title: Live-cell visualization of transmembrane protein oligomerization and membrane fusion using two-fragment haptoEGFP methodology.
abstract: Protein interactions play key roles throughout all subcellular compartments. In the present paper, we report the visualization of protein interactions throughout living mammalian cells using two oligomerizing MV (measles virus) transmembrane glycoproteins, the H (haemagglutinin) and the F (fusion) glycoproteins, which mediate MV entry into permissive cells. BiFC (bimolecular fluorescence complementation) has been used to examine the dimerization of these viral glycoproteins. The H glycoprotein is a type II membrane-receptor-binding homodimeric glycoprotein and the F glycoprotein is a type I disulfide-linked membrane glycoprotein which homotrimerizes. Together they co-operate to allow the enveloped virus to enter a cell by fusing the viral and cellular membranes. We generated a pair of chimaeric H glycoproteins linked to complementary fragments of EGFP (enhanced green fluorescent protein)--haptoEGFPs--which, on association, generate fluorescence. Homodimerization of H glycoproteins specifically drives this association, leading to the generation of a fluorescent signal in the ER (endoplasmic reticulum), the Golgi and at the plasma membrane. Similarly, the generation of a pair of corresponding F glycoprotein-haptoEGFP chimaeras also produced a comparable fluorescent signal. Co-expression of H and F glycoprotein chimaeras linked to complementary haptoEGFPs led to the formation of fluorescent fusion complexes at the cell surface which retained their biological activity as evidenced by cell-to-cell fusion.
authors.affiliation: School of Biological Sciences, Queen's University of Belfast, 97 Lisburn Road, Belfast BT9 7BL, UK.
authors.forename: Derek J
authors.initials: DJ
authors.lastname: Quinn
authors.forename: Neil V
authors.initials: NV
authors.lastname: McFerran
authors.forename: John
authors.initials: J
authors.lastname: Nelson
authors.forename: W Paul
authors.initials: WP
authors.lastname: Duprex
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D008561:Membrane Fusion; D008565:Membrane Proteins / Q000378:metabolism*; D018613:Microscopy, Confocal; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D059010:Single-Cell Analysis; D014709:Vero Cells; D014760:Viral Fusion Proteins / Q000096:biosynthesis
chemical_list: D008565:Membrane Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014760:Viral Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins