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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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Phaseolin accumulates in the Golgi complex in the presence of deletion mutants. (A) Localization of phaseolin in the presence of deletion mutants. Protoplasts were transformed with the indicated constructs, and localization of phaseolin was examined. Phaseolin was detected from the fixed protoplasts with antiphaseolin antibody, whereas the GFP signals of ST-GFP were directly observed. Arrowheads indicate overlaps between the indicated proteins. Bars, 20 μm. (B) EndoH resistance of the glycan moiety of phaseolin in the presence of AtRMR1 deletion mutants. Protein extracts were obtained from protoplasts transformed with the indicated constructs, treated with endoH, and analyzed by Western blotting using antiphaseolin and anti-HA antibodies that detect phaseolin and HA-tagged AtRMR1 deletion mutants, respectively. Single and double asterisks indicate deglycosylated forms of AtRMR1-HA and AtRMR1ΔCT-HA, respectively. C, control protein extracts; E, endoH-treated protein extracts. Dotted lines indicate that two separate images were brought together to generate a single composite image. (C) Secretion of phaseolin in the presence of coexpressed AtRMR1ΔLU-HA. Phaseolin and invertase-GFP were coexpressed in protoplasts together with the indicated AtRMR1 constructs. Proteins secreted from the protoplasts were prepared from the incubation medium (M). In addition, protein extracts were prepared from the transformed protoplasts (C). Phaseolin, endogenous aleurain, and invertase-GFP were detected using antiphaseolin, antialeurain, and anti-GFP antibodies, respectively. HA-tagged AtRMR1 deletion mutants were detected with anti-HA antibody. R6, an empty vector; WT, wild-type AtRMR1; ΔLU, AtRMR1ΔLU-HA; ΔCT, AtRMR1ΔCT-HA.
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fig5: Phaseolin accumulates in the Golgi complex in the presence of deletion mutants. (A) Localization of phaseolin in the presence of deletion mutants. Protoplasts were transformed with the indicated constructs, and localization of phaseolin was examined. Phaseolin was detected from the fixed protoplasts with antiphaseolin antibody, whereas the GFP signals of ST-GFP were directly observed. Arrowheads indicate overlaps between the indicated proteins. Bars, 20 μm. (B) EndoH resistance of the glycan moiety of phaseolin in the presence of AtRMR1 deletion mutants. Protein extracts were obtained from protoplasts transformed with the indicated constructs, treated with endoH, and analyzed by Western blotting using antiphaseolin and anti-HA antibodies that detect phaseolin and HA-tagged AtRMR1 deletion mutants, respectively. Single and double asterisks indicate deglycosylated forms of AtRMR1-HA and AtRMR1ΔCT-HA, respectively. C, control protein extracts; E, endoH-treated protein extracts. Dotted lines indicate that two separate images were brought together to generate a single composite image. (C) Secretion of phaseolin in the presence of coexpressed AtRMR1ΔLU-HA. Phaseolin and invertase-GFP were coexpressed in protoplasts together with the indicated AtRMR1 constructs. Proteins secreted from the protoplasts were prepared from the incubation medium (M). In addition, protein extracts were prepared from the transformed protoplasts (C). Phaseolin, endogenous aleurain, and invertase-GFP were detected using antiphaseolin, antialeurain, and anti-GFP antibodies, respectively. HA-tagged AtRMR1 deletion mutants were detected with anti-HA antibody. R6, an empty vector; WT, wild-type AtRMR1; ΔLU, AtRMR1ΔLU-HA; ΔCT, AtRMR1ΔCT-HA.

Mentions: Next, to understand how AtRMR1 deletion mutants inhibit trafficking to the PSV, we examined the identity of the organelle in which phaseolin accumulated in the presence of AtRMR1 deletion mutants. One possibility is that phaseolin may accumulate in the Golgi complex because these deletion mutants mainly localize there. Thus, protoplasts were cotransformed with phaseolin, AtRMR1ΔLU-HA, and ST-GFP or phaseolin, AtRMR1ΔCT-HA, and ST-GFP, and the localization of these proteins was examined. In both cases, the punctate stains of phaseolin that were detected with antiphaseolin antibody closely overlapped with the green fluorescent signal of ST-GFP at the Golgi complex (Fig. 5 A, e–l), which suggests that defective AtRMR1 mutants fail to deliver phaseolin to the PSV. Thus, phaseolin accumulates in the Golgi complex in the presence of AtRMR1 deletion mutants. To confirm this notion at the biochemical level, we examined the glycosylation pattern of phaseolin in the presence of deletion mutants. Phaseolin was resistant to endoH digestion in the presence of deletion mutants, which was similar to the presence of AtRMR1-HA (Fig. 5 B). In addition, a portion of phaseolin was subjected to proteolytic processing even in the presence of coexpressed AtRMR1ΔLU or AtRMR1ΔCT. This indicates that phaseolin is transported to the Golgi or post-Golgi compartments even in the presence of deletion mutants. Next, we examined the glycosylation pattern of AtRMR1 proteins. As expected from localization (Fig. 3 B), AtRMR1ΔCT-HA was also resistant to endoH (Fig. 5 B). As a control for endoH treatment, phaseolin was obtained from protoplasts coexpressing AtSar1[H74L], which is known to inhibit COPII-dependent anterograde trafficking (Takeuchi et al., 2000), and it was examined for sensitivity to endoH. It was found to be sensitive to endoH, as indicated by its faster migration in SDS gel (Fig. 5 B, asterisks). These results are consistent with the notion that phaseolin localizes at the Golgi complex in the presence of AtRMR1 deletion mutants.


