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 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.
Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 05610.
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Hammond
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Helenius
D017607:Aluminum Compounds / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport / Q000187:drug effects / Q000235:genetics; D016466:CHO Cells; D002135:Calcium-Binding Proteins / Q000378:metabolism; D037281:Calnexin; D002352:Carrier Proteins / Q000378:metabolism; D002451:Cell Compartmentation; D002458:Cell Fractionation; D006224:Cricetinae; D004229:Dithiothreitol / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure; D000091342:Endoplasmic Reticulum Chaperone BiP; D005455:Fluorescent Antibody Technique; D005459:Fluorides / Q000494:pharmacology; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D006360:Heat-Shock Proteins; D008562:Membrane Glycoproteins; D016253:Microscopy, Immunoelectron; D008954:Models, Biological; D018832:Molecular Chaperones; D009154:Mutation; D017510:Protein Folding; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism*
D017607:Aluminum Compounds; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; C028391:G protein, vesicular stomatitis virus; D006023:Glycoproteins; D006360:Heat-Shock Proteins; D008562:Membrane Glycoproteins; D018832:Molecular Chaperones; D014759:Viral Envelope Proteins; D037281:Calnexin; C050992:tetrafluoroaluminate; D005459:Fluorides; D004229:Dithiothreitol