Base-CP proteasome can serve as a platform for stepwise lid formation

Area of Science:

• Cellular Biology
• Molecular Biology
• Biochemistry

Background:

• The 26S proteasome, a eukaryotic protease, comprises a 20S core particle (CP) and a 19S regulatory particle (RP).
• The 19S RP has base and lid sub-complexes, with the lid further divided into module 1 and module 2.
• Regulatory particle non-ATPase 11 (Rpn11) is a key subunit within lid module 1.

Purpose of the Study:

• To investigate the role of Rpn11 in 26S proteasome assembly.
• To elucidate the sequential steps in proteasome lid formation and attachment to the core particle.
• To understand how proteasome intermediates form and contribute to holoenzyme assembly.

Main Methods:

• Gene expression suppression of RPN11.
• Analysis of proteasome assembly intermediates in wild-type and mutant strains (rpn11-m1).
• In vitro reconstitution of proteasome sub-complexes, including module 1.

Main Results:

• RPN11 suppression halted lid assembly but allowed base-CP pre-assembly.
• A mutant lacking functional Rpn11 formed an incomplete lid with module 1 subunits attached to base-CP.
• In vitro studies demonstrated stepwise module 1 assembly, initiated by Rpn11-Rpn8 heterodimerization, and its subsequent attachment to base-CP.

Conclusions:

• Rpn11 acts as a crucial bridge between lid modules 1 and 2, essential for complete lid assembly.
• The 20S core particle serves as a platform for the gradual assembly of the 19S regulatory particle lid.
• Understanding proteasome assembly intermediates clarifies the functional mechanisms of this holoenzyme.