Abstract
In endoplasmic reticulum-associated protein degradation (ERAD), membrane proteins are ubiquitinated, extracted from the membrane, and degraded by the proteasome. The cytosolic ATPase Cdc48 drives extraction by pulling on polyubiquitinated substrates. How hydrophobic transmembrane (TM) segments are moved from the phospholipid bilayer into cytosol, often together with hydrophilic and folded ER luminal protein parts, is not known. Using a reconstituted system with purified proteins from Saccharomyces cerevisiae, we show that the ubiquitin ligase Doa10 (Teb-4/MARCH6 in animals) is a retrotranslocase that facilitates membrane protein extraction. A substrate's TM segment interacts with the membrane-embedded domain of Doa10 and then passively moves into the aqueous phase. Luminal substrate segments cross the membrane in an unfolded state. Their unfolding occurs on the luminal side of the membrane by cytoplasmic Cdc48 action. Our results reveal how a membrane-bound retrotranslocase cooperates with the Cdc48 ATPase in membrane protein extraction.
Highlights
The endoplasmic reticulum (ER) is a major site for protein folding and maturation in the endomembrane system of the eukaryotic cell
We investigated ER-associated protein degradation (ERAD) mediated by the ubiquitin ligase Doa10 from S. cerevisiae
Mixing of the two liposome sets led to SNARE-mediated co-reconstitution of Ubc6 and Doa10 (Figure 1—figure supplement 1G). This approach ensures that Doa10 and Ubc6 only interact in the phospholipid bilayer, avoiding non-native interactions that can occur when membrane proteins are mixed in the presence of detergents for co-reconstitution
Summary
The endoplasmic reticulum (ER) is a major site for protein folding and maturation in the endomembrane system of the eukaryotic cell. ERAD is part of the ubiquitin proteasome system. Studies in the yeast Saccharomyces cerevisiae identified two universally conserved membrane-embedded ubiquitin ligases that ubiquitinate ERAD substrates, Hrd (SYVN1 in human) (Bordallo et al, 1998; Hampton et al, 1996; Kikkert et al, 2004; Nadav et al, 2003) and Doa (TEB-4/MARCH6 in animals, SUD-1 in Arabidopsis thaliana) (Doblas et al, 2013; Hassink et al, 2005; Swanson et al, 2001). A larger variety of ubiquitin ligases plays a role in ERAD (Olzmann et al, 2013)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
