Rha1, an Arabidopsis Rab5 Homolog, Plays a Critical Role in the Vacuolar Trafficking of Soluble Cargo Proteins

Sohn, Eun Ju; Kim, Eol Sun; Zhao, Min; Kim, Soo Jin; Kim, Hye‐Ran; Kim, Yong-Woo; Lee, Yong Jik; Hillmer, Stefan; Sohn, Uik; Jiang, Liwen; Hwang, Inhwan

doi:10.1105/tpc.009779

Cited by 188 publications

(235 citation statements)

References 71 publications

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“…In contrast, in tobacco leaf epidermal cells, AtRABF2b was localized to the late endosomal/pre-vacuolar compartment (PVC) (Kotzer et al, 2004). Localization of RABF proteins to the PVC was also seen in other cell types (Bolte et al, 2004a;Haas et al, 2007;Lee et al, 2004;Sohn et al, 2003). The contradiction regarding the localization of RABF1 and RABF2b, which is probably due to the various model systems employed, precludes the use of these proteins as specific markers to distinguish between early and late endosomes/PVC.…”

Section: Introductionmentioning

confidence: 80%

“…Three other groups have previously shown that the over-expression of dominant negative RABF proteins interferes with the correct sorting of lytic enzymes towards the vacuoles (Bolte et al, 2004a;Kotzer et al, 2004;Sohn et al, 2003). More recently, over-expression of a dominant negative form of RABF2b in Arabidopsis roots was reported to inhibit endocytosis and normal cell plate formation (Dhonukshe et al, 2006).…”

Section: Position Of the Pvc/mvb In The Endosomal Pathway In Arabidopmentioning

confidence: 99%

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Evidence for a sorting endosome in Arabidopsis root cells

et al. 2007

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SummaryIn eukaryotic cells, the endocytic and secretory pathways are key players in several physiological processes. These pathways are largely inter-connected in animal and yeast cells through organelles named sorting endosomes. Sorting endosomes are multi-vesicular compartments that redirect proteins towards various destinations, such as the lysosomes or vacuoles for degradation, the trans-Golgi network for retrograde transport and the plasma membrane for recycling. In contrast, cross-talk between the endocytic and secretory pathways has not been clearly established in plants, especially in terms of cargo protein trafficking. Here we show by co-localization analyses that endosomes labelled with the AtSORTING NEXIN1 (AtSNX1) protein overlap with the pre-vacuolar compartment in Arabidopsis root cells. In addition, alteration of the routing functions of AtSNX1 endosomes by drug treatments leads to mis-routing of endocytic and secretory cargo proteins. Based on these results, we propose that the AtSNX1 endosomal compartment represents a sorting endosome in root cells, and that this specialized organelle is conserved throughout eukaryotes.

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Section: Introductionmentioning

confidence: 80%

Section: Position Of the Pvc/mvb In The Endosomal Pathway In Arabidopmentioning

confidence: 99%

Evidence for a sorting endosome in Arabidopsis root cells

et al. 2007

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“…21), small GTP binding protein Rha1 (ref. 22) and actin filaments 23 have been studied in vacuolar trafficking. The protoplast isolation method and fluorescence-assisted cell sorting technique have been further applied to reveal genome-wide cell type-specific transcript profiles in Arabidopsis roots 24 .…”

Section: Applicationsmentioning

confidence: 99%

Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis

2007

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The transient gene expression system using Arabidopsis mesophyll protoplasts has proven an important and versatile tool for conducting cell-based experiments using molecular, cellular, biochemical, genetic, genomic and proteomic approaches to analyze the functions of diverse signaling pathways and cellular machineries. A well-established protocol that has been extensively tested and applied in numerous experiments is presented here. The method includes protoplast isolation, PEG-calcium transfection of plasmid DNA and protoplast culture. Physiological responses and high-throughput capability enable facile and cost-effective explorations as well as hypothesis-driven tests. The protoplast isolation and DNA transfection procedures take 6-8 h, and the results can be obtained in 2-24 h. The cell system offers reliable guidelines for further comprehensive analysis of complex regulatory mechanisms in whole-plant physiology, immunity, growth and development. INTRODUCTIONPlant protoplasts without cell walls offer a versatile cell-based experimental system. Macromolecules such as DNA, RNA and proteins can be delivered into protoplasts using various methods, e.g., PEG-calcium fusion, electroporation and microinjection. Signal transduction and metabolic pathways as well as transcription and translation machineries can be transiently manipulated to investigate cell-autonomous regulation and responses. The method is especially powerful in functional genomics analysis when combined with rich genetic resources, such as insertion libraries, available in the model plant Arabidopsis thaliana. We describe here a protocol for a system of transient expression in Arabidopsis mesophyll protoplast (TEAMP) and provide troubleshooting advice and detailed information on useful reporter constructs. Applications, advantages and limitations of the TEAMP assay are also discussed. More general information about plant protoplasts can be found in a review 1 and a live performance of the TEAMP procedure can be seen at the Sheen lab website (http://genetics. mgh.harvard.edu/sheenweb/protocols_reg.html).

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“…On the basis of uptake studies using electron-dense tracers, such MVBs have long been recognized as lying on the endocytic pathway in plant cells (Hillmer et al, 1986(Hillmer et al, , 1988Tanchak and Fowke, 1987;Galway et al, 1993). Moreover, recent FM4-64 uptake studies have confirmed their dual role in endocytosis and vacuolar protein transport by showing that the internalized dye reaches a VSR-enriched compartment (Sohn et al, 2003;Tse et al, 2004).…”

Section: Introductionmentioning

confidence: 96%

“…Recent investigations in tobacco (Nicotiana tabacum) BY-2 cells and Arabidopsis protoplasts have shown that PVCs are enriched in VSR proteins and are also characterized by the presence of Rha1, a Rab5 homolog, the t-SNARE Pep12p, and plant retromer homologs (Li et al, 2002;Sohn et al, 2003;Tse et al, 2004;Oliviusson et al, 2006). These organelles have a typical multivesiculate morphology (Tse et al, 2004;Mo et al, 2006;Oliviusson et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

Rice SCAMP1 Defines Clathrin-Coated,trans-Golgi–Located Tubular-Vesicular Structures as an Early Endosome in Tobacco BY-2 Cells

et al. 2007

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We recently identified multivesicular bodies (MVBs) as prevacuolar compartments (PVCs) in the secretory and endocytic pathways to the lytic vacuole in tobacco (Nicotiana tabacum) BY-2 cells. Secretory carrier membrane proteins (SCAMPs) are post-Golgi, integral membrane proteins mediating endocytosis in animal cells. To define the endocytic pathway in plants, we cloned the rice (Oryza sativa) homolog of animal SCAMP1 and generated transgenic tobacco BY-2 cells expressing yellow fluorescent protein (YFP)-SCAMP1 or SCAMP1-YFP fusions. Confocal immunofluorescence and immunogold electron microscopy studies demonstrated that YFP-SCAMP1 fusions and native SCAMP1 localize to the plasma membrane and mobile structures in the cytoplasm of transgenic BY-2 cells. Drug treatments and confocal immunofluorescence studies demonstrated that the punctate cytosolic organelles labeled by YFP-SCAMP1 or SCAMP1 were distinct from the Golgi apparatus and PVCs. SCAMP1-labeled organelles may represent an early endosome because the internalized endocytic markers FM4-64 and AM4-64 reached these organelles before PVCs. In addition, wortmannin caused the redistribution of SCAMP1 from the early endosomes to PVCs, probably as a result of fusions between the two compartments. Immunogold electron microscopy with high-pressure frozen/freeze-substituted samples identified the SCAMP1-positive organelles as tubular-vesicular structures at the trans-Golgi with clathrin coats. These early endosomal compartments resemble the previously described partially coated reticulum and trans-Golgi network in plant cells.

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Rha1, an Arabidopsis Rab5 Homolog, Plays a Critical Role in the Vacuolar Trafficking of Soluble Cargo Proteins

Cited by 188 publications

References 71 publications

Evidence for a sorting endosome in Arabidopsis root cells

Evidence for a sorting endosome in Arabidopsis root cells

Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis

Rice SCAMP1 Defines Clathrin-Coated,trans-Golgi–Located Tubular-Vesicular Structures as an Early Endosome in Tobacco BY-2 Cells

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