Njal Winther Jensen:
Investigating ER-associated Degradation with RNAi Screening - and Searching for Model Proteins to Do It with

Date: 01-10-2014    Supervisor: Lars Ellgaard

In eukaryotes, secretory proteins are translocated into the endoplasmic reticulum (ER) for folding assistance, acquisition of posttranslational modifications and sorting. Proteins that do not obtain their native conformation are eliminated by ER-associated degradation (ERAD). ERAD is a sophisticated pathway that recognizes misfolded proteins and targets them for degradation by the 26S proteasome residing in the cytosol. More than 60 diseases including Alzheimer’s disease, Huntington’s disease and Parkinson’s disease have been linked to the ERAD pathway underscoring its crucial role for cellular homeostasis.

The aim of this thesis has been to gain insight into ERAD. The experimental approach was RNAi screening, which is a fast and efficient method for initial evaluation of a large pool of genes. Since relatively few proteins routinely are used as ERAD substrates, the first goal of this project was to identify endogenous proteins suitable as model proteins. Unfortunately, the methodological approach proved unsuccessful. Instead, mutants of two polytopic membrane proteins were investigated for their potential as ERAD substrates and a mutant of one of these proteins, ATP13A2, turned out to possess the required properties.

RNAi screening was then performed to identify proteins of the ERAD machinery that was needed for the successful degradation of a HA-tagged version of the ATP13A2 mutant (HA-ATP13A2). After a validation phase, one of the identified proteins, Sec61α was verified as being important for the ERAD of HA-ATP13A2.

Future experiments are required to reveal the exact function of Sec61α in this intriguing pathway.