Localizing the movement proteins of Abutilon mosaic geminivirus in yeast by subcellular fractionation and freeze-fracture immuno-labelling

Arch Virol. 2002;147(1):103-17. doi: 10.1007/s705-002-8305-8.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Membrane / chemistry*
  • Cell Membrane / ultrastructure
  • Cloning, Molecular
  • Cytoplasmic Vesicles / chemistry*
  • Cytoplasmic Vesicles / ultrastructure
  • Freeze Fracturing*
  • Geminiviridae* / genetics
  • Glutathione Transferase
  • Microscopy, Electron
  • Plant Viral Movement Proteins
  • Recombinant Fusion Proteins / chemistry
  • Schizosaccharomyces / chemistry*
  • Schizosaccharomyces / genetics
  • Schizosaccharomyces / ultrastructure
  • Subcellular Fractions / chemistry*
  • Subcellular Fractions / ultrastructure
  • Viral Proteins / analysis*
  • Viral Proteins / genetics

Substances

  • Plant Viral Movement Proteins
  • Recombinant Fusion Proteins
  • Viral Proteins
  • Glutathione Transferase