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Ubiquitinated proteasome inhibitor is a component of the 26 S proteasome complex

X S Li1, J D Etlinger

  • 1Department of Cell Biology and Anatomy, New York Medical College, Valhalla, New York 10595.

Biochemistry
|December 8, 1992
PubMed

Insights

The 26 S proteasome complex contains an endogenous inhibitor. Ubiquitination of this inhibitor may regulate proteasome assembly and activity.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Background:

  • The 20 S proteasome is a major protease in eukaryotic cells.
  • The 26 S proteasome complex is involved in protein degradation.
  • An endogenous inhibitor of the 20 S proteasome has been identified.

Purpose of the Study:

  • To investigate the role of the endogenous inhibitor in the 26 S proteasome complex.
  • To determine if the inhibitor is ubiquitinated and how this affects its function.

Main Methods:

  • Western blot analysis using specific antibodies against the inhibitor and ubiquitin.
  • SDS-PAGE to analyze protein size.
  • Ubiquitin ligating assays.
  • Analysis of erythrocyte extracts under different ATP conditions.

Main Results:

  • The 240-kDa inhibitor antibody detected a 55-kDa component in the 26 S proteasome, distinct from the 40-kDa free inhibitor.
  • A ubiquitin antibody recognized the same 55-kDa component.
  • Ubiquitin ligating systems generated the 55-kDa species from the 40-kDa inhibitor.
  • In erythrocyte extracts, the inhibitor existed as a 55-kDa species with ATP and shifted to 40 kDa without ATP, indicating ubiquitin removal.

Conclusions:

  • The 55-kDa species represents ubiquitinated inhibitor within the 26 S proteasome complex.
  • Ubiquitination of the inhibitor is likely involved in regulating the assembly and/or activity of the 26 S proteasome.

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