Degradation of ornithine decarboxylase by the 26S proteasome

Ornithine decarboxylase (ODC) is a key enzyme in polyamine biosynthesis. Turnover of ODC is extremely rapid and highly regulated, and is accelerated when polyamine levels increase. Polyamine-stimulated ODC degradation is mediated by association with antizyme (AZ), an ODC inhibitory protein induced by polyamines. ODC, in association with AZ, is degraded by the 26S proteasome in an ATP-dependent, but ubiquitin-independent, manner. The 26S proteasome irreversibly inactivates ODC prior to its degradation. The inactivation, possibly due to unfolding, is coupled to sequestration of ODC within the 26S proteasome. This process requires AZ and ATP, but not proteolytic activity of the 26S proteasome. The carboxyl-terminal region of ODC presumably exposed by interaction with AZ plays a critical role for being trapped by the 26S proteasome. Thus, the degradation pathway of ODC proceeds as a sequence of multiple distinct processes, including recognition, sequestration, unfolding, translocation, and ultimate degradation mediated by the 26S proteasome.     

In vitro study of proteolytic degradation of rat histidine decarboxylase.

Mammalian ornithine decarboxylase (ODC) is a very unstable protein which is degraded in an ATP-dependent manner by proteasome 26S, after making contact with the regulatory protein antizyme. PEST regions are sequences described as signals for protein degradation. The C-terminal PEST region of mammalian ODC is essential for its degradation by proteasome 26S. Mammalian histidine decarboxylase (HDC) is also a short-lived protein. The full primary sequence of mammalian HDC contains PEST-regions at both the N- and C-termini. Rat ODC and different truncated and full versions of rat HDC were expressed in vitro. In vitro degradation of rat ODC and rat 1-512 HDC were compared. Like ODC, rat 1-512 HDC is degraded mainly by an ATP-dependent mechanism. However, antizyme has no effect on the degradation of 1-512 HDC. The use of the inhibitors MG-132 and lactacystine significantly inhibited the degradation of 1-512 HDC, suggesting that a ubiquitin-dependent, proteasome 26S proteolytic pathway is involved. Results obtained with the different modifications of rat HDC containing all three PEST regions (full version, 1-656 HDC), only the N-terminal PEST region (1-512 HDC), or no PEST region (69-512 HDC), indicate that the N-terminal (1-69) fragment, but not the C-terminal fragment, determines that the HDC protein is a proteasome substrate in vitro.     

Ubiquitin-independent mechanisms of mouse ornithine decarboxylase degradation are conserved between mammalian and fungal cells

The polyamine biosynthetic enzyme ornithine decarboxylase (ODC) is degraded by the 26 S proteasome via a ubiquitin-independent pathway in mammalian cells. Its degradation is greatly accelerated by association with the polyamine-induced regulatory protein antizyme 1 (AZ1). Mouse ODC (mODC) that is expressed in the yeast Saccharomyces cerevisiae is also rapidly degraded by the proteasome of that organism. We have now carried out in vivo and in vitro studies to determine whether S. cerevisiae proteasomes recognize mODC degradation signals. Mutations of mODC that stabilized the protein in animal cells also did so in the fungus. Moreover, the mODC degradation signal was able to destabilize a GFP or Ura3 reporter in GFP-mODC and Ura3-mODC fusion proteins. Co-expression of AZ1 accelerated mODC degradation 2-3-fold in yeast cells. The degradation of both mODC and the endogenous yeast ODC (yODC) was unaffected in S. cerevisiae mutants with various defects in ubiquitin metabolism, and ubiquitinylated forms of mODC were not detected in yeast cells. In addition, recombinant mODC was degraded in an ATP-dependent manner by affinity-purified yeast 26 S proteasomes in the absence of ubiquitin. Degradation by purified yeast proteasomes was sensitive to mutations that stabilized mODC in vivo, but was not accelerated by recombinant AZ1. These studies demonstrate that cell constituents required for mODC degradation are conserved between animals and fungi, and that both mammalian and fungal ODC are subject to proteasome-mediated proteolysis by ubiquitin-independent mechanisms.     

