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AtRMR1 functions as a cargo receptor for protein trafficking to the protein storage vacuole.

Park M, Lee D, Lee GJ, Hwang I - J. Cell Biol. (2005)

Bottom Line: The coexpression of AtRMR1 mutants that were localized to the Golgi complex strongly inhibited the trafficking of phaseolin to the PSV and caused accumulation of phaseolin in the Golgi complex or its secretion.Furthermore, phaseolin colocalized with AtRMR1 on its way to the PSV.Based on these results, we propose that AtRMR1 functions as the sorting receptor of phaseolin for its trafficking to the PSV.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular and Life Sciences, Center for Plant Intracellular Trafficking, Pohang University of Science and Technology, Pohang 790-784, Korea.

ABSTRACT
Organellar proteins are sorted by cargo receptors on the way to their final destination. However, receptors for proteins that are destined for the protein storage vacuole (PSV) are largely unknown. In this study, we investigated the biological role that Arabidopsis thaliana receptor homology region transmembrane domain ring H2 motif protein (AtRMR) 1 plays in protein trafficking to the PSV. AtRMR1 mainly colocalized to the prevacuolar compartment of the PSV, but a minor portion also localized to the Golgi complex. The coexpression of AtRMR1 mutants that were localized to the Golgi complex strongly inhibited the trafficking of phaseolin to the PSV and caused accumulation of phaseolin in the Golgi complex or its secretion. Co-immunoprecipitation and in vitro binding assays revealed that the lumenal domain of AtRMR1 interacts with the COOH-terminal sorting signal of phaseolin at acidic pH. Furthermore, phaseolin colocalized with AtRMR1 on its way to the PSV. Based on these results, we propose that AtRMR1 functions as the sorting receptor of phaseolin for its trafficking to the PSV.

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AtRMR1 localizes primarily to the DIP-positive organelle. (A) Localization of AtRMR1 and AtRMR1-HA. Untransformed (Non) or AtRMR1-HA–transformed (AtRMR1-HA) protoplasts were fixed and stained with anti-AtRMR1 or anti-HA antibodies, respectively. As a control, preimmune serum was used to stain untransformed protoplasts. Insets, bright field images. (B) Colocalization of AtRMR1 and AtRMR1-HA with DIP-myc. Protoplasts transformed with the indicated constructs were stained with anti-myc, anti-HA, or anti-AtRMR1 antibodies. AtRMR1, endogenous AtRMR1 detected with anti-AtRMR1 antibody; Non, untransformed protoplasts. Bars, 20 μm.
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fig1: AtRMR1 localizes primarily to the DIP-positive organelle. (A) Localization of AtRMR1 and AtRMR1-HA. Untransformed (Non) or AtRMR1-HA–transformed (AtRMR1-HA) protoplasts were fixed and stained with anti-AtRMR1 or anti-HA antibodies, respectively. As a control, preimmune serum was used to stain untransformed protoplasts. Insets, bright field images. (B) Colocalization of AtRMR1 and AtRMR1-HA with DIP-myc. Protoplasts transformed with the indicated constructs were stained with anti-myc, anti-HA, or anti-AtRMR1 antibodies. AtRMR1, endogenous AtRMR1 detected with anti-AtRMR1 antibody; Non, untransformed protoplasts. Bars, 20 μm.

