Abstract
Protein import across the endoplasmic reticulum membrane is physiologically regulated in a substrate-selective manner to ensure the protection of stressed ER from the overload of misfolded proteins. However, it is poorly understood how different types of substrates are accurately distinguished and disqualified during translocational regulation. In this study, we found poorly assembled translocon-associated protein (TRAP) complexes in stressed ER. Immunoaffinity purification identified calnexin in the TRAP complex in which poor assembly inhibited membrane insertion of the prion protein (PrP) in a transmembrane sequence-selective manner, through translocational regulation. This reaction was induced selectively by redox perturbation, rather than calcium depletion, in the ER. The liberation of ERp57 from calnexin appeared to be the reason for the redox sensitivity. Stress-independent disruption of the TRAP complex prevented a pathogenic transmembrane form of PrP (ctmPrP) from accumulating in the ER. This study uncovered a previously unappreciated role for calnexin in assisting the redox-sensitive function of the TRAP complex and provided insights into the ER stress-induced reassembly of translocon auxiliary components as a key mechanism by which protein translocation acquires substrate selectivity.
Highlights
The majority of secreted and membrane proteins synthesized in mammalian cells are co-translationally transported across or inserted into the endoplasmic reticulum (ER) membrane [1]
Synthesized secPrP and ctmPrP in pulse-labeled gNT and gTRAPα cells transiently transfected with wtPrP or N7a-prion protein (PrP)-AV3 constructs were analyzed by immunoprecipitation with the PrP-specific 3F4 antibody
We showed that the reassembly of translocon auxiliary components induced by ER
Summary
The majority of secreted and membrane proteins synthesized in mammalian cells are co-translationally transported across or inserted into the endoplasmic reticulum (ER) membrane (termed, “translocation”) [1] This event occurs at a specialized domain (termed, “translocon”), which serves as a gateway for nascent polypeptides entering the ER. The nascent polypeptide liberated from the SRP resumes translation and enters the ER through the central pore of the Sec complex [4]. Between these two steps, protein translocation can be regulated at the gating step and its efficiency appears to be determined by
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