title: Glycosylatable GFP as a compartment-specific membrane topology reporter.
abstract: Determination of the membrane topology is an essential step in structural and functional studies of integral membrane proteins, yet the choices of membrane topology reporters are limited and the experimental analysis can be laborious, especially in eukaryotic cells. Here, we present a robust membrane topology reporter, glycosylatable green fluorescent protein (gGFP). gGFP is fully fluorescent in the yeast cytosol but becomes glycosylated and does not fluoresce in the lumen of the endoplasmic reticulum (ER). Thus, by assaying fluorescence and the glycosylation status of C-terminal fusions of gGFP to target membrane proteins in whole-cell lysates, the localization of the gGFP moiety (and hence the fusion joint) relative to the ER membrane can be unambiguously determined.
authors.affiliation: School of Biological Sciences, Seoul National University, Seoul 151-747, South Korea.
authors.forename: Hunsang
authors.initials: H
authors.lastname: Lee
authors.forename: Jisoo
authors.initials: J
authors.lastname: Min
authors.forename: Gunnar
authors.initials: G
authors.lastname: von Heijne
authors.forename: Hyun
authors.initials: H
authors.lastname: Kim
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005453:Fluorescence; D006031:Glycosylation; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D015202:Protein Engineering; D012441:Saccharomyces cerevisiae / Q000378:metabolism
chemical_list: D049452:Green Fluorescent Proteins