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Braun, Sigurd (2005): ER-associated degradation (ERAD): Novel components and cellular regulation. Dissertation, LMU München: Faculty of Biology
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Abstract

The majority of eukaryotic proteins are degraded by the ubiquitin-proteasome system. In this pathway, cytosolic substrates are first earmarked for degradation by modification with ubiquitin ('ubiquitylation') and subsequently degraded by the 26S pro-teasome, a large protease residing in both the cytosol and the nucleus. ER-resident proteins are similarly degraded but take the route of a specialized pathway coined ER-associated degradation (ERAD). In order to reach the cytosolic ubiquitin/proteasome system, these substrates must first relocate from the ER to the cytosol, possibly with the help of protein conducting membrane channels. Previous work has shown that specific ubiquitin-conjugating enzymes (e.g. Ubc6, Ubc7) and ubiquitin ligases (e.g. Hrd1) con-tribute to ERAD, but how the substrates reach the proteasome remained to be clarified. Besides its function as a quality control system in recognizing and eliminating aberrant proteins, ERAD appears also to play a part in regulatory pathways. This study focuses on the identification of novel components contributing to ERAD. It could be demonstrated that the yeast protein Cdc48 (p97 in mammals), to-gether with its co-factors Ufd1 and Npl4, plays a key role in this process. Cdc48 belongs to the large family of AAA-type ATPases and is believed to function as a chaperone-like enzyme. Previous work has shown that the Cdc48 complex specifically acts on ubiquitylated substrates. This study indicates that the Cdc48 complex takes part in mo-bilization of ERAD substrates from the ER membrane for proteasomal targeting. Fur-thermore, degradation of some ERAD substrates involves the multiubiquitylation factor E4/Ufd2 and proteasome targeting factors of the Rad23 protein family. Another aspect of this work addresses the regulatory functions of ERAD. The fatty acid desaturase Ole1, an integral membrane protein of the ER, was identified as a novel ERAD substrate. Intriguingly, ERAD of Ole1 is specifically regulated since the protein is particularly short lived in the presence of high levels of unsaturated fatty acids, the products of Ole1. Thus, this feedback loop provides an additional mechanism, by which the cell regulates the amount of unsaturated fatty acids. The t-SNARE (syntaxin) protein Ufe1 was characterized as another substrate of ERAD. This protein is required for homotypic membrane fusion of ER vesicles. Notably, Ufe1 degradation is negatively controlled by its binding partner Sly1, a member of the SM (Sec1/Munc18) protein fam-ily. Reciprocal mutations in the Ufe1-Sly1 interaction face result in rapid degradation of Ufe1 by ERAD. Conversely, strong overproduction of Ufe1 was found to be detrimental for cellular growth. These findings suggest that one important function of Sly1 is to con-trol Ufe1 SNARE levels in order to ensure cellular homeostasis. In conclusion, analysis of the degradation of Ole1 and Ufe1 revealed an important contribution of ERAD to es-sential regulatory pathways.