CLEAVAGE MOTIFS OF THE YEAST 20S PROTEASOME BETA-SUBUNITS DEDUCED FROM DIGESTS OF ENOLASE-1

The 436-amino acid protein enolase 1 from yeast was degraded in vitro hy purified wild-type and mutant yeast 20S proteasome particles. Analy sis of the cleavage products at different times revealed a processive degradation mechanism and a length distribution of fragments ranging f rom 3 to 25 amino acids with an average length of 7 to 8 amino acids. Surprisingly, the average fragment length was very similar between wil d-type and mutant 20S proteasomes with reduced numbers of active sites . This implies that the fragment length is not influenced by the dista nce between the active sites, as previously postulated. A detailed ana lysis of the cleavages also allowed the identification of certain amin o acid characteristics in positions flanking the cleavage site that gu ide the selection of the pi residues by the three active beta subunits . Because yeast and mammalian proteasomes are highly homologous, simil ar cleavage moths might be used by mammalian proteasomes. Therefore, o ur data provide a basis for predicting proteasomal degradation product s from which peptides are sampled by major histocompatibility complex class I molecules for presentation to cytotoxic T cells.