SPECIFIC INTERACTIONS BETWEEN ATPASE SUBUNITS OF THE 26-S-PROTEASE

The regulatory complex of the 26 S protease contains at least 15 disti nct subunits, Six of these subunits (S4, S6, S6', S7, S8, and S10b) be long to a novel subfamily of presumptive nucleotidases that we call su bunit 4 (S4)like ATPases, Each of these putative ATPases was synthesiz ed in reticulocyte lysate containing [S-35]methionine, and the radiola beled proteins were used in binding studies, S4, S6, S10b, and S6' dis played specific binding to components of the regulatory complex separa ted by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) or two-dimens ional PAGE, S4 bound to S7, and S6 bound two proteins: S8 and centract in, a component of the dynactin complex, S10b bound to S6' and bound m uch more weakly to SI and p50, another component of the dynactin compl ex, S6' bound to S10b, Two subunits, S7 and S8, did not bind any compo nents present on nitrocellulose membranes, presumably because S7 and S 8 are already oligomeric following synthesis, Go-translation and sucro se gradient sedimentation of S-35-labeled ATPases demonstrated the for mation of S6'-S10b dimers in solution but revealed more complex associ ations, namely the formation of trimers and tetramers, among S4, S6, S 7, and S8. Progressive COOH-terminal deletions that removed as much as 300 amino acids from S4 had no effect on the binding of S4 to S7. In striking contrast, truncation of 85 NH2-terminal amino acids from S4 a brogated binding, clearly implicating the NH2, terminus of S4 in its s pecific interaction with S7, Since S4-like ATPases contain putative co iled-coils within the first 150 NH2-terminal amino acids, we propose t hat coiled-coil interactions are responsible for the specificity of th e observed subunit associations and that these associations are import ant for self-assembly of the regulatory complex.