Quaternary structure of the ATPase complex of human 26S proteasomes determined by chemical cross-linking

The 26S proteasome is the major protease responsible for nonlysosomal prote in degradation in eukaryotic cells. The enzyme is composed of two subpartic les: the 20S proteasome, and a 19S regulatory particle (PA700) which binds to the ends of the 20S proteasome cylinder and accounts for ATP dependence and substrate specificity. Among the approximately 18 sub-units of PA700 re gulator, six are ATPases, The ATPases presumably recognize, unfold, and tra nslocate substrates into the interior of the 26S proteasome. It is generall y believed that the ATPases form a hexameric ring. By means of chemical cro ss-linking, immunoprecipitation, and blotting, we have determined that the ATPases are organized in the order S6-S6'-S10b-S8-S4-S7. Additionally, we f ound cross-links between the ATPase S10b and the 20S proteasome subunit alp ha6. Together with the previously known interaction between S8 and alpha1 a nd between S4 and alpha7, these data establish the relative orientations of ATPases with respect to the 20S proteasome. (C) 2001 Academic Press.