Hybrid proteasomes. Induction by interferon-gamma and contribution to ATP-dependent proteolysis

Eukaryotic cells contain various types of proteasomes. Core 20 S proteasomes (abbreviated 20 S below) have two binding sites for the regulatory particles, PA700 and PA28. PA700-20 S-PA700 complexes are known as 26 S proteasomes and are ATP-dependent machines that degrade cell proteins. PA28 is found both in previously described complexes of the type PA28-20 S-PA28 and in complexes that also contain PA700, as PA700-20 S-PA28. We refer to the latter as "hybrid proteasomes." The relative amounts of the various types of proteasomes in HeLa extracts were determined by a combination of immunoprecipitation and immunoblotting. Hybrid proteasomes accounted for about a fourth of all proteasomes in the extracts. Association of PA28 and proteasomes proved to be ATP-dependent. Hybrid proteasomes catalyzed ATP-dependent degradation of ornithine decarboxylase (ODC) without ubiquitinylation, as do 26 S proteasomes. In contrast, the homo-PA28 complex (PA28-20 S-PA28) was incapable of degrading ODC. Intriguingly, a major immunomodulatory cytokine, interferon-gamma, appreciably enhanced the ODC degradation in HeLa and SW620 cells through induction of the hybrid proteasome, which may also be responsible for the immunological processing of intracellular antigens. Taken together, we report here for the first time the existence of two types of ATP-dependent proteases, the 26 S proteasome and the hybrid proteasome, which appear to share the ATP-dependent proteolytic pathway in mammalian cells.     

Characterization of a counterpart to Mammalian ornithine decarboxylase antizyme in prokaryotes

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.     

Determinants of proteasome recognition of ornithine decarboxylase,a ubiquitin-independent substrate

Ornithine decarboxylase (ODC) is regulated by its metabolic products through a feedback loop that employs a second protein, antizyme 1 (AZ1). AZ1 accelerates the degradation of ODC by the proteasome. We used purified components to study the structural elements required for proteasomal recognition of this ubiquitin-independent substrate. Our results demonstrate that AZ1 acts on ODC to enhance the association of ODC with the proteasome, not the rate of its processing. Substrate-linked or free polyubiquitin chains compete for AZ1-stimulated degradation of ODC. ODC-AZ1 is therefore recognized by the same element(s) in the proteasome that mediate recognition of polyubiquitin chains. The 37 C-terminal amino acids of ODC harbor an AZ1-modulated recognition determinant. Within the ODC C terminus, three subsites are functionally distinguishable. The five terminal amino acids (ARINV, residues 457-461) collaborate with residue C441 to constitute one recognition element, and AZ1 collaborates with additional constituents of the ODC C terminus to generate a second recognition element.     

The C terminus of Rpt3, an ATPase subunit of PA700 (19 S) regulatory complex, is essential for 26 S proteasome assembly but not for activation

PA700, the 19 S regulatory subcomplex of the 26 S proteasome, contains a heterohexameric ring of AAA subunits (Rpt1 to -6) that forms the binding interface with a heteroheptameric ring of α subunits (α1 to -7) of the 20 S proteasome. Binding of these subcomplexes is mediated by interactions of C termini of certain Rpt subunits with cognate binding sites on the 20 S proteasome. Binding of two Rpt subunits (Rpt2 and Rpt5) depends on their last three residues, which share an HbYX motif (where Hb is a hydrophobic amino acid) and open substrate access gates in the center of the α ring. The relative roles of other Rpt subunits for proteasome binding and activation remain poorly understood. Here we demonstrate that the C-terminal HbYX motif of Rpt3 binds to the 20 S proteasome but does not promote proteasome gating. Binding requires the last three residues and occurs at a dedicated site on the proteasome. A C-terminal peptide of Rpt3 blocked ATP-dependent in vitro assembly of 26 S proteasome from PA700 and 20 S proteasome. In HEK293 cells, wild-type Rpt3, but not Rpt3 lacking the HbYX motif was incorporated into 26 S proteasome. These results indicate that the C terminus of Rpt3 was required for cellular assembly of this subunit into 26 S proteasome. Mutant Rpt3 was assembled into intact PA700. This result indicates that intact PA700 can be assembled independently of association with 20 S proteasome and thus may be a direct precursor for 26 S proteasome assembly under normal conditions. These results provide new insights to the non-equivalent roles of Rpt subunits in 26 S proteasome function and identify specific roles for Rpt3.     

