The 26S proteasome degrades mouse and yeast ornithine decarboxylase in yeast cells.

Eukaryotic cells possess two high-molecular-mass proteases, the 700 kDa, 20S proteasome, as well as the even larger 1,400 kDa, 26S proteasome. It has been demonstrated that ornithine decarboxylase is degraded, in vitro, by the 26S proteasome that contains the 20S protease as its catalytic core, but not by the free 20S proteasome. Recently, by demonstrating severe inhibition of mouse and yeast ODC degradation in a mutant yeast cell line, defective in the chymotripsin-like activity of the yeast 20S proteasome, we implicated the 20S proteasome in the degradation of ODC, in vivo, in yeast cells. Here we show that the degradation of ODC is also severely inhibited in the mutant yeast cell lines, cim3-1 and cim5-1, containing a specific lesion in subunits that are unique to the yeast 26S proteasome. We therefore, conclude, that as illustrated in vitro, also in intact cells, it is the 26S proteasome, not the free 20S proteasome, that degrades ODC. We also demonstrate, that while deficiency in the proteasome chymotrypsine-like activity (in the yeast pre1-1 mutant) inhibits the degradation of both yeast and mouse ODCs, deficiency in the peptidyl-glutamyl-peptide-hydrolyzing (PGPH) activity inhibits only yeast ODC degradation. Similarly, we have noted that whereas the putative ATPase activity of both the CIM3 and CIM5 subunits is essential for the degradation of mouse ODC, only that of the CIM3 subunit is required for the degradation of yeast ODC. These results suggest differential utilization of individual proteasomal subunits in the recognition and degradation of individual short-lived proteins.

A unified pathway for the degradation of ornithine decarboxylase in reticulocyte lysate requires interaction with the polyamine-induced protein, ornithine decarboxylase antizyme.

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 polyamine-induced 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.

The 20S proteasome mediates the degradation of mouse and yeast ornithine decarboxylase in yeast cells.

Ornithine decarboxylase (ODC), a key enzyme in the biosynthesis of polyamines, is one of the most rapidly degraded proteins in mammalian cells. Recently it has been demonstrated that mammalian ODC is degraded in vitro by the 26S protease that contains the 20S proteasome as its catalytic core, in a reaction that does not require ubiquitin. Here, we show that yeast and mouse ODC are both rapidly degraded in yeast cells and that their degradation severely inhibited in a mutant yeast cell line defective in the chymotryptic activity of proteinase yscE, the yeast 20S proteasome. These results provide compelling genetic support to previous biochemical studies suggesting the involvement of the 20S proteasome in the degradation of ornithine decarboxylase.

Gly387 of murine ornithine decarboxylase is essential for the formation of stable homodimers.

In its active form mammalian ornithine decarboxylase (ODC) is a homodimer composed of two 53-kDa subunits while the monomer retains no enzymic activity. In the present study we demonstrate that Gly387 of mouse ODC plays an important role in enabling dimer formation. Gly387 of mouse ODC, an evolutionary conserved residue, was converted to all possible 19 amino acids using site-directed mutagenesis. With the exception of alanine, all other substitutions of Gly387 completely abolished enzymic activity. Cross-linking analysis and fractionation through a Superose-12 sizing column have demonstrated that mutant subunits are detected only in their monomeric form. These results strongly suggest that the primary lesion of substitution at position 387 of mouse ODC is the inability of mutant subunits to associate with each other to form the active homodimers. In agreement with this conclusion, G387A, the only mutant that retained partial activity, displayed reduced dimerization. The degradation rate of ODC mutants in which Gly387 was substituted by aspartic acid or alanine was enhanced compared to the wild-type enzyme, suggesting that monomers may be more susceptible to degradation.