In vitro reconstitution of calreticulin-substrate interactions.
Calreticulin is a soluble, endoplasmic reticulum-resident protein and a molecular chaperone for glycoproteins. We have reconstituted the binding of recombinant calreticulin to two glycoprotein substrates, vesicular stomatitis virus G protein and influenza hemagglutinin, in vitro. The binding was found to be direct and to require monoglucosylated, asparagine-linked oligosaccharides on the substrate glycoprotein but no other cellular factors. The binding could be modulated in vitro by incubation of substrate with purified preparations of the glycan modifying enzymes glucosidase II and the UDP-glucose:glycoprotein glucosyltransferase, thus recapitulating the regulation of calreticulin-binding by glycan modification that occurs in vivo. Using the purified ER enzymes and the recombinant calreticulin, an assay was established for reconstituting a complex, multicomponent chaperone binding cycle in vitro. We demonstrated, moreover, that the acidic C-terminal 62 residues of calreticulin are dispensable for substrate binding whereas further deletions inhibit substrate binding.
Department of Cell Biology, Yale School of Medicine, New Haven, CT 06510, USA. peterson@bcmp.med.harvard.edu
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Peterson
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Helenius
D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D037282:Calreticulin; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005947:Glucose / Q000737:chemistry / Q000378:metabolism; D005964:Glucosyltransferases / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000737:chemistry / Q000378:metabolism; D066298:In Vitro Techniques; D037102:Lectins; D008099:Liver / Q000737:chemistry / Q000201:enzymology; D008562:Membrane Glycoproteins; D018832:Molecular Chaperones / Q000378:metabolism*; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D017510:Protein Folding; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism*; D013379:Substrate Specificity; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000378:metabolism; D000520:alpha-Glucosidases / Q000378:metabolism
D002135:Calcium-Binding Proteins; D037282:Calreticulin; C028391:G protein, vesicular stomatitis virus; D006023:Glycoproteins; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D037102:Lectins; D008562:Membrane Glycoproteins; D018832:Molecular Chaperones; D009844:Oligosaccharides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D014759:Viral Envelope Proteins; D005964:Glucosyltransferases; C034251:4-nitrophenyl-alpha-glucosidase; D000520:alpha-Glucosidases; D005947:Glucose