摘要：SummaryThe mammalian endoplasmic reticulum (ER) harbors more than 20 members of the protein disulfide isomerase (PDI) family that act to maintain proteostasis. Herein, we developed anin vitrosystem for directly monitoring PDI- or ERp46-catalyzed disulfide bond formation in ribosome-associated nascent chains of human serum albumin. The results indicated that ERp46 more efficiently introduced disulfide bonds into nascent chains with a short segment exposed outside the ribosome exit site than PDI. Single-molecule analysis by high-speed atomic force microscopy further revealed that PDI binds nascent chains persistently, forming a stable face-to-face homodimer, whereas ERp46 binds for a shorter time in monomeric form, indicating their different mechanisms for substrate recognition and disulfide bond introduction. Thus, ERp46 serves as a more potent disulfide introducer especially during the early stages of translation, whereas PDI can catalyze disulfide formation when longer nascent chains emerge out from ribosome.Graphical abstractDisplay OmittedHighlights•We developed anin vitrosystem for monitoring nascent-chain disulfide formation•High-speed AFM visualized PDI and ERp46 molecules acting on nascent chains•PDI persistently holds nascent chains via dimerization for disulfide introduction•ERp46 rapidly introduces disulfide bonds to nascent chains via short-time bindingCell Biology; Molecular Biology; Structural Biology
