The 436-amino acid protein enolase 1 from yeast was degraded in vitro by purified wild-type and mutant yeast 20S proteasome particles. Analysis of the cleavage products at different times revealed a processive degradation mechanism and a length distribution of fragments ranging from 3 to 25 amino acids with an average length of 7 to 8 amino acids. Surprisingly, the average fragment length was very similar between wild-type and mutant 20S proteasomes with reduced numbers of active sites. This implies that the fragment length is not influenced by the distance between the active sites, as previously postulated. A detailed analysis of the cleavages also allowed the identification of certain amino acid characteristics in positions flanking the cleavage site that guide the selection of the P1 residues by the three active ␤ subunits. Because yeast and mammalian proteasomes are highly homologous, similar cleavage motifs might be used by mammalian proteasomes. Therefore, our data provide a basis for predicting proteasomal degradation products from which peptides are sampled by major histocompatibility complex class I molecules for presentation to cytotoxic T cells.The eukaryotic 20S proteasome represents the catalytic core particle of the 26S proteasome, which is an essential component of the ubiquitin-dependent protein degradation pathway. The 20S particle is composed of 14 different but related subunits. Two outer disks, each containing seven ␣ subunits, guard the two inner heptameric ␤ subunit rings, which contain three proteolytically active sites each (1). How substrate molecules gain access to the active sites at the inner surface of the ␤ rings is not known. It has been hypothesized that on association of the 20S particle with regulatory complexes, such as the 19S cap structure or PA28, an opening in the middle of the ␣ subunit rings is induced allowing unfolded proteins to be fed into the 20S particle.Probably as a consequence of this closed structure, very few proteins have been found to be degraded by 20S proteasomes in vitro, and only very few individual fragments and cleavage sites generated during the degradation of proteins by eukaryotic 20S proteasomes have been analyzed until now. This kind of analysis is of interest for several reasons.First, it still has to be confirmed that the specificities of the proteasomal ␤ subunits observed in experiments using peptide substrates (3-8), summarized as trypsin-like, chymotrypsin (ChT)-like, and peptidylglutamyl-peptide hydrolyzing activity, correspond to cleavages performed in intact proteins, a situation physiologically more relevant.Second, the mechanism of protein degradation by the proteasome is not known. For the archebacterial proteasome, which is a less complex structure consisting of 14 identical ␣ and ␤ subunits (9), a processive model has been proposed (2, 10). Because of the high structural homology with the archebacterial particle a similar mechanism can be expected for eukaryotic proteasomes. Furthermore, it has been postulated that the 20S pro...

Proteasomes generate peptides that can be presented by major histocompatibility complex (MHC) class I molecules in vertebrate cells. Using yeast 20 S proteasomes carrying different inactivated ␤-subunits, we investigated the specificities and contributions of the different ␤-subunits to the degradation of polypeptide substrates containing MHC class I ligands and addressed the question of additional proteolytically active sites apart from the active ␤-subunits. We found a clear correlation between the contribution of the different subunits to the cleavage of fluorogenic and long peptide substrates, with ␤5/Pre2 cleaving after hydrophobic, ␤2/Pup1 after basic, and ␤1/Pre3 after acidic residues, but with the exception that ␤2/Pup1 and ␤1/Pre3 can also cleave after some hydrophobic residues. All proteolytic activities including the "branched chain amino acid-preferring" component are associated with ␤5/Pre2, ␤1/Pre3, or ␤2/ Pup1, arguing against additional proteolytic sites. Because of the high homology between yeast and mammalian 20 S proteasomes in sequence and subunit topology and the conservation of cleavage specificity between mammalian and yeast proteasomes, our results can be expected to also describe most of the proteolytic activity of mammalian 20 S proteasomes leading to the generation of MHC class I ligands.

Nonclassical HLA-G molecules present peptides essentially in the same way as classical HLA molecules do. We determined the peptide motif that is specifically recognized by HLA-G; its basic features are described by the sequence XI/LPXXXXXL: This information should help to elucidate the physiological role of HLA-G molecules at the fetal-maternal interface. Most likely, this role is to protect fetal cells from lysis by natural killer cells, and possibly to present foreign peptides to a class of T cells that has not yet been identified.