Proteins are unfolded on the surface of the ATPase ring before transport into the proteasome

Area of Science:

• Molecular Biology
• Biochemistry
• Cellular Biology

Background:

• The 26S proteasome is a crucial cellular machine responsible for protein degradation.
• Its 19S regulatory particle, composed of six ATPase subunits, plays a key role in substrate unfolding and translocation into the 20S core particle.
• Understanding this process is vital for comprehending cellular protein homeostasis and disease mechanisms.

Purpose of the Study:

• To elucidate the mechanism by which ATPase subunits of the 26S proteasome unfold and translocate proteins.
• To investigate the role of substrate modifications and orientation in proteasome-mediated degradation.

Main Methods:

• Utilized the archaebacterial proteasome-regulatory ATPase complex PAN as a model system.
• Employed Green Fluorescent Protein fused with a degradation tag (GFP-SsrA) as a model substrate.
• Experimentally attached different moieties (Biotin, Avidin) to the substrate's N and C termini to assess their impact on unfolding and translocation.

Main Results:

• ATP-dependent unfolding of GFP-SsrA occurred on the surface of the PAN complex.
• Substrate translocation into the proteasome required a specific C-terminal to N-terminal threading direction.
• Attachment of a large moiety (Avidin) to the C-terminus blocked translocation and degradation, while still allowing ATP-dependent unfolding and PAN binding.

Conclusions:

• Protein translocation is a consequence of, not the cause of, ATP-dependent unfolding.
• The 19S proteasome unfolds substrates on its surface before translocation.
• Directionality (C-terminus to N-terminus) is critical for successful substrate translocation and degradation by the proteasome.