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.
