title: Localizing the movement proteins of Abutilon mosaic geminivirus in yeast by subcellular fractionation and freeze-fracture immuno-labelling.
abstract: The movement proteins BC1 and BV1 of Abutilon mosaic geminivirus fused to glutathion-S-transferase (GST) and Flag-peptide were expressed in fission yeast (Schizosaccharomyces pombe) cells to analyse the fundamental intracellular distribution of these proteins in an eukaryotic cell in the absence of plant-specific factors. Most of BC1 protein sedimented rapidly after cell lysis and differential centrifugation. Using freeze-fracture immuno-labelling, the protein was detected in situ predominantly at plasma membranes and to a lower extent at cytoplasmic vesicles but not in the cytoplasm, the nuclei, or the mitochondria. Anti-BC1, anti-GST, and anti-Flag antibodies tagged smooth flecks only at the protoplasmic faces of the plasma membrane. The consequences of the BC1 behaviour for its use in two-hybrid analysis in yeast are discussed. In contrast, BV1 was detected mainly in the nucleus and partially in the cytoplasm but never associated with membranes.
authors.affiliation: Department of Molecular Biology and Plant Virology, University of Stuttgart, Germany.
authors.forename: H J
authors.initials: HJ
authors.lastname: Aberl
authors.forename: M L
authors.initials: ML
authors.lastname: Rütz
authors.forename: M
authors.initials: M
authors.lastname: Karayavuz
authors.forename: S
authors.initials: S
authors.lastname: Frischmuth
authors.forename: C
authors.initials: C
authors.lastname: Wege
authors.forename: D
authors.initials: D
authors.lastname: Hülser
authors.forename: H
authors.initials: H
authors.lastname: Jeske
mesh_terms: D002462:Cell Membrane / Q000737:chemistry* / Q000648:ultrastructure; D003001:Cloning, Molecular; D022162:Cytoplasmic Vesicles / Q000737:chemistry* / Q000648:ultrastructure; D005614:Freeze Fracturing; D017791:Geminiviridae / Q000235:genetics; D005982:Glutathione Transferase; D008854:Microscopy, Electron; D053830:Plant Viral Movement Proteins; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012568:Schizosaccharomyces / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D013347:Subcellular Fractions / Q000737:chemistry* / Q000648:ultrastructure; D014764:Viral Proteins / Q000032:analysis* / Q000235:genetics
chemical_list: D053830:Plant Viral Movement Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D005982:Glutathione Transferase