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)

Phaseolin accumulates in the Golgi complex in the presence of deletion mutants. (A) Localization of phaseolin in the presence of deletion mutants. Protoplasts were transformed with the indicated constructs, and localization of phaseolin was examined. Phaseolin was detected from the fixed protoplasts with antiphaseolin antibody, whereas the GFP signals of ST-GFP were directly observed. Arrowheads indicate overlaps between the indicated proteins. Bars, 20 μm. (B) EndoH resistance of the glycan moiety of phaseolin in the presence of AtRMR1 deletion mutants. Protein extracts were obtained from protoplasts transformed with the indicated constructs, treated with endoH, and analyzed by Western blotting using antiphaseolin and anti-HA antibodies that detect phaseolin and HA-tagged AtRMR1 deletion mutants, respectively. Single and double asterisks indicate deglycosylated forms of AtRMR1-HA and AtRMR1ΔCT-HA, respectively. C, control protein extracts; E, endoH-treated protein extracts. Dotted lines indicate that two separate images were brought together to generate a single composite image. (C) Secretion of phaseolin in the presence of coexpressed AtRMR1ΔLU-HA. Phaseolin and invertase-GFP were coexpressed in protoplasts together with the indicated AtRMR1 constructs. Proteins secreted from the protoplasts were prepared from the incubation medium (M). In addition, protein extracts were prepared from the transformed protoplasts (C). Phaseolin, endogenous aleurain, and invertase-GFP were detected using antiphaseolin, antialeurain, and anti-GFP antibodies, respectively. HA-tagged AtRMR1 deletion mutants were detected with anti-HA antibody. R6, an empty vector; WT, wild-type AtRMR1; ΔLU, AtRMR1ΔLU-HA; ΔCT, AtRMR1ΔCT-HA.
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fig5: Phaseolin accumulates in the Golgi complex in the presence of deletion mutants. (A) Localization of phaseolin in the presence of deletion mutants. Protoplasts were transformed with the indicated constructs, and localization of phaseolin was examined. Phaseolin was detected from the fixed protoplasts with antiphaseolin antibody, whereas the GFP signals of ST-GFP were directly observed. Arrowheads indicate overlaps between the indicated proteins. Bars, 20 μm. (B) EndoH resistance of the glycan moiety of phaseolin in the presence of AtRMR1 deletion mutants. Protein extracts were obtained from protoplasts transformed with the indicated constructs, treated with endoH, and analyzed by Western blotting using antiphaseolin and anti-HA antibodies that detect phaseolin and HA-tagged AtRMR1 deletion mutants, respectively. Single and double asterisks indicate deglycosylated forms of AtRMR1-HA and AtRMR1ΔCT-HA, respectively. C, control protein extracts; E, endoH-treated protein extracts. Dotted lines indicate that two separate images were brought together to generate a single composite image. (C) Secretion of phaseolin in the presence of coexpressed AtRMR1ΔLU-HA. Phaseolin and invertase-GFP were coexpressed in protoplasts together with the indicated AtRMR1 constructs. Proteins secreted from the protoplasts were prepared from the incubation medium (M). In addition, protein extracts were prepared from the transformed protoplasts (C). Phaseolin, endogenous aleurain, and invertase-GFP were detected using antiphaseolin, antialeurain, and anti-GFP antibodies, respectively. HA-tagged AtRMR1 deletion mutants were detected with anti-HA antibody. R6, an empty vector; WT, wild-type AtRMR1; ΔLU, AtRMR1ΔLU-HA; ΔCT, AtRMR1ΔCT-HA.
Mentions: Next, to understand how AtRMR1 deletion mutants inhibit trafficking to the PSV, we examined the identity of the organelle in which phaseolin accumulated in the presence of AtRMR1 deletion mutants. One possibility is that phaseolin may accumulate in the Golgi complex because these deletion mutants mainly localize there. Thus, protoplasts were cotransformed with phaseolin, AtRMR1ΔLU-HA, and ST-GFP or phaseolin, AtRMR1ΔCT-HA, and ST-GFP, and the localization of these proteins was examined. In both cases, the punctate stains of phaseolin that were detected with antiphaseolin antibody closely overlapped with the green fluorescent signal of ST-GFP at the Golgi complex (Fig. 5 A, e–l), which suggests that defective AtRMR1 mutants fail to deliver phaseolin to the PSV. Thus, phaseolin accumulates in the Golgi complex in the presence of AtRMR1 deletion mutants. To confirm this notion at the biochemical level, we examined the glycosylation pattern of phaseolin in the presence of deletion mutants. Phaseolin was resistant to endoH digestion in the presence of deletion mutants, which was similar to the presence of AtRMR1-HA (Fig. 5 B). In addition, a portion of phaseolin was subjected to proteolytic processing even in the presence of coexpressed AtRMR1ΔLU or AtRMR1ΔCT. This indicates that phaseolin is transported to the Golgi or post-Golgi compartments even in the presence of deletion mutants. Next, we examined the glycosylation pattern of AtRMR1 proteins. As expected from localization (Fig. 3 B), AtRMR1ΔCT-HA was also resistant to endoH (Fig. 5 B). As a control for endoH treatment, phaseolin was obtained from protoplasts coexpressing AtSar1[H74L], which is known to inhibit COPII-dependent anterograde trafficking (Takeuchi et al., 2000), and it was examined for sensitivity to endoH. It was found to be sensitive to endoH, as indicated by its faster migration in SDS gel (Fig. 5 B, asterisks). These results are consistent with the notion that phaseolin localizes at the Golgi complex in the presence of AtRMR1 deletion mutants.

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