Recent studies have provided convincing evidence to add to a number of earlier observations suggesting that the rapid intracellular degradation of mammalian ornithine decarboxylase (ODC) is further accelerated by the action of ornithine decarboxylase antizyme (ODC-Az), a polyamineinduced protein. However, the mechanism whereby ODC-Az exerts its effect in this proteolytic process is mostly unknown. Here, by using reticulocyte-lysate-based synthesis and degradation systems, we demonstrate that interaction of ODC-Az with ODC results in two related outcomes: (a) ODC is inactivated as a result of its monomerization, and (b) ODC degradation is dramatically accelerated. While ODC inactivation requires the integrity of the ODC-Az binding site of ODC and the ODC binding site of ODC-Az, acceleration in ODC degradation also requires the previously characterized carboxyl-terminal destabilizing segment of ODC and a specific segment of ODC-Az that may be functionally distinct from that required for ODC binding. Interestingly, an active ODC variant with a mutant ODC-Az binding site is stable under basal degradation conditions. This, together with the ability of anti-(ODC-Az) antibody to specifically inhibit the basal degradation of ODC in the lysate, suggests that ODC-Az is an essential general mediator of ODC degradation. Based on these observations, we propose a model for the degradation of ODC which always require interaction with antizyme.Short intracellular half-life is a characteristic feature shared by highly regulated proteins, which enables their efficient modulation by upstream cellular regulatory mechanisms [l]. Ornithine decarboxylase (ODC) is a highly regulated enzyme in the biosynthesis of polyamines, which is one of the most labile cellular proteins [l]. Studies performed, both in intact cells and in vitro in a reticulocyte-lysate-based degradation system, revealed that ODC is degraded via a non-lysosomal, ATP-dependent, but ubiquitin-independent, proteolytic pathway [2, 31. Furthermore, genetically engineered ODC variants revealed that the integrity of a segment, encompassing the most carboxyl-terminal 37 amino acids of mouse ODC is required for maintaining its rapid degradation both in cells [4, 51 and in vitro [5].Recent studies have suggested that ODC degradation may be mediated by two distinct proteolytic machinery's [6, 71. According to these studies, the first proteolytic machinery is engaged in degrading ODC under basal metabolic conditions [6, 71 ; the second, that is stimulated by high concentration of polyamines, requires interaction of ODC with a small protein, termed ornithine decarboxylase antizyme (ODC-Az) [6, 71. ODC-Az is a 26-kDa protein that is induced in cells by polyamines [S-lo]. It binds ODC with high affinity and Correspondence to C. Kahana, Department of Molecular Genetics and Virology, The Weizmann Institute of Science, IL-Rehovot IL-76100, IsraelAbbreviations. ODC, ornithine decarboxylase; ODC-Az, ornithine decarboxylase antizyme ; GST, glutathione S-transferase. inhibits its activity ...