Structural studies of ER-associated protein quality control systems

Thesis

ER-associated protein degradation (ERAD) is an important degradation pathway for the removal of misfolded proteins from the endoplasmic reticulum (ER). This pathway encompasses a set of evolutionary conserved mechanisms for the selection, ubiquitination and retrotranslocation of membrane and lumenal proteins from the ER to the cytosol. Recent structural studies of the yeast Hrd1 E3 ligase complex identified a possible mechanism by which ERAD substrates are retrotranslocated across the ER membrane. Yeast Hrd1 forms a heterodimer with the rhomboid pseudoprotease Der1, each of which is proposed to contribute a half-channel in the membrane for retrotranslocation. Crosslinking studies also support this hypothesis. However, the mechanisms for selection of membrane substrates are still unknown and the identity of the translocon in other ERAD pathways remains elusive. This is further hindered by a lack of structural studies, especially in mammalian systems.

This thesis presents a bottom-up screen for the identification of mammalian ERAD factors amenable to structure determination by cryo-electron microscopy (cryo-EM). High-resolution structures of the mammalian E3 ligase Hrd1 were determined and two distinct dimeric states identified. These are termed the “open” and “closed” state due to rearrangements at the dimer interface that lead to repositioning of the E3 ligase RING domains within the dimer. These structures are compared with their yeast counterpart and analysed to understand key differences that may be important in ERAD. A mammalian expression system was developed for reconstitution of Hrd1 complexes containing additional binding partners. Complexes containing Hrd1 and its core interactors were isolated and imaged by cryo-EM. Other ERAD complexes were also purified and imaged by cryo-EM. This thesis provides a foundation for future structural studies of mammalian ERAD complexes.

Authors: Robson-Tull, J

• DOI: 10.5287/ora-9ezpxze09
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Subjects: Quality Control; Cryo-EM; Endoplasmic Reticulum; Protein Degradation; Structural Biology