Involvement of the secretory pathway and the cytoskeleton in intracellular targeting and tubule assembly of Grapevine fanleaf virus movement protein in tobacco BY-2 cells

Plant Cell. 2003 Sep;15(9):2058-75. doi: 10.1105/tpc.013896.

Abstract

Grapevine fanleaf virus (GFLV) is one of a large class of plant viruses whose cell-to-cell transport involves the passage of virions through tubules composed of virus-encoded movement protein (MP). The tubules are embedded within modified plasmodesmata, but the mechanism of targeting of MP to these sites is unknown. To study intracellular GFLV MP trafficking, a green fluorescent protein-MP fusion (GFP:MP) was expressed in transgenic tobacco BY-2 suspension cells under the control of an inducible promoter. We show that GFP:MP is targeted preferentially to calreticulin-labeled foci within the youngest cross walls, where it assembles into tubules. During cell division, GFP:MP colocalizes in the cell plate with KNOLLE, a cytokinesis-specific syntaxin, and both proteins are linked physically, as shown by coimmunoprecipitation of the two proteins from the same microsomal fraction. In addition, treatment with various drugs has revealed that a functional secretory pathway, but not the cytoskeleton, is required for tubule formation. However, correct GFP:MP targeting to calreticulin-labeled foci seems to be cytoskeleton dependent. Finally, biochemical analyses have revealed that at least a fraction of the MP behaves as an intrinsic membrane protein. These findings support a model in which GFP:MP would be transported to specific sites via Golgi-derived vesicles along two different pathways: a microtubule-dependent pathway in normal cells and a microfilament-dependent default pathway when microtubules are depolymerized.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actin Cytoskeleton / physiology
  • Calreticulin / pharmacology
  • Cell Division / physiology
  • Cells, Cultured
  • Cytoskeleton / drug effects
  • Cytoskeleton / metabolism*
  • Golgi Apparatus / metabolism
  • Green Fluorescent Proteins
  • Immunohistochemistry
  • Luminescent Proteins / metabolism
  • Microscopy, Confocal
  • Microscopy, Electron
  • Microtubules / physiology
  • Nepovirus / growth & development*
  • Plant Viral Movement Proteins
  • Plants, Genetically Modified
  • Precipitin Tests
  • Protein Transport / physiology
  • Recombinant Fusion Proteins / metabolism
  • Secretory Vesicles / drug effects
  • Secretory Vesicles / metabolism*
  • Tobacco / metabolism
  • Tobacco / ultrastructure
  • Tobacco / virology
  • Transfection
  • Viral Proteins / genetics
  • Viral Proteins / metabolism*

Substances

  • Calreticulin
  • Luminescent Proteins
  • Plant Viral Movement Proteins
  • Recombinant Fusion Proteins
  • Viral Proteins
  • Green Fluorescent Proteins