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 protein degradation in eukaryotic cells. The enzyme is composed of two subparticles: 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 subunits of PA700 regulator, six are ATPases. The ATPases presumably recognize, unfold, and translocate substrates into the interior of the 26S proteasome. It is generally believed that the ATPases form a hexameric ring. By means of chemical cross-linking, immunoprecipitation, and blotting, we have determined that the ATPases are organized in the order S6-S6′-S10b-S8-S4-S7. Additionally, we found cross-links between the ATPase S10b and the 20S proteasome subunit α6. Together with the previously known interaction between S8 and α1 and between S4 and α7, these data establish the relative orientations of ATPases with respect to the 20S proteasome.