2) immunoprecipitated complexes made up of α and precursor β but no processed β subunits. These complexes possess short half‐lives, are enzymatically inactive and their molecular weight is ∼300 kDa. Radioactivity chases from these complexes into mature, long‐lived ∼700 kDa proteasomes. Therefore, these structures represent precursor proteasomes and are probably made up of two rings: one containing α subunits and the other, precursor β subunits. The assembly of precursor proteasomes occurs in at least two stages, with precursor β subunits PSMB2 (C7‐I), PSMB3 (C10‐II), PSMB7 (Z), PSMB9 (LMP2) and PSMB10 (LMP10) being incorporated before others [PSMB1 (C5), PSMB6 (delta), and PSMB8 (LMP7)]. Proteasome maturation (processing of the β subunits and juxtaposition of the two β rings) is accompanied by conformational changes in the (outer) α rings, and may be inefficient. Finally, interferon‐γ had no significant effect on the half‐lives or total amounts of precursor or mature proteasomes.