Newly synthesized human delta opioid receptors retained in the endoplasmic reticulum are retrotranslocated to the cytosol, deglycosylated, ubiquitinated, and degraded by the proteasome.
We have previously shown that only a fraction of the newly synthesized human delta opioid receptors is able to leave the endoplasmic reticulum (ER) and reach the cell surface (Petäjä-Repo, U. E, Hogue, M., Laperrière, A., Walker, P., and Bouvier, M. (2000) J. Biol. Chem. 275, 13727-13736). In the present study, we investigated the fate of those receptors that are retained intracellularly. Pulse-chase experiments revealed that the disappearance of the receptor precursor form (M(r) 45,000) and of two smaller species (M(r) 42,000 and 39,000) is inhibited by the proteasome blocker, lactacystin. The treatment also promoted accumulation of the mature receptor form (M(r) 55,000), indicating that the ER quality control actively routes a significant proportion of rescuable receptors for proteasome degradation. In addition, degradation intermediates that included full-length deglycosylated (M(r) 39,000) and ubiquitinated forms of the receptor were found to accumulate in the cytosol upon inhibition of proteasome function. Finally, coimmunoprecipitation experiments with the beta-subunit of the Sec61 translocon complex revealed that the receptor precursor and its deglycosylated degradation intermediates interact with the translocon. Taken together, these results support a model in which misfolded or incompletely folded receptors are transported to the cytoplasmic side of the ER membrane via the Sec61 translocon, deglycosylated and conjugated with ubiquitin prior to degradation by the cytoplasmic 26 S proteasomes.
Petaja-Repo
U E
UE
Département de Biochimie, Université de Montréal, Montréal, Quebec H3C 3J7, Canada. bouvier@bcm.umontreal.ca
Hogue
M
M
Laperriere
A
A
Bhalla
S
S
Walker
P
P
Bouvier
M
M
D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D006868:Hydrolysis; D009097:Multienzyme Complexes / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D021381:Protein Transport; D017465:Receptors, Opioid, delta / Q000378:metabolism*; D014452:Ubiquitins / Q000378:metabolism*
D009097:Multienzyme Complexes; D009844:Oligosaccharides; D017465:Receptors, Opioid, delta; D014452:Ubiquitins; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex