Slippery sequences stall the 26S proteasome at multiple points along the translocation pathway

Area of Science:

• Cellular Biology
• Molecular Mechanisms
• Protein Degradation

Background:

• The ubiquitin-proteasome system (UPS) degrades intracellular proteins in eukaryotes.
• Proteins are tagged with ubiquitin, recognized by the 19S regulatory particle (RP) of the 26S proteasome, unfolded, and degraded by the 20S core particle (CP).
• Aromatic paddles on the RP's Rpt subunits are crucial for gripping and unfolding substrate proteins.

Purpose of the Study:

• To investigate the spatial requirements for substrate unfolding by the 26S proteasome.
• To determine how the location of glycine-rich sequences affects protein unfolding, particularly near the N-terminus.
• To map the spacing of interactions between substrates and the proteasome during unfolding.

Main Methods:

• Experimental manipulation of glycine-rich tract locations relative to folded protein domains.
• Assessing the impact of these insertions on the unfolding efficiency by the 26S proteasome.
• Mapping affected regions to predicted substrate-proteasome interaction sites.

Main Results:

• Inserting glycine-rich sequences closer to a folded domain reduced unfolding ability more than when positioned further away.
• The most significant effects on unfolding occurred at locations corresponding to predicted aromatic paddle interactions.
• Observed effects extended up to 67 amino acids from the folded domain, indicating broader substrate-proteasome interactions.

Conclusions:

• The precise spacing of low-complexity sequences relative to folded domains is critical for efficient proteasomal unfolding.
• Beyond the aromatic paddles, additional interactions facilitate substrate translocation through the proteasome channel.
• This research enhances understanding of the mechanical forces governing protein unfolding and degradation by the 26S proteasome.