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.
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.
Laporte
Céline
C
Institut de Biologie Moléculaire des Plantes, 67084 Strasbourg Cedex, France.
Vetter
Guillaume
G
Loudes
Anne-Marie
AM
Robinson
David G
DG
Hillmer
Stefan
S
Stussi-Garaud
Christiane
C
Ritzenthaler
Christophe
C
D008841:Actin Cytoskeleton / Q000502:physiology; D037282:Calreticulin / Q000494:pharmacology; D002455:Cell Division / Q000502:physiology; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins; D007150:Immunohistochemistry; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D008870:Microtubules / Q000502:physiology; D017860:Nepovirus / Q000254:growth & development*; D053830:Plant Viral Movement Proteins; D030821:Plants, Genetically Modified; D011233:Precipitin Tests; D021381:Protein Transport / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D022142:Secretory Vesicles / Q000187:drug effects / Q000378:metabolism*; D014026:Nicotiana / Q000378:metabolism / Q000648:ultrastructure / Q000821:virology; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D037282:Calreticulin; D008164:Luminescent Proteins; D053830:Plant Viral Movement Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins