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

Abstract

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.

Evidence for a sorting endosome in Arabidopsis root cells

“…This is deduced from the lack of co-localization between AtSNX1-mRFP or YFP-BP80 and specific markers of GNOM endosomes and the TGN, and by the insensitivity of these organelles to Wm. Interestingly, in contrast to our observations, Lam et al (2007) recently reported that, in tobacco BY-2 cells, the PVC/MVB is not found in BFA compartments, although it is sensitive to Wm, and that the TGN fuses to the PVC/MVB after Wm treatment. This discrepancy between our data and those of Lam et al underlines the variability that endomembrane systems may display depending on cell types.…”

“…Recently, the TGN was reported to be the first compartment labelled with FM4-64 in Arabidopsis root cells (Dettmer et al, 2006). This was confirmed in cultured tobacco BY-2 cells (Lam et al, 2007). Whether FM4-64 transits first through AtSNX1 endosomes to then reach the TGN or directly accumulates in the TGN without trafficking via endosomal structures remains an unsolved question.…”

“…In BY-2 cells, Wm was previously reported to lead to vacuolation of YFP-BP80-bearing organelles (Tse et al, 2004), and a similar effect was recently described for the TGN (Lam et al, 2007). To investigate which organelles in Arabidopsis root cells are similarly sensitive to Wm, we treated a collection of Arabidopsis transgenic lines expressing fluorescent-tagged organelle markers with Wm (see Supplementary Table S1).…”

“…These observations indicate that GFP-Lti6b traffics only partially through the Wm-sensitive AtSNX1 endosomes, and that GFP-Lti6b uses other endocytic pathways that are Wminsensitive, such as those involving GNOM endosomes (Jaillais et al, 2006) or the TGN. Indeed, the TGN has recently been proposed to be an early endosomal compartment, as it is the first compartment to be labelled with FM4-64 both in Arabidopsis root cells (Dettmer et al, 2006) and cultured tobacco BY-2 cells (Lam et al, 2007). HIGH BORON REQUIRING 1 (BOR1) is a boron transporter that is localized at the PM in the absence of boron, but is rapidly routed to vacuoles for degradation in the presence of boron (Takano et al, 2002(Takano et al, , 2005 (Figure 6g,i, and see also Supplementary Figure S7).…”

Isolation and proteomic analysis of the SYP61 compartment reveal its role in exocytic trafficking in Arabidopsis

“…YFP fusions of plant SCAMPs have been localized to many endomembrane compartments, including EE, PM, secretory vesicles and the cell plate [58,59]. Moreover, ES1 treatment showed that trafficking of SCAMP1 in Arabidopsis is TGN dependent (Figure 4F), validating its colocalization with SYP61 in TGN vesicles.…”