Abstract
The degradation of mammalian ornithine decarboxylase (ODC) (EC 4.1.1.17) by 26 S proteasome, is accelerated by the ODC antizyme (AZ), a trigger protein involved in the specific degradation of eukaryotic ODC. In prokaryotes, AZ has not been found. Previously, we found that in Selenomonas ruminantium, a strictly anaerobic and Gram-negative bacterium, a drastic degradation of lysine decarboxylase (LDC; EC 4.1.1.18), which has decarboxylase activities toward both L-lysine and L-ornithine with similar K(m) values, occurs upon entry into the stationary phase of cell growth by protease together with a protein of 22 kDa (P22). Here, we show that P22 is a direct counterpart of eukaryotic AZ by the following evidence. (i) P22 synthesis is induced by putrescine but not cadaverine. (ii) P22 enhances the degradation of both mouse ODC and S. ruminantium LDC by a 26 S proteasome. (iii) S. ruminantium LDC degradation is also enhanced by mouse AZ replacing P22 in a cell-free extract from S. ruminantium. (iv) Both P22 and mouse AZ bind to S. ruminantium LDC but not to the LDC mutated in its binding site for P22 and AZ. In this report, we also show that P22 is a ribosomal protein of S. ruminantium.
Highlights
In mammalian cells and tissues, turnover of ornithine decarboxylase (ODC)3 (EC 4.1.1.17) is very rapid and highly regulated
We show that protein of 22 kDa (P22) is a direct counterpart of eukaryotic AZ by the following evidence. (i) P22 synthesis is induced by putrescine but not cadaverine. (ii) P22 enhances the degradation of both mouse ODC and S. ruminantium lysine decarboxylase (LDC) by a 26 S proteasome. (iii) S. ruminantium LDC degradation is enhanced by mouse AZ replacing P22 in a cell-free extract from S. ruminantium. (iv) Both P22 and mouse AZ bind to S. ruminantium LDC but not to the LDC mutated in its binding site for P22 and AZ
Degradation of S. ruminantium LDC in a P22-free Cytoplasmic Fraction by Mouse AZ—Previously, we reported that the purified P22 protein strongly enhances LDC proteolysis, which requires ATP in cell-free extracts of S. ruminantium [15] and suggested that P22 is a mouse AZ-like enhancer for degradation of S. ruminantium LDC/ODC because of many similarities between LDC degradation and that of mouse ODC degraded by 26 S proteasome in the presence of mouse AZ
Summary
Materials—Plasmid vectors, pMW119 and pET15b, were purchased from Nippon gene (Tokyo, Japan) and Novagen (Madison, WI), respectively. The purified P22 or mouse AZ preparation was added to a reaction mixture containing 0.1 M Tris-HCl (pH 7.5), 0.1 mM pyridoxal phosphate and either the purified S. ruminantium LDC or a mouse ODC preparation. To remove endogenous P22, S. ruminantium cell extract (200 g of protein) was incubated in a reaction mixture (200 l) containing 1 mM dithiothreitol and anti-P22 antiserum in 20 mM potassium phosphate, pH 6.5, at 4 °C for 60 min. For the degradation of S. ruminantium LDC in 26 S proteasome, which was confirmed to be free from ubiquitin (Ub) using anti-Ub antibody, the basal reaction mixture (Mixture B) contained 40 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 2 mM dithiothreitol, 0.5 mM ATP, 10 mM phosphocreatine, 5 g of creatine kinase, 0.1 mM cycloheximide, 50 nM [35S]LDC, and 20 g of 26 S proteasome in a total volume of 100 l. Nucleotide Sequence Accession Number—The nucleotide sequences of the p22 gene and seven open reading frames have been deposited in the GenBankTM/EMBL/DDBJ data bases under accession number AB100500
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