Rapid endoplasmic reticulum (ER) stress‐induced export (RESET) is undoubtedly beneficial in that it eliminates misfolded prion protein (PrP) from a stressed ER. Considering that RESET induces rapid endocytosis of misfolded PrP for degradation, it is questionable whether RESET is beneficial when its product amount overwhelms the capacity of subsequent clearance pathways. We require a strategy to monitor the endocytic flux rate of misfolded PrPs. Here, we stabilized misfolded PrPs by inserting red fluorescent protein (RFP) and indirectly determined this rate by monitoring the lysosomal free RFP. We discovered a surveillance mechanism that limits endocytosis of misfolded PrPs through plasma membrane quality control (pmQC). pmQC was regulated by the thiol‐disulfide status of misfolded PrPs and consequently accumulates nonpathogenic PrP variants at the plasma membrane. This variant alleviated prion proteotoxicity induced by persistent RESET. Thus, PrP endocytosis is regulated by pmQC to ensure the safety of endolysosomal pathway from persistent internalization of misfolded PrP.—Lee, D., Lee, S., Shin, Y., Song, Y., Kang, S.‐W. Thiol‐disulfide status regulates quality control of prion protein at the plasma membrane. FASEB J. 33, 11567–11578 (2019). http://www.fasebj.org

Regulating protein import across the endoplasmic reticulum (ER) membrane occasionally results in the synthesis of topologically unnatural variants, and their accumulation often leads to proteotoxicity. However, since this is a regulated process, it is questionable whether the topological rearrangement really has adverse consequences. In the present study, we provide an insight into the functional benefit of translocational regulation by illustrating mutant-selective topologic conversion (MSTC) and demonstrate that MSTC contributes to selective degradation of a membrane-anchored prion protein isoform (ctmPrP). We find that ctmPrP is inherently short-lived and topologically competent for degradation rather than accumulation. MSTC achieves, cotranslationally, the unique topology of ctmPrP during translocation, facilitating selective ctmPrP degradation from the ER via the proteasome-dependent pathway before entering the secretory pathway. At this time, the N-terminal polycationic cluster is essential for MSTC, and its cytosolic exposure acquires "ERAD-degron"-like activity for ctmPrP. Bypassing MSTC delays ctmPrP degradation, thus increasing prion proteotoxicity. Thus, topological rearrangement is used for the MSTC as a part of the protein quality control pathway to ensure the safety of the secretory pathway from misfolded PrP.