Regulation of the Hrd1 Ubiquitin ligase during protein degradation at the endoplasmic reticulum

by Benjamin Zaremba née Gnoth
Doctoral thesis
Date of Examination:2024-04-15
Date of issue:2024-05-16
Advisor:Dr. Alexander Stein
Referee:Dr. Alexander Stein
Referee:Prof. Dr. Kai Tittmann
Persistent Address: http://dx.doi.org/10.53846/goediss-10486

 

 

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Abstract

English

Misfolded or redundant proteins of the endoplasmic reticulum are degraded via ER associated degradation: They are recognized, undergo a retrotranslocation into the cytosol, become ubiquitinated, are extracted from the ER and are finally degraded via the proteasome. Luminal and some membrane bound proteins are degraded via the Hrd1 E3 ligase complex. Although components of the complex are known and a structure of the complex has been publishes, there are open questions remaining, such as the role of autoubiquitination and how the complex is regulated. In this thesis, the substrate degradation of the luminal substrate CPY* and of the membrane bound substrate Hmg2 by the Hrd1 E3 ligase complex in different constitutions has been characterized in vivo in Saccharomyces cerevisiae. Findings in this thesis suggest that the requirement for autoubiquitination of Hrd1 is strongly linked to the dimerization of the complex, which is mediated by Usa1. A Hrd1-Usa1 fusion protein was confirmed to be biologically active and substrate degradation was shown to be autoubiquitination independent. Besides substrate degradation, the composition of the complex has been analyzed in dependence on Hrd1 autoubiquitination. It could not be confirmed that components of the Hrd1 E3 ligase complex, such as Hrd3 or Der1, leave the complex. Crosslinking experiments were carried out to identify Hrd1 dimers. A dimerization of Hrd1 was observed to only minor amounts, contradicting recent publications and questioning the role of autoubiquitination in dimer disassembly. Additionally, an overexpression protocol for Hrd1 complexes has been established and native-like complexes have been purified from Saccharomyces cerevisiae for the first time. The complexes were tested for in vitro autoubiquitination and substrate ubiquitination. Thereby it was shown that autoubiquitination is strongly regulated by the presence of either, Hrd3 and Usa1. After successful reconstitution into ApoE nanodiscs, the protein complexes were subjected to CPY* binding assays.
Keywords: Hrd1; E3 ligase; ERAD; protein degradation