Localization of Green Fluorescent Protein Fusions with the Seven Arabidopsis Vacuolar Sorting Receptors to Prevacuolar Compartments in Tobacco BY-2 Cells

Yansong Miao,Liwen Jiang,Inhwan Hwang,Pak Kan Yan,Hyeran Kim

doi:10.1104/pp.106.083618

Abstract

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 (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 the previously proven 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 green fluorescent protein (GFP) 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 (Oryza sativa), pea (Pisum sativum), and mung bean (Vigna radiata). 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.

Highlights

We have previously demonstrated that vacuolar sorting receptor (VSR) proteins are concentrated on prevacuolar compartments (PVCs) in plant cells
Phylogenetic analysis (Fig. 1A) based on full-length VSR protein sequences indicated that AtVSR1 and AtVSR2 are closely related to the pumpkin PV72 and black gram (Vigna mungo) VmVSR, while AtVSR3 and AtVSR4 are more closely related to the pea BP-80, and the other three AtVSR members are grouped into another cluster depending on their evolutional relationship
To study the subcellular localization of the seven AtVSR proteins, we generated various chimeric constructs that contain sp sequences from the barley (Hordeum vulgare) proaleurain linked to green fluorescent protein (GFP) and transmembrane domain (TMD)/cytoplasmic tail (CT) sequences of individual AtVSR

Summary

Introduction

We have previously demonstrated that vacuolar sorting receptor (VSR) proteins are concentrated on prevacuolar compartments (PVCs) in plant cells. VSR protein isolated from pea (Pisum sativum) due to its interaction with a vacuolar sorting determinant Asn-Pro-Ile-Arg (Kirsch et al, 1994), and several in vitro and in vivo studies demonstrated that BP-80 may function as a sorting receptor for transporting the Cys protease aleurain from Golgi to lytic vacuole in plant cells (Jiang and Rogers, 1998; Humair et al, 2001; Paris and Neuhaus, 2002; Neuhaus and Paris, 2005). In plant cells, BP-80 is believed to function as a VSR that selects cargo proteins at the TGN for subsequent packaging into CCVs before delivery to the vacuole via a PVC, where the receptor is recycled back to TGN for another round of binding/selection of cargoes

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