Mentions: It has been shown previously that the A. thaliana genome encodes multiple isoforms of RMR (Jiang et al., 2000; Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200504112/DC1). When detected with anti-RMR antibodies raised against an AtRMR isoform (the RMR isoform encoded by the cDNA clone JR702; Jiang et al., 2000), RMR yields a punctate staining pattern and colocalizes with DIP, which is an isoform of TIP (Jiang et al., 2000), in root tip cells and mature seed cells. The punctate stains of these proteins were located within the PSV that was marked by δ- and α-TIP. To investigate the biological role played by AtRMR1 (Fig. S1), we examined its expression and localization in leaf cells. RT-PCR analysis using AtRMR1-specific primers revealed that AtRMR1 is expressed in most A. thaliana tissues regardless of the growth stage of the plant (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200504112/DC1). To examine the localization of AtRMR1 in leaf cells, we generated an antibody against the COOH-terminal domain of AtRMR1 (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200504112/DC1). In addition, we generated HA epitope–tagged AtRMR1 (AtRMR1-HA; Fig. S3). We examined AtRMR1 localization in leaf cells by immunohistochemistry using anti-AtRMR1 and anti-HA antibodies. We prepared two different samples—untransformed and AtRMR1-HA–transformed protoplasts—and stained them with anti-AtRMR1 and anti-HA antibodies, respectively (Frigerio et al., 2001; Sohn et al., 2003). Both anti-AtRMR1 and anti-HA antibodies gave punctate staining patterns in untransformed and AtRMR1-HA–transformed protoplasts, respectively (Fig. 1 A). As a control, we stained untransformed protoplasts with control preimmune serum and anti-HA antibody but did not observe any punctate stains (Fig. 1 A, b and d). To identify AtRMR1-positive organelles, we examined whether AtRMR1 colocalizes with DIP. DIP, a homologue of tonoplastic water channel, is expressed in root tip cells and seed cells from tobacco and gives punctate staining patterns. In mature seed and root tip cells, these punctate stains were detected within the PSV. In contrast, in immature seed cells, these punctate stains were observed outside the PSV. However, the function of DIP has not been clearly defined yet. We expressed myc- or HA-tagged tobacco DIP (DIP-myc and DIP-HA) in leaf cell protoplasts (Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200504112/DC1). Both anti-myc and anti-HA antibodies gave the same punctate staining pattern in the transformed protoplasts (Fig. 1 B, a and c). Furthermore, punctate stains that were observed with the anti-myc antibody closely overlapped with those detected with the anti-HA antibody (Fig. 1 B, d–g). Next, we examined colocalization of AtRMR1 and AtRMR1-HA with DIP-myc in protoplasts. A majority of endogenous AtRMR1-positive punctate stains that were detected with anti-AtRMR1 antibody colocalized with the DIP-myc stains (Fig. 1 B, h–k). In addition, a majority of punctate stains of transiently expressed AtRMR1-HA colocalized with those of DIP-myc (Fig. 1 B, l–o). These results indicate that a majority of both endogenous and transiently expressed AtRMR1 colocalizes with DIP-myc in protoplasts. Furthermore, these results imply that transiently expressed AtRMR1-HA behaves in the same way as endogenous AtRMR1.


AtRMR1 functions as a cargo receptor for protein trafficking to the protein storage vacuole.

Park M, Lee D, Lee GJ, Hwang I - J. Cell Biol. (2005)

AtRMR1 localizes primarily to the DIP-positive organelle. (A) Localization of AtRMR1 and AtRMR1-HA. Untransformed (Non) or AtRMR1-HA–transformed (AtRMR1-HA) protoplasts were fixed and stained with anti-AtRMR1 or anti-HA antibodies, respectively. As a control, preimmune serum was used to stain untransformed protoplasts. Insets, bright field images. (B) Colocalization of AtRMR1 and AtRMR1-HA with DIP-myc. Protoplasts transformed with the indicated constructs were stained with anti-myc, anti-HA, or anti-AtRMR1 antibodies. AtRMR1, endogenous AtRMR1 detected with anti-AtRMR1 antibody; Non, untransformed protoplasts. Bars, 20 μm.
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Related In: Results  -  Collection

