Quality control in the secretory pathway: retention of a misfolded viral membrane glycoprotein involves cycling between the ER, intermediate compartment, and Golgi apparatus.

TLDR

Proteins are normally exported from the ER to the Golgi only after achieving a fully folded conformation. The study investigated how misfolded proteins are selectively retained by tracking the long‑term fate of a temperature‑sensitive G protein from tsO45 vesicular stomatitis virus. The authors used immunofluorescence, immunoelectron microscopy, Nycodenz gradient centrifugation, folding and recycling inhibitors, and calnexin co‑immunoprecipitation to monitor the G protein through the ER, intermediate compartment, and Golgi. The misfolded G protein cycles between the ER, intermediate compartment, and cis‑Golgi, being retrieved back to the ER where it associates with BiP/GRP78, showing that retention involves calnexin binding and selective retrieval from later compartments.

Abstract

Proteins synthesized in the ER are generally transported to the Golgi complex and beyond only when they have reached a fully folded and assembled conformation. To analyze how the selective retention of misfolded proteins works, we monitored the long-term fate of a membrane glycoprotein with a temperature-dependent folding defect, the G protein of tsO45 vesicular stomatitis virus. We used indirect immunofluorescence, immunoelectron microscopy, and a novel Nycodenz gradient centrifugation procedure for separating the ER, the intermediate compartment, and the Golgi complex. We also employed the folding and recycling inhibitors dithiothreitol and AIF4-, and coimmunoprecipitation with calnexin antibodies. The results showed that the misfolded G protein is not retained in the ER alone; it can move to the intermediate compartment and to the cis-Golgi network but is then recycled back to the ER. In the ER it is associated with calnexin and BiP/GRP78. Of these two chaperones, only BiP/GRP78 seems to accompany it through the recycling circuit. Thus, the retention of this misfolded glycoprotein is the result of multiple mechanisms including calnexin binding in the ER and selective retrieval from the intermediate compartment and the cis-Golgi network.