Real-Time Fluorescence Detection of ERAD Substrate Retrotranslocation in a Mammalian In Vitro System

Abstract

SummarySecretory proteins unable to assemble into their native states in the endoplasmic reticulum (ER) are transported back or “retrotranslocated” into the cytosol for ER-associated degradation (ERAD). To examine the roles of different components in ERAD, one fluorescence-labeled ERAD substrate was encapsulated with selected lumenal factors inside mammalian microsomes. After mixing microsomes with fluorescence-quenching agents and selected cytosolic proteins, the rate of substrate efflux was monitored continuously in real time by the decrease in fluorescence intensity as cytosolic quenchers contacted dye-labeled substrates. The retrotranslocation kinetics of nonglycosylated pro-α factor were not significantly altered by replacing all lumenal proteins with only protein disulfide isomerase or all cytosolic proteins with only PA700, the 19S regulatory particle of the 26S proteasome. Retrotranslocation was blocked by antibodies against a putative retrotranslocation channel protein, derlin-1, but not Sec61α. In addition, pro-α factor photocrosslinked derlin-1, but not Sec61α. Thus, derlin-1 appears to be involved in pro-α factor retrotranslocation.

Protein Folding and Quality Control in the ER

“…Derlin-1 was initially reported to be important for the extraction of MHC class I molecules from the ER membrane in cytomegalovirus-infected cells (Lilley and Ploegh 2004;Ye et al 2004). Reconstitution assay using a fluorescently labeled substrate also showed that Derlin-1 is involved in substrate dislocation, which is independent of Sec61a (Wahlman 2007). The yeast Hrd1p E3 ligase, which interacts with the ERAD substrate via its trans-membrane region, is also a possible candidate (Carvalho et al 2010).…”

Derlin-1 Facilitates the Retro-Translocation of Cholera Toxin

“…Juxtaposing the ER factors that prime the misfolded substrates for retro-translocation to the channel itself enables an efficient means of transport. Of note, retro-translocation of the nonglycosylated prepro-␣-factor requires PDI (Gillece et al, 1999;Wahlman et al, 2007) and Derlin-1 (Wahlman et al, 2007). Similarly, the ER-to-cytosol transport of the murine polyomavirus also relies on PDI and Derlin-2 (Lilley et al, 2006).…”

“…Indeed, PDI has been implicated in the retro-translocation of misfolded proteins (Gillece et al, 1999;Molinari et al, 2002;Wahlman et al, 2007). …”

Sequential Actions of the AAA-ATPase Valosin-containing Protein (VCP)/p97 and the Proteasome 19 S Regulatory Particle in Sterol-accelerated, Endoplasmic Reticulum (ER)-associated Degradation of 3-Hydroxy-3-methylglutaryl-coenzyme A Reductase

“…Binding of the base to the 20 S CP activates proteolysis, and the AAA-ATPases of the base facilitate substrate unfolding and delivery into the proteolytic chamber for degradation. In addition, the 19 S RP has been implicated in cytosolic dislocation of ERAD substrates (17,(35)(36)(37); however, the majority of these studies focused on soluble substrates sequestered within the ER lumen. In some cases, dislocation of these substrates was independent of ubiquitination and the action of VCP/p97.…”