Range of sizes of peptide products generated during degradation of different proteins by archaeal proteasomes

The 20 S proteasome processively degrades cell proteins to peptides, Information on the sizes and nature of these products is essential for understanding the proteasome's degradative mechanism, the subsequent steps in protein turnover, and major histocompatibility complex class I antigen presentation, Using proteasomes from Thermoplasma acidophilum and four unfolded polypeptides as substrates (insulin-like growth factor, lactalbumin, casein, and alkaline phosphatase, whose lengths range from 71 to 471 residues), we demonstrate that the number of cuts made in a polypeptide and the time needed to degrade it increase with length, The average size of peptides generated from these four polypeptides was 8 +/- 1 residues, but ranged from 6 to 10 residues, depending on the protein, as determined by two new independent methods. However, the individual peptide products ranged in length from approximately 3 to 30 residues, as demonstrated by mass spectrometry and size-exclusion chromatography. The sizes of individual peptides fit a lognormal distribution, No length was predominant, and more than half were shorter than 10 residues, Peptide abundance decreased with increasing length, and less than 10% exceeded 20 residues, These findings indicate that: 1) the proteasome does not generate peptides according to the "molecular ruler" hypothesis, and 2) other peptidases must function after the proteasome to complete the turnover of cell proteins to amino acids.