Specific interactions between ATPase subunits of the 26 S protease.
The regulatory complex of the 26 S protease contains at least 15 distinct subunits. Six of these subunits (S4, S6, S6', S7, S8, and S10b) belong to a novel subfamily of presumptive nucleotidases that we call subunit 4 (S4)-like ATPases. Each of these putative ATPases was synthesized in reticulocyte lysate containing [35S]methionine, and the radiolabeled proteins were used in binding studies. S4, S6, S10b, and S6' displayed specific binding to components of the regulatory complex separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) or two-dimensional PAGE. S4 bound to S7, and S6 bound two proteins: S8 and centractin, a component of the dynactin complex. S10b bound to S6' and bound much more weakly to S1 and p50, another component of the dynactin complex. S6' bound to S10b. Two subunits, S7 and S8, did not bind any components present on nitrocellulose membranes, presumably because S7 and S8 are already oligomeric following synthesis. Co-translation and sucrose gradient sedimentation of 35S-labeled ATPases demonstrated the formation of S6'-S10b dimers in solution but revealed more complex associations, namely the formation of trimers and tetramers, among S4, S6, S7, 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 abrogated binding, clearly implicating the NH2 terminus of S4 in its specific interaction with S7. Since S4-like ATPases contain putative coiled-coils within the first 150 NH2-terminal amino acids, we propose that coiled-coil interactions are responsible for the specificity of the observed subunit associations and that these associations are important for self-assembly of the regulatory complex.
Richmond
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Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah 84132, USA.
Gorbea
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Rechsteiner
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D000251:Adenosine Triphosphatases / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D010447:Peptide Hydrolases / Q000737:chemistry*; D010449:Peptide Mapping; D046988:Proteasome Endopeptidase Complex; D013329:Structure-Activity Relationship
D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D000251:Adenosine Triphosphatases