INTERMEDIATES IN THE FORMATION OF MOUSE 20S PROTEASOMES - IMPLICATIONS FOR THE ASSEMBLY OF PRECURSOR BETA-SUBUNITS

The assembly of individual proteasome subunits into catalytically acti ve mammalian 20S proteasomes is not well understood, Using subunit-spe cific antibodies, we characterized both precursor and mature proteasom e complexes, Antibodies to PSMA4 (C9) immunoprecipitated complexes com posed of alpha, precursor beta and processed beta subunits, However, a ntibodies to PSMA3 (C8) and PSMBB (LMP2) immunoprecipitated complexes made up of alpha and precursor beta but no processed beta subunits. Th ese complexes possess short half-lives, are enzymatically inactive and their molecular weight is similar to 300 kDa. Radioactivity chases fr om these complexes into mature, long-lived similar to 700 kDa proteaso mes, Therefore, these structures represent precursor proteasomes and a re probably made up of two rings: one containing a subunits and the ot her, precursor beta subunits, The assembly of precursor proteasomes oc curs in at least two stages, with precursor beta subunits PSMB2 (C7-I) , PSMB3 (C10-II), PSMB7 (Z), PSMB9 (LMP2) and PSMB10 (LMP10) being inc orporated before others [PSMB1 (C5), PSMB6 (delta), and PSMB8 (LMP7)], Proteasome maturation (processing of the beta subunits and juxtaposit ion of the two beta rings) is accompanied by conformational changes in the (outer) a rings, and may be inefficient, Finally, interferon-gamm a had no significant effect on the half-lives or total amounts of prec ursor or mature proteasomes.