Antizyme, a mediator of ubiquitin-independent proteasomal degradation

Ornithine decarboxylase (ODC) is among the small set of proteasome substrates that is not ubiquitinated. It is instead degraded in conjunction with the protein antizyme (AZ). ODC and AZ are participants in a regulatory circuit that restricts pools of polyamines, the downstream products of ODC enzymatic activity. Functional studies using directed mutagenesis have identified regions of ODC and AZ required for the process of ODC degradation. Within ODC, there is a region that is required for AZ binding which lies on the surface of an alpha-beta barrel forming one domain of the ODC monomer. A carboxy-terminal ODC domain is needed for both AZ-dependent and AZ-independent degradation. Within AZ, the carboxy-terminal half molecule is sufficient for binding to ODC, but an additional domain found within the AZ amino terminus must be present for stimulation of ODC degradation by the proteasome. Recently, the AZs have been found to consist of an ancient gene family. Within vertebrate species, multiple isoforms are found, with distinct functions that remain to be sorted out. Although AZ homologs have been found in some yeast species, homology searches have failed to identify an AZ homolog in Saccharomyces cerevisiae. Nevertheless, the close parallel between polyamine-induced ODC degradation in S. cerevisiae and in animal cells suggests that this organism will also be found to harbor an AZ-like protein.     

Subcellular localization and phosphorylation of antizyme 2

Antizymes (AZs) are polyamine‐induced proteins that negatively regulate cellular polyamine synthesis and uptake. Three antizyme isoforms are conserved among mammals. AZ1 and AZ2 have a broad tissue distribution, while AZ3 is testis specific. Both AZ1 and AZ2 inhibit ornithine decarboxylase (ODC) activity by binding to ODC monomer and target it to the 26S proteasome at least in vivo. Both also inhibit extra‐cellular polyamine uptake. Despite their being indistinguishable by these criteria, we show here using enhanced green fluorescent protein (EGFP)‐AZ2 fusion protein that in mammalian cells, the subcellular location of AZ2 is mainly in the nucleus, and is different from that of AZ1. The C‐terminal part of AZ2 is necessary for the nuclear distribution. Within a few hours, a shift in the distribution of EGFP‐AZ2 fusion protein from cytoplasm to the nucleus or from nucleus to cytoplasm is observable in NIH3T3 cells. In addition, we found that in cells a majority of AZ2, but not AZ1, is phosphorylated at Ser‐186, likely by protein kinase CK2. There may be a specific function of AZ2 in the nucleus. J. Cell. Biochem. 108: 1012–1021, 2009. © 2009 Wiley‐Liss, Inc.     

Mechanisms of Protein Degradation: An Odyssey with ODC

Intracellular proteolysis plays an important role in regulating fundamental cellularprocesses such as cell cycle, immune and inflammation responses, development,differentiation, and transformation. The ubiquitin-proteasome system accounts for thedegradation of the majority of cellular short-lived proteins. This system involves theconjugation of multiple ubiquitin residues to the target protein and its recognition by the26S proteasome through the poly-ubiquitin chain. Studies on the degradation of ornithinedecarboxylase (ODC) demonstrated that poly-ubiquitin is not the only signal recognizedby the 26S proteasome. The recognition of ODC by the 26S proteasome is mediated byinteraction with a polyamine-induced protein termed, antizyme (Az). While thedegradation of ODC is ubiquitin-independent, the degradation of its regulator Az, and ofantizyme-inhibitor (AzI), an ODC homologous protein that regulates Az availability, areubiquitin dependent. Interestingly, ODC undergoes another type of ubiquitin-independentdegradation by the 20S proteasome that is regulated by NAD(P)H quinoneoxidoreductase 1 (NQO1). Considering the prevalence of the ubiquitin system in theprocess of cellular protein degradation it is rather remarkable that a key cellular enzymeis subjected to two different proteolytic pathways that are different from the ubiquitindependent one. This exceptional behavior of ODC provides us with valuable insightsregarding protein degradation in general.     

