Role of ribosome and translocon complex during folding of influenza hemagglutinin in the endoplasmic reticulum of living cells.
Protein folding in the living cell begins cotranslationally. To analyze how it is influenced by the ribosome and by the translocon complex during translocation into the endoplasmic reticulum, we expressed a mutant influenza hemagglutinin (a type I membrane glycoprotein) with a C-terminal extension. Analysis of the nascent chains by two-dimensional SDS-PAGE showed that ribosome attachment as such had little effect on ectodomain folding or trimer assembly. However, as long as the chains were ribosome bound and inside the translocon complex, formation of disulfides was partially suppressed, trimerization was inhibited, and the protein protected against aggregation.
Chen
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Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520-8002, USA.
Helenius
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D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D006224:Cricetinae; D003513:Cycloheximide / Q000494:pharmacology; D004220:Disulfides / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D018870:Endoplasmic Reticulum, Rough / Q000737:chemistry* / Q000201:enzymology / Q000378:metabolism*; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation / Q000187:drug effects; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D026563:LDL-Receptor Related Protein-Associated Protein; D009154:Mutation / Q000235:genetics; D011233:Precipitin Tests; D011485:Protein Binding; D014176:Protein Biosynthesis / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011691:Puromycin / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism*; D014415:Tunicamycin / Q000494:pharmacology
D002352:Carrier Proteins; D004220:Disulfides; D006023:Glycoproteins; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D026563:LDL-Receptor Related Protein-Associated Protein; D011500:Protein Synthesis Inhibitors; D011993:Recombinant Fusion Proteins; D014415:Tunicamycin; D011691:Puromycin; D003513:Cycloheximide