title: Subcellular localization and self-interaction of plant-specific Nt-4/1 protein.
abstract: The Nicotiana tabacum Nt-4/1 protein is a plant-specific protein of unknown function. Analysis of bacterially expressed Nt-4/1 protein in vitro revealed that the protein secondary structure is mostly alpha-helical and suggested that it could consist of three structural domains. Earlier studies of At-4/1, the Arabidopsis thaliana-encoded ortholog of Nt-4/1, demonstrated that GFP-fused At-4/1 was capable of polar localization in plant cells, association with plasmodesmata, and cell-to-cell transport. Together with the At-4/1 ability to interact with a plant virus movement protein, these data supported the hypothesis of the At-4/1 protein involvement in viral transport through plasmodesmata. Studies of the Nt-4/1-GFP fusion protein reported in this paper revealed that the protein was localized to cytoplasmic bodies, which were co-aligned with actin filaments and capable of actin-dependent intracellular movement. The Nt-4/1-GFP bodies, being non-membrane structures, were found in association with the plasma membrane, the tubular endoplasmic reticulum and endosome-like structures. Bimolecular fluorescence complementation experiments and inhibition of nuclear export showed that the Nt-4/1 protein was capable of nuclear-cytoplasmic transport. The nuclear export signal (NES) was identified in the Nt-4/1 protein by site-directed mutagenesis. The Nt-4/1 NES mutant was localized to the nucleoplasm forming spherical bodies. Immunogold labeling and electron microscopy of cytoplasmic Nt-4/1-containing bodies and nuclear structures containing the Nt-4/1 NES mutant revealed differences in their fine structure. In mammalian cells, Nt-4/1-GFP formed cytoplasmic spherical bodies similar to those found for the Nt-4/1 NES mutant in plant cell nuclei. Using dynamic laser light scattering and electron microscopy, the Nt-4/1 protein was found to form multimeric complexes in vitro.
authors.affiliation: A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Chochlova Str. 1, 119992 Moscow, Russia.
authors.forename: A G
authors.initials: AG
authors.lastname: Solovyev
authors.forename: E A
authors.initials: EA
authors.lastname: Minina
authors.forename: S S
authors.initials: SS
authors.lastname: Makarova
authors.forename: T N
authors.initials: TN
authors.lastname: Erokhina
authors.forename: V V
authors.initials: VV
authors.lastname: Makarov
authors.forename: I B
authors.initials: IB
authors.lastname: Kaplan
authors.forename: L
authors.initials: L
authors.lastname: Kopertekh
authors.forename: J
authors.initials: J
authors.lastname: Schiemann
authors.forename: K R
authors.initials: KR
authors.lastname: Richert-Pöggeler
authors.forename: S Y
authors.initials: SY
authors.lastname: Morozov
mesh_terms: D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D006224:Cricetinae; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008854:Microscopy, Electron; D016297:Mutagenesis, Site-Directed; D049790:Nuclear Export Signals; D010940:Plant Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000378:metabolism*
chemical_list: D049790:Nuclear Export Signals; D010940:Plant Proteins; D049452:Green Fluorescent Proteins