C termini of proteasomal ATPases play nonequivalent roles in cellular assembly of mammalian 26 S proteasome

The 26 S proteasome comprises two multisubunit subcomplexes as follows: 20 S proteasome and PA700/19 S regulatory particle. The cellular mechanisms by which these subcomplexes assemble into 26 S proteasome and the molecular determinants that govern the assembly process are poorly defined. Here, we demonstrate the nonequivalent roles of the C termini of six AAA subunits (Rpt1-Rpt6) of PA700 in 26 S proteasome assembly in mammalian cells. The C-terminal HbYX motif (where Hb is a hydrophobic residue, Y is tyrosine, and X is any amino acid) of each of two subunits, Rpt3 and Rpt5, but not that of a third subunit Rpt2, was essential for assembly of 26 S proteasome. The C termini of none of the three non-HbYX motif Rpt subunits were essential for cellular 26 S proteasome assembly, although deletion of the last three residues of Rpt6 destabilized the 20 S-PA700 interaction. Rpt subunits defective for assembly into 26 S proteasome due to C-terminal truncations were incorporated into intact PA700. Moreover, intact PA700 accumulated as an isolated subcomplex when cellular 20 S proteasome content was reduced by RNAi. These results indicate that 20 S proteasome is not an obligatory template for assembly of PA700. Collectively, these results identify specific structural elements of two Rpt subunits required for 26 S proteasome assembly, demonstrate that PA700 can be assembled independently of the 20 S proteasome, and suggest that intact PA700 is a direct intermediate in the cellular pathway of 26 S proteasome assembly.     

Crystal structure of human ornithine decarboxylase at 2.1 A resolution: structural insights to antizyme binding

The polyamines spermidine and spermine are ubiquitous and required for cell growth and differentiation in eukaryotes. Ornithine decarboxylase (ODC, EC 4.1.1.17) performs the first step in polyamine biosynthesis, the decarboxylation of ornithine to putrescine. Elevated polyamine levels can lead to down-regulation of ODC activity by enhancing the translation of antizyme mRNA, resulting in subsequent binding of antizyme to ODC monomers which targets ODC for proteolysis by the 26S proteasome. The crystal structure of ornithine decarboxylase from human liver has been determined to 2.1 A resolution by molecular replacement using truncated mouse ODC (Delta425-461) as the search model and refined to a crystallographic R-factor of 21.2% and an R-free value of 28.8%. The human ODC model includes several regions that are disordered in the mouse ODC crystal structure, including one of two C-terminal basal degradation elements that have been demonstrated to independently collaborate with antizyme binding to target ODC for degradation by the 26S proteasome. The crystal structure of human ODC suggests that the C terminus, which contains basal degradation elements necessary for antizyme-induced proteolysis, is not buried by the structural core of homodimeric ODC as previously proposed. Analysis of the solvent-accessible surface area, surface electrostatic potential, and the conservation of primary sequence between human ODC and Trypanosoma brucei ODC provides clues to the identity of potential protein-binding-determinants in the putative antizyme binding element in human ODC.     

Degradation of oxidized proteins by the 20S proteasome

Oxidatively modified proteins are continuously produced in cells by reactive oxygen and nitrogen species generated as a consequence of aerobic metabolism. During periods of oxidative stress, protein oxidation is significantly increased and may become a threat to cell survival. In eucaryotic cells the proteasome has been shown (by purification of enzymatic activity, by immunoprecipitation, and by antisense oligonucleotide studies) to selectively recognize and degrade mildly oxidized proteins in the cytosol, nucleus, and endoplasmic reticulum, thus minimizing their cytotoxicity. From in vitro studies it is evident that the 20S proteasome complex actively recognizes and degrades oxidized proteins, but the 26S proteasome, even in the presence of ATP and a reconstituted functional ubiquitinylating system, is not very effective. Furthermore, relatively mild oxidative stress rapidly (but reversibly) inactivates both the ubiquitin activating/conjugating system and 26S proteasome activity in intact cells, but does not affect 20S proteasome activity. Since mild oxidative stress actually increases proteasome-dependent proteolysis (of oxidized protein substrates) the 20S 'core' proteasome complex would appear to be responsible. Finally, new experiments indicate that conditional mutational inactivation of the E1 ubiquitin-activating enzyme does not affect the degradation of oxidized proteins, further strengthening the hypothesis that oxidatively modified proteins are degraded in an ATP-independent, and ubiquitin-independent, manner by the 20S proteasome. More severe oxidative stress causes extensive protein oxidation, directly generating protein fragments, and cross-linked and aggregated proteins, that become progressively resistant to proteolytic digestion. In fact these aggregated, cross-linked, oxidized proteins actually bind to the 20S proteasome and act as irreversible inhibitors. It is proposed that aging, and various degenerative diseases, involve increased oxidative stress (largely from damaged and electron 'leaky' mitochondria), and elevated levels of protein oxidation, cross-linking, and aggregation. Since these products of severe oxidative stress inhibit the 20S proteasome, they cause a vicious cycle of progressively worsening accumulation of cytotoxic protein oxidation products.     

