ATP- and antizyme-dependent endoproteolysis of ornithine decarboxylase to oligopeptides by the 26 S proteasome.
Previously we reported that ornithine decarboxylase (ODC) is degraded ATP-dependently by the 26 S proteasome in the presence of antizyme (AZ), an ODC inhibitor (Murakami, Y., Matsufuji, S., Kameji, T., Hayashi, S., Igarashi, K., Tamura, T., Tanaka, K., and Ichihara, A. (1992) Nature 360, 597-599). Here we examined the cleavage of ODC by the 26 S proteasome. When ODC purified from ODC-overproducing cells was incubated with the 26 S proteasome and with AZ fused with maltose-binding protein (MBP) in the presence of ATP, ODC was degraded specifically without appreciable breakdown of MBP-AZ. The major degradation products of ODC, which were separated by high performance liquid chromatography on a reverse-phase column, were identified by N-terminal amino acid sequencing. The 26 S proteasome generated a variety of short peptides of 5-11 amino acid residues derived from regions throughout the ODC sequence. No detectable amounts of free amino acid residues were produced, indicating endoproteolytic degradation of ODC by the 26 S proteasome. Their major sites for cleavage of ODC by the 26 S proteasome were on the carboxyl sides of neutral/hydrophobic amino acid residues, but a few were on those of acidic or basic amino acid residues. These results demonstrate that the 26 S proteasome causes exhaustive endoproteolysis of the naturally occurring short-lived protein ODC in a multicatalytic and ATP-dependent manner.
Department of Life Science, Faculty of Science, Himeji Institute of Technology, Hyogo, Japan.
F
F
Tokunaga
T
T
Goto
T
T
Koide
Y
Y
Murakami
S
S
Hayashi
T
T
Tamura
K
K
Tanaka
A
A
Ichihara
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D006868:Hydrolysis; D051379:Mice; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D009842:Oligopeptides / Q000096:biosynthesis* / Q000378:metabolism; D009955:Ornithine Decarboxylase / Q000378:metabolism*; D065108:Ornithine Decarboxylase Inhibitors; D010447:Peptide Hydrolases / Q000378:metabolism*; D010449:Peptide Mapping; D046988:Proteasome Endopeptidase Complex; D011506:Proteins / Q000378:metabolism*; D016415:Sequence Alignment
D009097:Multienzyme Complexes; D009842:Oligopeptides; D065108:Ornithine Decarboxylase Inhibitors; D011506:Proteins; C031017:ornithine decarboxylase antizyme; D000255:Adenosine Triphosphate; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D009955:Ornithine Decarboxylase