QUALITY-CONTROL IN THE SECRETORY PATHWAY - RETENTION OF A MISFOLDED VIRAL MEMBRANE GLYCOPROTEIN INVOLVES CYCLING BETWEEN THE ER, INTERMEDIATE COMPARTMENT, AND GOLGI-APPARATUS

Proteins synthesized in the ER are generally transported to the Golgi complex and beyond only when they have reached a fully folded and asse mbled conformation. To analyze how the selective retention of misfolde d proteins works, we monitored the longterm fate of a membrane glycopr otein with a temperature-dependent folding defect, the G protein of ts O45 vesicular stomatitis virus. We used indirect immunofluorescence, i mmunoelectron microscopy, and a novel Nycodenz gradient centrifugation procedure for separating the ER, the intermediate compartment, and th e Golgi complex. We also employed the folding and recycling inhibitors dithiothreitol and AIF(4-), and coimmunoprecipitation with calnexin a ntibodies. The results showed that the misfolded G protein is not reta ined 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 E R it is associated with calnexin and BiP/GRP78. Of these two chaperone s, only BiP/GRP78 seems to accompany it through the recycling circuit. Thus, the retention of this misfolded glycoprotein is the result of m ultiple mechanisms including calnexin binding in the ER and selective retrieval from the intermediate compartment and the cis-Golgi network.