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

Area of Science:

• Molecular Biology
• Cell Biology
• Protein Degradation

Background:

• Misfolded proteins in the endoplasmic reticulum (ER) are eliminated via ER-associated degradation (ERAD).
• The Hrd1 ubiquitin ligase is central to the ERAD pathway, mediating the degradation of soluble, lumenal ERAD targets.
• Understanding Hrd1's mechanism is crucial for elucidating protein quality control in the ER.

Purpose of the Study:

• To investigate the functional roles of specific residues and regions within the Hrd1 protein.
• To identify Hrd1 components exclusively required for the degradation of lumenal ERAD substrates.
• To elucidate how Hrd1 mediates protein retrotranslocation across the ER membrane.

Main Methods:

• Deep mutational scanning was employed to identify functionally important residues in Hrd1.
• In vivo and in vitro experiments were conducted to analyze Hrd1 function.
• Structural and functional analyses focused on disordered regions within Hrd1.

Main Results:

• Several regions of Hrd1 were identified as critical for its various functions.
• Two cytosolic regions of Hrd1 were found to be essential for lumenal ERAD substrate degradation.
• Disordered regions within Hrd1 are required for its autoubiquitination and substrate binding.
• These disordered regions control Hrd1 activation and the directionality of substrate retrotranslocation.

Conclusions:

• Disordered cytosolic regions of Hrd1 play a pivotal role in lumenal ERAD substrate degradation.
• Hrd1 activation and directional retrotranslocation are regulated by these specific protein regions.
• The findings provide new insights into the molecular mechanisms governing ER-associated degradation.