The endoplasmic reticulum (ER) is an intracellular membranous system of all eukaryotic cells where most of the secretory proteins acquire their native tertiary structure. The ER contains a highly active quality control system. Malfolded proteins are selectively recognized in the ER and transported back to the cytoplasm, where they are finally degraded by the 26S proteasome in an ubiquitin dependent manner. The latter process is known as ER associated degradation (ERAD). Failure of this process

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... eads to formation of protein aggregates and results in impaired cell function. To understand how ERAD related components cooperatively participate in ER degradation and to get a better insight if Sec61p is really the pore-forming unit of the protein retrotranslocon, Blue Native Electrophoresis (BN) and co-immunoprecipitation techniques were applied in this study. To determine the composition of the retrotranslocon and to obtain new information on the entire process involved in ERAD, a series of CPY* fusion protein substrates with special structural characteristics were constructed and investigated. Using Blue Native electrophoresis, a stable Der3-Hrd3p complex was detected. The finding of the Der3-Hrd3p complex together with soluble malfolded substrates as CPY* or CPY*GFP and membrane substrates as CT* or CTG*, indicated that Der3-Hrd3p complex acts as a part of a retrotranslocation machinery. The AAA ATPase Cdc48p was also found in this Der3-Hrd3p complex when using Blue Native electrophoresis, indicating the existence of a Cdc48-Der3-Hrd3p complex. The finding of the interaction between Cdc48 and the Der3-Hrd3p complex by co-immunoprecipitation experiments confirmed this complex composition. Blue Native electrophoresis showed two stable Sec61-Sec62-Sec63p complexes (430kDa and 480kDa), the composition of which did not undergo changes in any of the ERAD mutants, indicating that these might constitute the protein import complex. Further insights into the retrotranslocation process was intended to reach with the help of new [...]