title: Topology of Endoplasmic Reticulum-Associated Cellular and Viral Proteins Determined with Split-GFP.
abstract: The split green fluorescent protein (GFP) system was adapted for investigation of the topology of ER-associated proteins. A 215-amino acid fragment of GFP (S1-10) was expressed in the cytoplasm as a free protein or fused to the N-terminus of calnexin and in the ER as an intraluminal protein or fused to the C-terminus of calnexin. A 16-amino acid fragment of GFP (S11) was fused to the N- or C-terminus of the target protein. Fluorescence occurred when both GFP fragments were in the same intracellular compartment. After validation with the cellular proteins PDI and tapasin, we investigated two vaccinia virus proteins (L2 and A30.5) of unknown topology that localize to the ER and are required for assembly of the viral membrane. Our results indicated that the N- and C-termini of L2 faced the cytoplasmic and luminal sides of the ER, respectively. In contrast both the N- and C-termini of A30.5 faced the cytoplasm. The system offers advantages for quickly determining the topology of intracellular proteins: the S11 tag is similar in length to commonly used epitope tags; multiple options are available for detecting fluorescence in live or fixed cells; transfection protocols are adaptable to numerous expression systems and can enable high throughput applications.
authors.affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.|Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, USA.
authors.forename: Seong-In
authors.initials: SI
authors.lastname: Hyun
authors.affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Liliana
authors.initials: L
authors.lastname: Maruri-Avidal
authors.affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Bernard
authors.initials: B
authors.lastname: Moss
mesh_terms: D000818:Animals; D037281:Calnexin / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014616:Vaccinia virus / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D014764:Viral Proteins; D037281:Calnexin; D049452:Green Fluorescent Proteins
keywords: calnexin; endoplasmic reticulum; green fluorescent protein; membrane topology; poxvirus; protein disulfide isomerase; tapasin; vaccinia virus