Membrane glycoprotein folding, oligomerization and intracellular transport: effects of dithiothreitol in living cells.
Using influenza hemagglutinin (HA0) and vesicular stomatitis virus G protein as model proteins, we have analyzed the effects of dithiothreitol (DTT) on conformational maturation and transport of glycoproteins in the secretory pathway of living cells. While DTT caused reduction of folding intermediates and misfolded proteins in the endoplasmic reticulum (ER), it did not affect molecules that had already acquired a mature trimeric conformation, whether present in the ER or elsewhere. The conversion to DTT resistance was therefore a pre-Golgi event. Reduction of folding intermediates was dependent on the intactness of the ER and on metabolic energy, suggesting cooperativity between DTT and ER folding factors. DTT did not inhibit most cellular functions, including ATP synthesis and protein transport within the secretory pathway. The results established DTT as an effective tool for analyzing the folding and compartmental distribution of proteins with disulfide bonds.
Tatu
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Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06510.
Braakman
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Helenius
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D016466:CHO Cells; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D006224:Cricetinae; D004229:Dithiothreitol / Q000494:pharmacology*; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral / Q000737:chemistry / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D017510:Protein Folding; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000378:metabolism
C028391:G protein, vesicular stomatitis virus; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral; D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins; D000255:Adenosine Triphosphate; D004229:Dithiothreitol