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 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 beta 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.
Institut für Zellbiologie, Abteilung Immunologie, Universität Tübingen, Auf der Morgenstelle 15, D-72076 Tübingen, Germany.
A K
AK
Nussbaum
T P
TP
Dick
W
W
Keilholz
M
M
Schirle
S
S
Stevanović
K
K
Dietz
W
W
Heinemeyer
M
M
Groll
D H
DH
Wolf
R
R
Huber
H G
HG
Rammensee
H
H
Schild
D000595:Amino Acid Sequence; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism*; D010751:Phosphopyruvate Hydratase / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
D009097:Multienzyme Complexes; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D010751:Phosphopyruvate Hydratase