Deep mutational scanning highlights a new role for cytosolic regions in Hrd1 function

Area of Science:

• Cell Biology
• Protein Degradation
• Molecular Mechanisms

Background:

• Misfolded proteins in the endoplasmic reticulum (ER) are cleared via ER-associated degradation (ERAD).
• The Hrd1 ubiquitin ligase is essential for ERAD, particularly for degrading soluble lumenal ERAD substrates.
• Understanding Hrd1's precise molecular functions in substrate retrotranslocation and degradation is critical.

Approach:

• Developed a deep mutational scanning method to systematically identify functional residues in Hrd1.
• Employed in vivo and in vitro experiments to investigate Hrd1's autoubiquitination and substrate interactions.
• Focused on characterizing the roles of specific cytosolic regions within Hrd1.

Key Points:

• Identified distinct functional regions within Hrd1, including novel cytosolic domains essential for lumenal ERAD.
• Disordered regions between Hrd1's structural elements are critical for autoubiquitination and substrate binding.
• These cytosolic regions regulate Hrd1 activation and direct the retrotranslocation of lumenal substrates across the ER membrane.

Conclusions:

• Disordered cytosolic regions of Hrd1 play a surprising and vital role in lumenal ERAD substrate degradation.
• These regions are key regulators of Hrd1's enzymatic activity and substrate handling.
• The findings provide new insights into the mechanistic control of protein retrotranslocation in the ER.