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fig1: AtRMR1 localizes primarily to the DIP-positive organelle. (A) Localization of AtRMR1 and AtRMR1-HA. Untransformed (Non) or AtRMR1-HA–transformed (AtRMR1-HA) protoplasts were fixed and stained with anti-AtRMR1 or anti-HA antibodies, respectively. As a control, preimmune serum was used to stain untransformed protoplasts. Insets, bright field images. (B) Colocalization of AtRMR1 and AtRMR1-HA with DIP-myc. Protoplasts transformed with the indicated constructs were stained with anti-myc, anti-HA, or anti-AtRMR1 antibodies. AtRMR1, endogenous AtRMR1 detected with anti-AtRMR1 antibody; Non, untransformed protoplasts. Bars, 20 μm.
Mentions: It has been shown previously that the A. thaliana genome encodes multiple isoforms of RMR (Jiang et al., 2000; Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200504112/DC1). When detected with anti-RMR antibodies raised against an AtRMR isoform (the RMR isoform encoded by the cDNA clone JR702; Jiang et al., 2000), RMR yields a punctate staining pattern and colocalizes with DIP, which is an isoform of TIP (Jiang et al., 2000), in root tip cells and mature seed cells. The punctate stains of these proteins were located within the PSV that was marked by δ- and α-TIP. To investigate the biological role played by AtRMR1 (Fig. S1), we examined its expression and localization in leaf cells. RT-PCR analysis using AtRMR1-specific primers revealed that AtRMR1 is expressed in most A. thaliana tissues regardless of the growth stage of the plant (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200504112/DC1). To examine the localization of AtRMR1 in leaf cells, we generated an antibody against the COOH-terminal domain of AtRMR1 (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200504112/DC1). In addition, we generated HA epitope–tagged AtRMR1 (AtRMR1-HA; Fig. S3). We examined AtRMR1 localization in leaf cells by immunohistochemistry using anti-AtRMR1 and anti-HA antibodies. We prepared two different samples—untransformed and AtRMR1-HA–transformed protoplasts—and stained them with anti-AtRMR1 and anti-HA antibodies, respectively (Frigerio et al., 2001; Sohn et al., 2003). Both anti-AtRMR1 and anti-HA antibodies gave punctate staining patterns in untransformed and AtRMR1-HA–transformed protoplasts, respectively (Fig. 1 A). As a control, we stained untransformed protoplasts with control preimmune serum and anti-HA antibody but did not observe any punctate stains (Fig. 1 A, b and d). To identify AtRMR1-positive organelles, we examined whether AtRMR1 colocalizes with DIP. DIP, a homologue of tonoplastic water channel, is expressed in root tip cells and seed cells from tobacco and gives punctate staining patterns. In mature seed and root tip cells, these punctate stains were detected within the PSV. In contrast, in immature seed cells, these punctate stains were observed outside the PSV. However, the function of DIP has not been clearly defined yet. We expressed myc- or HA-tagged tobacco DIP (DIP-myc and DIP-HA) in leaf cell protoplasts (Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200504112/DC1). Both anti-myc and anti-HA antibodies gave the same punctate staining pattern in the transformed protoplasts (Fig. 1 B, a and c). Furthermore, punctate stains that were observed with the anti-myc antibody closely overlapped with those detected with the anti-HA antibody (Fig. 1 B, d–g). Next, we examined colocalization of AtRMR1 and AtRMR1-HA with DIP-myc in protoplasts. A majority of endogenous AtRMR1-positive punctate stains that were detected with anti-AtRMR1 antibody colocalized with the DIP-myc stains (Fig. 1 B, h–k). In addition, a majority of punctate stains of transiently expressed AtRMR1-HA colocalized with those of DIP-myc (Fig. 1 B, l–o). These results indicate that a majority of both endogenous and transiently expressed AtRMR1 colocalizes with DIP-myc in protoplasts. Furthermore, these results imply that transiently expressed AtRMR1-HA behaves in the same way as endogenous AtRMR1.

Bottom Line: The coexpression of AtRMR1 mutants that were localized to the Golgi complex strongly inhibited the trafficking of phaseolin to the PSV and caused accumulation of phaseolin in the Golgi complex or its secretion.Furthermore, phaseolin colocalized with AtRMR1 on its way to the PSV.Based on these results, we propose that AtRMR1 functions as the sorting receptor of phaseolin for its trafficking to the PSV.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular and Life Sciences, Center for Plant Intracellular Trafficking, Pohang University of Science and Technology, Pohang 790-784, Korea.

ABSTRACT
Organellar proteins are sorted by cargo receptors on the way to their final destination. However, receptors for proteins that are destined for the protein storage vacuole (PSV) are largely unknown. In this study, we investigated the biological role that Arabidopsis thaliana receptor homology region transmembrane domain ring H2 motif protein (AtRMR) 1 plays in protein trafficking to the PSV. AtRMR1 mainly colocalized to the prevacuolar compartment of the PSV, but a minor portion also localized to the Golgi complex. The coexpression of AtRMR1 mutants that were localized to the Golgi complex strongly inhibited the trafficking of phaseolin to the PSV and caused accumulation of phaseolin in the Golgi complex or its secretion. Co-immunoprecipitation and in vitro binding assays revealed that the lumenal domain of AtRMR1 interacts with the COOH-terminal sorting signal of phaseolin at acidic pH. Furthermore, phaseolin colocalized with AtRMR1 on its way to the PSV. Based on these results, we propose that AtRMR1 functions as the sorting receptor of phaseolin for its trafficking to the PSV.

Show MeSH
Related in: MedlinePlus