Structure and function of proteasome in higher plants

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The 26S proteasome complex catalyzing ATP-dependent breakdown of ubiquitin-ligated proteins was purified from spinach leaves to near homogeneity by chromatography on DEAE-cellurose, gel filtration on Biogel A-1.5m and glycerol density gradient cenrifugation. The purified enzyme was shown to degrade multi-ubiquitinated, but not unmodified, lysozyme in an ATP-dependent fashion, coupled with ATPase activity supplying energy for proteolysis, and isopeptidase activity to generate free ubiquitin. By non-denaturing electrophoresis, the puried enzyme was separated into to distinct forms of the 26S complex, named 26Salpha and 26Sbeta proteasomes, with different electorophoretic mobilities. The 26S proteasome was found to consist of multiple polypeptides with molecular masses of 23-35 and 39-115kDa, which were thought to be those of a 20S proteasome with multicatalytic proteinase activity and an associated regulatory part with ATPase and de-ubiquitinating activities, respectively. the subunit multiplicity of the spinach 26S proteasome dosely resembled that of rat liver with minor differences in certain components. No sulfhydryl bond was involved in the assembly of this multi-component polypeptide complex. Electron microscopy showed that the 26S proteasome complex had a "caterpillar" like shape, consisting of four central protein layrs, assumed to be the 20S proteasome, with asymmetric V-shaped layrs of each end. These structural and functional characteristics of the spinach 26S proteasome showed marked similarity to those of the mammalian 26S proteasomes reported recently, suggesting that the 26S proteasome is widely distributed in eukaryotic cells and is of general importance for catalyzing the soluble energy-and ubiquitin-dependent proteolytic pathway.