MOLECULAR CHARACTERIZATION OF THE 26S PROTEASOME COMPLEX FROM RAT-LIVER

The molecular properties of an ATP/ubiquitin-dependent ''26S'' proteas ome complex purified from rat liver were examined by physicochemical, biochemical, and morphological analyses. On ultracentrifugation, the p roteasome complex sedimented as almost a single component with a sedim entation coefficient of 30.3S. Dynamic light-scattering measurements i ndicated that it has a diffusion coefficient of 1.38 x 10(-7) cm(2)/se c and a Stokes radius of 15.5 nm. From these two coefficients, the pro tein complex was estimated to have the high molecular weight of 2.02 x 10(6). Static light-scattering analysis indicated a molecular weight of 1.91 x 10(6) and a radius of gyration of 16.8 nm. The proteasome co mplex was found to be composed of multiple subunits of the 20S proteas ome with molecular weights of 2.1-3.1 x 10(4) and 15-20 protein specie s with molecular weights of 3.5-11.0 x 10(4), which were directly asso ciated with the 20S proteasome. The electron micrographic finding that the 26S proteasome complex had a caterpillar shape, direct electron-m icroscopic observations on the subunit arrangement of the 20S proteaso me, and classification of the subunits of the latter into two groups w ith respect to sequence homology suggested that the 26S complex is a s ymmetrical assembly of two domains, each containing a large terminal s ubset and half the central 20S subset of components. For clarification of the molecular structure of the 26S proteasome complex in solution, its physicochemical parameters were calculated theoretically using a model based on this caterpillar-shaped complex. The values obtained fo r the Stokes radius and radius of gyration of 12.2 and 14.9 nm were co nsistent with the experimental values. These results provide evidence that the 26S proteasome complex is a cylindrical caterpillar-like stru cture of ''30S'' in solution, consisting of a 20S proteasome component with proteolytic function and multiple other components, which possib ly have regulatory roles. (C) 1993 Academic Press, Inc.