Ornithine decarboxylase-antizyme is rapidly degraded through a mechanism that requires functional ubiquitin-dependent proteolytic activity.

Antizyme is a polyamine-induced cellular protein that binds to ornithine decarboxylase (ODC), and targets it to rapid ubiquitin-independent degradation by the 26S proteasome. However, the metabolic fate of antizyme is not clear. We have tested the stability of antizyme in mammalian cells. In contrast with previous studies demonstrating stability in vitro in a reticulocyte lysate-based degradation system, in cells antizyme is rapidly degraded and this degradation is inhibited by specific proteasome inhibitors. While the degradation of ODC is stimulated by the presence of cotransfected antizyme, degradation of antizyme seems to be independent of ODC, suggesting that antizyme degradation does not occur while presenting ODC to the 26S proteasome. Interestingly, both species of antizyme, which represent initiation at two in-frame initiation codons, are rapidly degraded. The degradation of both antizyme proteins is inhibited in ts20 cells containing a thermosensitive ubiquitin-activating enzyme, E1. Therefore we conclude that in contrast with ubiquitin-independent degradation of ODC, degradation of antizyme requires a functional ubiquitin system.     

Initiation of Cyclin B Degradation by the 26S Proteasome upon Egg Activation

Immediately before the transition from metaphase to anaphase, the protein kinase activity of maturation or M-phase promoting factor (MPF) is inactivated by a mechanism that involves the degradation of its regulatory subunit, cyclin B. The availability of biologically active goldfish cyclin B produced in Escherichia coli and purified goldfish proteasomes (a nonlysosomal large protease) has allowed the role of proteasomes in the regulation of cyclin degradation to be examined for the first time. The 26S, but not the 20S proteasome, digested recombinant 49-kD cyclin B at lysine 57 (K57), producing a 42-kD truncated form. The 42-kD cyclin was also produced by the digestion of native cyclin B forming a complex with cdc2, a catalytic subunit of MPF, and a fragment transiently appeared during cyclin degradation when eggs were released from metaphase II arrest by egg activation. Mutant cyclin at K57 was resistant to both digestion by the 26S proteasome and degradation at metaphase/anaphase transition in Xenopus egg extracts. The results of this study indicate that the destruction of cyclin B is initiated by the ATP-dependent and ubiquitin-independent proteolytic activity of 26S proteasome through the first cutting in the NH₂ terminus of cyclin (at K57 in the case of goldfish cyclin B). We also surmise that this cut allows the cyclin to be ubiquitinated for further destruction by ubiquitin-dependent activity of the 26S proteasome that leads to MPF inactivation.     

The ubiquitin-proteasome pathway plays an essential role in proteolysis during Trypanosoma cruzi remodeling

Here, we document for the first time the presence of the 26S proteasome and the ubiquitin pathway in a protozoan parasite that is in an early branch in the eukaryotic lineage. The 26S proteasome of Trypanosoma cruzi epimastigotes was identified as a high molecular weight complex (1400 kDa) with an ATP-dependent chymotrypsin-like activity against the substrate Suc-LLVY-Amc. This activity was inhibited by proteasome inhibitors and showed same electrophorectic migration pattern as yeast 26S proteasome in nondenaturating gels. About 30 proteins in a range of 25-110 kDa were detected in the purified T. cruzi 26S proteasome. Antibodies raised against the AAA family of ATPases from eukaryotic 26S proteasome and the T. cruzi 20S core specifically recognized components of T. cruzi 26S. To confirm the biological role of 26S in this primitive eukaryotic parasite, we analyzed the participation of the ubiquitin (Ub)-proteasome system in protein degradation during the time of parasite remodeling. Protein turnover in trypomastigotes was proteasome and ATP-dependent and was enhanced during the transformation of the parasites into amastigotes. If 20S proteasome activity is inhibited, ubiquitinated proteins accumulate in the parasites. As expected from the profound morphological changes that occur during transformation, cytoskeletal proteins associated with the flagellum are targets of the ubiquitin-proteasome pathway.     

