Involvement of the 20S proteasome in the degradation of ornithine decarboxylase

Abstract

Eukaryotic cells have been shown to contain two high-molecular-mass proteases of 700 kDa and 1400kDa (20s and 26s proteases, respectively). It has been suggested that the 20s protease, also known as proteasome, may constitute the catalytic core of the 26s protease. While the role of the free 20s protease in intracellular protein degradation is unclear, the 26s protease is implicated in the degradation of ubiquinated proteins. We have recently demonstrated, that ornithine decarboxylase (ODC), one of the most labile proteins in mammalian cells, is degraded via an ATP-dependent but ubiquitin-independent proteolytic pathway. Here we extend these observations by demonstrating that in reticulocyte lysate ODC degradation is inhibited by antibodies raised against the C9 subunit of rat proteasome. Partial fractionation of the lysate demonstrated preferential degradation of ODC in the fraction of the lysate proteins that are precipitated by 38% ammonium sulfate. Since it was demonstrated that the 26s protease precipitates at this concentration of ammonium sulfate while the 20s proteasome remains soluble, our results suggest that the 26s protease is the one degrading ODC.Two high-molecular-mass proteases have been described in a variety of eukaryotic cells including rabbit reticulocytes [l, 21. The first is a 700-kDa proteolytic complex termed proteasome or 20s protease (for review see . It is a cylinder-shaped particle [8, 91 composed of a large number of typical low-molecular-mass subunits [lo, 111, exhibiting various proteolytic activities specific to proteins and peptides [4,12, 131. The physiological role of this protease is still unknown, mainly because of contradictory reports concerning its ATP-dependence [l, 2, 141 which does not reconcile with the absolute requirement for energy in the process of intracellular protein breakdown [15]. The second, is an even larger 1400-kDa proteolytic complex known as the 26s protease. Recent studies have demonstrated that this ATP-dependent protease has clear preference for ubiquinated proteins [l, 21. It has been recently suggested that the 20s proteasome may constitute the catalytic core of the larger 26s protease [ 16 -181.Ornithine decarboxylase (ODC) is a highly regulated enzyme in the biosynthesis pathway of polyamines [19], whose activity is crucial for the process of cellular proliferation [20-231. ODC is one of the most rapidly turned over proteins in mammalian cells [24, 251. We have recently demonstrated that in cells, and in an in v i m reticulocyte-lysatebased degradation mix, ODC degradation occurs via a nonlysosomal, ATP-dependent but ubiquitin-independent pathway [26, 271. In the present study we have further characCorrespondence to C . Kahana, Department of Molecular Genetics and Virology, The Weizmann Institute of Science, Rehovot 76100, Israel Abbreviation. ODC, omithine decarboxylase.Enzyme. Ornithine decarboxylase (EC 4.1.1.17).terized ODC degradation in reticulocyte lysate. We show here that highly specific rabbit polyclonal antibodies raised against the C9...

Degradation of Ornithine Decarboxylase by the Mammalian and Yeast 26S Proteasome Complexes Requires all the Components of the Protease

“…The inhibitor was used as a glutathione transferase fusion protein. 3 pg mouse ODC cDNA subcloned into pGEM-1 transcription vector (Promega) under the control of T7 RNA polymerase was linearized with BarnHI and transcribed and translated as described previously [37]. A cDNA clone that encodes the T brucei ODC [pBHS2.2; 201 was linearized with HincII, transcribed with T7 RNA polymerase, and translated as described [37,381.…”

“…3 pg mouse ODC cDNA subcloned into pGEM-1 transcription vector (Promega) under the control of T7 RNA polymerase was linearized with BarnHI and transcribed and translated as described previously [37]. A cDNA clone that encodes the T brucei ODC [pBHS2.2; 201 was linearized with HincII, transcribed with T7 RNA polymerase, and translated as described [37,381. The cDNAs that encode truncated mouse ODC [des(F425 -V461)-ODC(mouse)], and trypanosome N-terminal mouse C-terminal chimeric protein (residues 1-395 of trypanosome and residues 376-461 of the mouse enzymes) are described elsewhere [20, 211.…”

Proteasomal Degradation of Spermidine/Spermine N 1-Acetyltransferase Requires the Carboxyl-terminal Glutamic Acid Residues

“…locyte Lysate System-The degradation of SSAT was studied by using a reticulocyte lysate system shown previously to degrade ODC in a physiologically relevant manner (29,33,35). Labeled SSAT was synthesized using the TNT synthesis system with pSAT9.3, and aliquots were added to reticulocyte lysates.…”

“…The turnover of ODC, which is mediated by a polyamine-inducible protein termed antizyme, has been studied extensively (25)(26)(27)(28)(29), but there is no published information available on the mechanism of degradation of S-adenosylmethionine decarboxylase and SSAT.…”

Feedback repression of polyamine transport is mediated by antizyme in mammalian tissue-culture cells

“…Substantial progress has been made in understanding feedback repression of polyamine biosynthesis, which appears to centre around the polyamine-stimulated production of a labile protein, antizyme, that binds specifically and reversibly to the initial enzyme in polyamine synthesis, ornithine decarboxylase (ODC) [5][6][7][8][9][10]. Experiments in vitro have revealed that this ODC-antizyme complex is sensitive to rapid degradation by the 26 S proteasome [11,12]. In recent studies Murakami et al [13] firmly established that the polyamine feedback on ODC activity in intact cells is indeed mediated by antizyme.…”