Plant Physiology Preview
Published on September 15, 2006; 10.1104/pp.106.083618
OPEN ACCESS ARTICLE
Received May 15, 2006
Accepted September 8, 2006
Localization of GFP Fusions with the Seven Arabidopsis Vacuolar Sorting Receptors to Prevacuolar Compartments in Tobacco BY-2 Cells
Yansong Miao , Pak Kan Yan , Hyeran Kim , Inhwan Hwang , and Liwen Jiang *
Department of Biology and Molecular Biotechnology Program, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China
Department of Life Science, Pohang University of Science and Technology, Pohang, Korea
* Corresponding author; email: ljiang{at}cuhk.edu.hk.
We have previously demonstrated that vacuolar sorting receptor (VSR) proteins are concentrated on prevacuolar compartments (PVCs) in plant cells. PVCs in tobacco (Nicotiana tabacum) BY-2 cells are multivesicular bodies (MVBs) as defined by VSR proteins and the BP-80 reporter, where the transmembrane domain (TMD) and cytoplasmic tail (CT) sequences of BP-80 are sufficient and specific for correct targeting of the reporter to PVCs. The genome of Arabidopsis thaliana contains seven VSR proteins, but little is known about their individual subcellular localization and function. Here we study the subcellular localization of the seven Arabidopsis VSR proteins (AtVSR1-7) based on previously proved hypothesis that the TMD and CT sequences correctly target individual VSR to its final destination in transgenic tobacco BY-2 cells. Toward this goal, we have generated seven chimeric constructs containing signal peptide (sp) linked to GFP (green fluorescent protein) and TMD/CT sequences (sp-GFP-TMD/CT) of the seven individual AtVSR. Transgenic tobacco BY-2 cell lines expressing these seven sp-GFP-TMD-CT fusions all exhibited typical punctate signals colocalizing with VSR proteins by confocal immunofluorescence. In addition, wortmannin caused the GFP-marked prevacuolar organelles to form small vacuoles and VSR antibodies labeled these enlarged MVBs in transgenic BY-2 cells. Wortmannin also caused VSR-marked PVCs to vacuolate in other cell types including Arabidopsis, rice, pea, and mungbean. Therefore, the seven AtVSRs are localized to MVBs in tobacco BY-2 cells and wortmannin-induced vacuolation of PVCs is a general response in plants.
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