Reconstitution of Doa10-mediated ER-associated protein degradation with purified components

by Claudia C Schmidt
Cumulative thesis
Date of Examination:2019-11-25
Date of issue:2020-11-09
Advisor:Dr. Alexander Stein
Referee:Prof. Dr. Blanche Schwappach-Pignataro
Referee:Prof. Dr. Holger Stark
Referee:Dr. Alex Faesen
Referee:Prof. Dr. Peter Rehling
Referee:Prof. Dr. Henning Urlaub
Persistent Address: http://dx.doi.org/10.53846/goediss-8286

 

 

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Abstract

English

ER-associated protein degradation (ERAD) is a protein quality control pathway at the ER membrane mediated by membrane-embedded ubiquitin ligase complexes. During ERAD, membrane and luminal proteins are ubiquitinated and retrotranslocated back into the cytosol where they are degraded by the proteasome. The conserved ubiquitin ligase Doa10 mediates degradation of ER membrane proteins as well as cytosolic and nuclear proteins. It acts with the E2 enzymes Ubc6 and Ubc7. How Doa10 substrates are recognized, ubiquitinated and retrotranslocated is not well understood. In this thesis, I have established a system to investigate the mechanism of Doa10-mediated ERAD using purified components from S. cerevisiae. Membrane proteins are hereby reconstituted into separate sets of phospholipid vesicles and co-reconstituted upon SNARE-mediated fusion. Using this approach, I was able to recapitulate ubiquitination and extraction of the tail-anchored membrane protein Ubc6 which is itself unstable and degraded in a Doa10-dependent manner. In the presence of Doa10, I observe spontaneous retrotranslocation of the transmembrane (TM) anchor of Ubc6. A folded luminal domain attached to Ubc6 inhibits spontaneous retrotranslocation. Extraction can be restored upon ubiquitination and extraction by the Cdc48/UN complex. My results show that Doa10 is a retrotranslocase and indicate that Doa10 and Cdc48 cooperate in extraction of membrane proteins. In order to characterize structural elements in Doa10, I tested Doa10 truncations as well as previously described Doa10 mutants in the reconstituted system. My results show that the Doa10 TM domain is required for ubiquitination and spontaneous retrotranslocation of Ubc6. The tested Doa10 mutants did not show any differences. Thus, to identify interaction sites between the TM domains of Ubc6 and Doa10, a site-specific photocrosslinking approach was optimized. To extend conclusions obtained from Ubc6 to another Doa10 substrate, ERAD of the tail-anchored membrane protein Sbh2 was investigated. To characterize the machinery required for ERAD of Sbh2, we first performed a screen, as the machinery for cytosolic, but not membrane-bound Doa10 substrates has been characterized in screens until now. The screen identified described ERAD components as the most conclusive hits. Thus, I next characterized Sbh2 in a reconstituted system and identified a minimal machinery for Doa10-mediated ubiquitination of Sbh2. In conclusion, this thesis provides mechanistic insights into Doa10-mediated ERAD of membrane proteins. It also provides a tool for co-reconstituting membrane proteins for studying ERAD as well as other membrane-associated processes in the future.
Keywords: ER-associated protein degradation; Protein quality control; In vitro reconstitution; Ubiquitination
 
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