Plant cells may contain two functionally distinct vacuolar compartments. Membranes of protein storage vacuoles (PSV) are marked by the presence of α-tonoplast intrinsic protein (TIP), whereas lytic vacuoles (LV) are marked by the presence of γ-TIP. Mechanisms for sorting integral membrane proteins to the different vacuoles have not been elucidated. Here we study a chimeric integral membrane reporter protein expressed in tobacco suspension culture protoplasts whose traffic was assessed biochemically by following acquisition of complex Asn-linked glycan modifications and proteolytic processing, and whose intracellular localization was determined with confocal immunofluorescence. We show that the transmembrane domain of the plant vacuolar sorting receptor BP-80 directs the reporter protein via the Golgi to the LV prevacuolar compartment, and attaching the cytoplasmic tail (CT) of γ-TIP did not alter this traffic. In contrast, the α-TIP CT prevented traffic of the reporter protein through the Golgi and caused it to be localized in organelles separate from ER and from Golgi and LV prevacuolar compartment markers. These organelles had a buoyant density consistent with vacuoles, and α-TIP protein colocalized in them with the α-TIP CT reporter protein when the two were expressed together in protoplasts. These results are consistent with two separate pathways to vacuoles for membrane proteins: a direct ER to PSV pathway, and a separate pathway via the Golgi to the LV.
Integral Membrane Protein Sorting to Vacuoles in Plant Cells: Evidence for Two Pathways
Address correspondence to J.C. Rogers, Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340. Tel.: (509) 335-2773. Fax: (509) 335-7643. E-mail: [email protected]
We greatly appreciate the assistance from S. Rogers (Washington State University, Pullman, WA) in many ways during the course of this work and thank M.J. Chrispeels (University of California, San Diego, CA) for sharing the α-TIP cDNA and anti–α-TIP protein antiserum, and S.J. Coughlan (Pioneer Hi-Bred International, Johnston, IA), D.J. Meyer (Washington State University), and A. Bennet (University of California, Davis, CA), and A. Sturm (Friedrich Miescher Institute, Basel, Switzerland) for sharing other antisera used in this study. Special thanks are given to X.-F. Cao and G.-Y. Jauh (both from Washington State University) for making available the 17F9 monoclonal antibody to BP-80 and polyclonal antibodies to the α-TIP CT peptide.
This research was supported by grants from the National Institutes of Health (GM 52427) and the Department of Energy (DE-FG 95ER 20165).
Liwen Jiang, John C. Rogers; Integral Membrane Protein Sorting to Vacuoles in Plant Cells: Evidence for Two Pathways . J Cell Biol 30 November 1998; 143 (5): 1183–1199. doi: https://doi.org/10.1083/jcb.143.5.1183
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