SummaryInterferon (IFN) y induces replacements of the proteasomal subunits X and Y by LMP7 and LMP2, respectively, resulting in an alteration of the proteolytic specificity. We found a third pair ofproteasome subunits expressed reciprocally in response to IFN-y. Molecular cloning of a cDNA encoding one subunit designated as Z, downregulated by IFN-% showed that it is a novel proteasomal subunit with high homology to MECL1, which is markedly induced by IFN-% Thus, IFN-~/induces subunit replacements of not only X and Y by LMP7 and LMP2, respectively, but also of Z by MECL1, producing proteasomes responsible for immunological processing of endogenous antigens. When processed from their precursors, three pairs of the 10 homologous, but distinct, [3-type subunits of eukaryotic proteasomes, that is, X/LMP7, Y/LMP2, and Z/MECL1, have an NH2-terminal threonine residue, assumed to be part of a catalytic center. These findings suggest that the altered molecular organization of the proteasome induced by IFN-y may be responsible for acquisition of its functional change.

In human 20S proteasomes two copies of each of seven different ␣-type and seven different ␤-type subunits are assembled to form a stack of four sevenmembered rings, giving the general structure ␣ 1-7 , ␤ 1-7 , ␤ 1-7 , ␣ 1-7 . By means of immunoelectron microscopy and chemical crosslinking of neighboring subunits, we have determined the positions of the individual subunits in the proteasome. The topography shows that for the trypsin-like, the chymotrypsin-like, and the postglutamyl cleaving activities, the pairs of ␤ type subunits, which are thought to form active sites, are nearest neighbors.

No abstract

Proteasomes catalyze the non-lysosomal, An-dependent selective breakdown of ubiquitinated proteins and are thought to be responsible for MHC class I-restricted antigen presentation. Recently, we reported that gamma interferon (IFN-y) induced not only marked synthesis of the MHC-encoded proteasome subunits LMP2 and LMP7, but also ahnost complete loss of two unidentified proteasome subunits tentatively designated as X and Y in various human cells. Here, we show that subunit X is a new proteasomai subunit highly homologous to LMW, and that subunit Y is identical to the LM~-related proteasomal subunit delta. Thus, IFN-y appears to induce subunit replacements of X and Y by LMP7 and LMPZ, respectively, producing 'Juno-prot~omes' with the functional diversity responsible for processing of endogenous antigens.

The proteasome or multicatalytic endopeptidase from eukaryotic cells consists of at least 14 subunits that fall into two families, alpha and beta. Subunit-specific monoclonal antibodies against ten different subunits of human proteasomes have been produced, together with an antibody that reacts with a motif (prosbox 1), common to alpha-type subunits. Four of the subunit-specific antibodies were able to precipitate proteasomes. The subunit composition of HeLa-cell proteasomes precipitated with these four different antibodies were identical, as judged from two-dimensional electrophoresis. One of the four antibodies was used to obtain proteasomes from cell lines (HeLa, Daudi, IMR90 and BSC-1) and human tissues (placenta, kidney, and liver). Electrophoretic analysis of these proteasomes, combined with peptide mapping of some subunits, suggests that they all contain 14 types of subunits as their major constituents. However, one subunit was present in two isoelectric isoforms in all cells examined. Two other subunits occurred in two or three isoelectric isoforms in placenta, liver and kidney, but not in the cell cultures. Extracts of human cells (HeLa, IMR90, Daudi and erythrocytes) were analysed by non-denaturing electrophoresis and immunoblotting. All of the 11 subunits detected by antibodies were present in a pair of ATP-stabilized protein complexes, presumed to be the 26 S proteinase, and in a doublet of complexes which migrated more slowly than purified proteasomes. Besides being present in proteasomes, one subunit was also found to occur in the free state in cell extracts.