Structural Models for Interactions between the 20S Proteasome and Its PAN/19S Activators

Proteasome activity is regulated by sequestration of its proteolytic centers in a barrel-shaped structure that limits substrate access. Substrates enter the proteasome by means of activator complexes that bind to the end rings of proteasome α subunits and induce opening of an axial entrance/exit pore. The PA26 activator binds in a pocket on the proteasome surface using main chain contacts of its C-terminal residues and uses an internal activation loop to trigger gate opening by repositioning the proteasome Pro-17 reverse turn. Subunits of the unrelated PAN/19S activators bind with their C termini in the same pockets but can induce proteasome gate opening entirely from interactions of their C-terminal peptides, which are reported to cause gate opening by inducing a rocking motion of proteasome α subunits rather than by directly contacting the Pro-17 turn. Here we report crystal structures and binding studies of proteasome complexes with PA26 constructs that display modified C-terminal residues, including those corresponding to PAN. These findings suggest that PA26 and PAN/19S C-terminal residues bind superimposably and that both classes of activator induce gate opening by using direct contacts to residues of the proteasome Pro-17 reverse turn. In the case of the PAN and 19S activators, a penultimate tyrosine/phenylalanine residue contacts the proteasome Gly-19 carbonyl oxygen to stabilize the open conformation.     

Differential impairment of 20S and 26S proteasome activities in human hematopoietic K562 cells during oxidative stress

The 20S proteasome and the 26S proteasome are major components of the cytosolic and nuclear proteasomal proteolytic systems. Since proteins are known to be highly susceptible targets for reactive oxygen species, the effect of H(2)O(2) treatment of K562 human hematopoietic cells toward the activities of 20S and 26S proteasomes was investigated. While the ATP-independent degradation of the fluorogenic peptide suc-LLVY-MCA was not affected by H(2)O(2) concentrations of up to 5 mM, the ATP-stimulated degradation of suc-LLVY-MCA by the 26S proteasome began to decline at 400 microM and was completely abolished at 1 mM oxidant treatment. A combination of nondenaturing electrophoresis and Western blotting let us believe that the high oxidant susceptibility of the 26S proteasome is due to oxidation of essential amino acids in the proteasome activator PA 700 which mediates the ATP-dependent proteolysis of the 26S-proteasome. The activity of the 26S-proteasome could be recovered within 24 h after exposure of cells to 1 mM H(2)O(2) but not after 2 mM H(2)O(2). In view of the specific functions of the 26S proteasome in cell cycle control and other important physiological functions, the consequences of the higher susceptibility of this protease toward oxidative stress needs to be considered.     

Polyamines regulate their synthesis by inducing expression and blocking degradation of ODC antizyme

Polyamines are essential organic cations with multiple cellular functions. Their synthesis is controlled by a feedback regulation whose main target is ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. In mammals, ODC has been shown to be inhibited and targeted for ubiquitin-independent degradation by ODC antizyme (AZ). The synthesis of mammalian AZ was reported to involve a polyamine-induced ribosomal frameshifting mechanism. High levels of polyamine therefore inhibit new synthesis of polyamines by inducing ODC degradation. We identified a previously unrecognized sequence in the genome of Saccharomyces cerevisiae encoding an orthologue of mammalian AZ. We show that synthesis of yeast AZ (Oaz1) involves polyamine-regulated frameshifting as well. Degradation of yeast ODC by the proteasome depends on Oaz1. Using this novel model system for polyamine regulation, we discovered another level of its control. Oaz1 itself is subject to ubiquitin-mediated proteolysis by the proteasome. Degradation of Oaz1, however, is inhibited by polyamines. We propose a model, in which polyamines inhibit their ODC-mediated biosynthesis by two mechanisms, the control of Oaz1 synthesis and inhibition of its degradation.