title: Oligomerization and interacellular localization of the glycoprotein receptor ERGIC-53 is independent of disulfide bonds.
abstract: ERGIC-53 is a type I transmembrane lectin facilitating the efficient export of a subset of secretory glycoproteins from the endoplasmic reticulum. Previous results have shown that ERGIC-53 is present as reduction-sensitive homo-oligomers, i.e. as a balanced mixture of disulfide-linked hexamers and dimers, with the two cysteine residues located close to the transmembrane domain playing a crucial role in oligomerization. Here, we demonstrate, using sucrose gradient sedimentation, cross-linking analyses, and non-denaturing gel electrophoresis, that ERGIC-53 is present exclusively as a hexameric complex in cells. However, the hexamers exist in two forms, one as a disulfide-linked, Triton X-100, perfluoro-octanic acid, and SDS-resistant complex, and the other as a non-covalent, Triton X-100, perfluoro-octanoic acid-resistant, but SDS-sensitive, complex made up of three disulfide-linked dimers that are likely to interact through the coiled-coil domains present in the luminal part of the protein. In contrast to what was previously believed, neither of the membrane-proximal cysteine residues plays an essential role in the formation, or maintenance, of the latter form of hexamers. Subcellular fractionation revealed that the double-cysteine mutant was present in the endoplasmic reticulum-Golgi-intermediate compartment, indicating that the two cysteine residues are not essential for the intracellular distribution of ERGIC-53. Based on these results, we present a model for the formation of the two hexameric forms.
authors.affiliation: Ludwig Institute for Cancer Research, Stockholm Branch, Karolinska Institutet, Box 240, S-17177 Stockholm, Sweden.
authors.forename: Etienne P A
authors.initials: EP
authors.lastname: Neve
authors.forename: Ulla
authors.initials: U
authors.lastname: Lahtinen
authors.forename: Ralf F
authors.initials: RF
authors.lastname: Pettersson
mesh_terms: D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D004220:Disulfides / Q000378:metabolism*; D004586:Electrophoresis; D006023:Glycoproteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D037241:Mannose-Binding Lectins; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D011489:Protein Denaturation; D020836:Protein Structure, Quaternary; D021381:Protein Transport; D033921:Vesicular Transport Proteins
chemical_list: D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D004220:Disulfides; C494448:ERGIC1 protein, human; D006023:Glycoproteins; C501458:Lman1 protein, rat; D037241:Mannose-Binding Lectins; D008565:Membrane Proteins; D033921:Vesicular Transport Proteins; D003545:Cysteine