Characterization of organelles in the vacuolar-sorting pathway by visualization with GFP in tobacco BY-2 cells.
We have shown the localization and mobilization of modified green fluorescent proteins (GFPs) with various signals in different compartments in a vacuolar-sorting system of tobacco BY-2 cells. In contrast to the efficient secretion of GFP from the transformed cells expressing SP-GFP composed of a signal peptide and GFP, accumulation of GFP in the vacuoles was observed in the cells expressing SP-GFP fused with the C-terminal peptide of pumpkin 2S albumin. This indicated that this peptide is sufficient for vacuolar targeting. Interestingly, the fluorescence in the vacuoles disappeared sharply at 7 d after inoculation of the cells, but it appeared again after re-inoculation into a new culture medium. When SP-GFP was fused with the region, termed PV72C, including a transmembrane domain and a cytosolic tail of a vacuolar-sorting receptor PV72, GFP-PV72C was detected in the Golgi-complex-like small particles. Prolonged culture showed that GFP-PV72C that reached the prevacuolar compartments was cleaved off the PV72C region to produce GFP, that arrived at the vacuoles to be diffused. These findings suggested that the vacuolar-sorting receptor might be recycled between the Golgi complex and prevacuolar compartments.
Mitsuhashi
N
N
Department of Cell Biology, National Institute for Basic Biology, Okazaki, Japan.
Shimada
T
T
Mano
S
S
Nishimura
M
M
Hara-Nishimura
I
I
D001483:Base Sequence; D002460:Cell Line; D017930:Genes, Reporter; D006056:Golgi Apparatus / Q000502:physiology / Q000648:ultrastructure; D049452:Green Fluorescent Proteins; D007425:Intracellular Membranes / Q000502:physiology / Q000648:ultrastructure; D007700:Kinetics; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D015388:Organelles / Q000502:physiology* / Q000648:ultrastructure; D010940:Plant Proteins / Q000378:metabolism; D010947:Plants, Toxic; D021382:Protein Sorting Signals; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014026:Nicotiana / Q000166:cytology / Q000502:physiology*; D014162:Transfection; D014617:Vacuoles / Q000502:physiology* / Q000648:ultrastructure
D008164:Luminescent Proteins; D009838:Oligodeoxyribonucleotides; C111597:PAC vesicle 72-kDa protein, Cucurbita; D010940:Plant Proteins; D021382:Protein Sorting Signals; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins