Localizing the movement proteins of Abutilon mosaic geminivirus in yeast by subcellular fractionation and freeze-fracture immuno-labelling.
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.
Aberl
H J
HJ
Department of Molecular Biology and Plant Virology, University of Stuttgart, Germany.
Rütz
M L
ML
Karayavuz
M
M
Frischmuth
S
S
Wege
C
C
Hülser
D
D
Jeske
H
H
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
D053830:Plant Viral Movement Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D005982:Glutathione Transferase