Fluorescent-detected retrotranslocation of an endoplasmic reticulum - associated degradation (ERAD) substrate in a mammalian in vitro system
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Secretory proteins that are unable to assemble into native proteins in the endoplasmic reticulum (ER) are transported back into the cytosol for degradation. Many cytosolic and ER resident proteins have been identified so far as being involved in this retrotranslocation process, but it is difficult to determine whether these proteins have a direct or indirect effect. Interpretations are further complicated if the loss of a specific protein is obscured by the presence of another protein that is partially or wholly redundant. To overcome these limitations, a mammalian in vitro system was developed that allowed to monitor retrotranslocation synchronously and in real time in the absence of concurrent translocation. To examine the roles of different components in ER-associated degradation (ERAD), well-defined and homogeneous mammalian ER microsomes were prepared biochemically by encapsulating a fluorescent-labeled ERAD substrate with specific lumenal components. After mixing ATP, specific cytosolic proteins, and specific fluorescence quenching agents with microsomes, substrate retrotranslocation was initiated. The rate of substrate efflux from microsomes was monitored spectroscopically and continuously in real time by the reduction in fluorescence intensity as the fluorescent substrates passed through the ER membrane and were exposed to the quenching agents. Retrotranslocation kinetics were not significantly altered by replacing all lumenal proteins with only protein disulfide isomerase, or all cytosolic proteins with only the 19S proteasome cap. Retrotranslocation was blocked by affinity-purified antibodies against Derlin1, but not by affinity-purified antibodies against Sec61α or by membrane-bound ribosomes. Since the substrate also photocrosslinked Derlin1, but not Sec61α or TRAM, retrotranslocation of this ERAD substrate apparently involves Derlin1, but not the translocon. By labeling either the C- or N-terminus, it was revealed that the N-terminus of one ERAD substrate leaves the ER lumen first. This finding suggests that the protein is retrotranslocated as a linear polymer in a preferred direction. When RRMs were reconstituted with a fluorescent-labeled ERAD substrate and various ions. Ca2+ ions in the ER lumen increased the rate and extent of retrotranslocation, while Ca2+ ions in the cytosol decreased retrotranslocation. This approach therefore provides the first direct evidence of the involvement and importance of specific ionic requirements for ERAD.
Wahlman, Judit (2007). Fluorescent-detected retrotranslocation of an endoplasmic reticulum - associated degradation (ERAD) substrate in a mammalian in vitro system. Doctoral dissertation, Texas A&M University. Available electronically from