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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 deletion mutants inhibit the trafficking of phaseolin to the PSV. (A) Inhibition of phaseolin trafficking to the PSV by AtRMR1 deletion mutants. Protoplasts were transformed with the indicated constructs, and localization of the reporter proteins was examined. GFP signals of AALP-GFP and sporamin-GFP were observed from intact protoplasts, whereas phaseolin was detected from fixed protoplasts by immunostaining with antiphaseolin antibody. Un, untransformed protoplasts; CH, chloroplasts. Insets, bright field images of protoplasts. Bars, 20 μm. (B) Quantification of trafficking efficiency. To estimate the trafficking efficiency, transformed protoplasts were counted based on their GFP or immunostaining patterns. More than 100 protoplasts were counted for each transformation in a triplicate experiment. The numbers and error bars indicate the means and SEM, respectively. (C) Western blot analysis of vacuolar trafficking of AALP-GFP and sporamin-GFP. Protein extracts were prepared from protoplasts transformed with the indicated constructs and used for Western blot analysis using anti-HA or anti-GFP antibodies. WT, wild-type AtRMR1; ΔLU, AtRMR1ΔLU-HA; ΔCT, AtRMR1ΔCT-HA; R6, an empty vector used to balance the amount of plasmid DNA that was introduced into protoplasts. Single arrowhead, precursor; double arrowhead, proteolytically processed form.
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fig4: AtRMR1 deletion mutants inhibit the trafficking of phaseolin to the PSV. (A) Inhibition of phaseolin trafficking to the PSV by AtRMR1 deletion mutants. Protoplasts were transformed with the indicated constructs, and localization of the reporter proteins was examined. GFP signals of AALP-GFP and sporamin-GFP were observed from intact protoplasts, whereas phaseolin was detected from fixed protoplasts by immunostaining with antiphaseolin antibody. Un, untransformed protoplasts; CH, chloroplasts. Insets, bright field images of protoplasts. Bars, 20 μm. (B) Quantification of trafficking efficiency. To estimate the trafficking efficiency, transformed protoplasts were counted based on their GFP or immunostaining patterns. More than 100 protoplasts were counted for each transformation in a triplicate experiment. The numbers and error bars indicate the means and SEM, respectively. (C) Western blot analysis of vacuolar trafficking of AALP-GFP and sporamin-GFP. Protein extracts were prepared from protoplasts transformed with the indicated constructs and used for Western blot analysis using anti-HA or anti-GFP antibodies. WT, wild-type AtRMR1; ΔLU, AtRMR1ΔLU-HA; ΔCT, AtRMR1ΔCT-HA; R6, an empty vector used to balance the amount of plasmid DNA that was introduced into protoplasts. Single arrowhead, precursor; double arrowhead, proteolytically processed form.

Mentions: Next, we examined the effect of these AtRMR1 deletion mutants on the trafficking of phaseolin, which is a storage protein of the common bean (Frigerio et al., 1998). Phaseolin has been shown to be targeted to the PSV when it is transiently expressed in leaf protoplasts (Park et al., 2004). In A. thaliana leaf protoplasts that express phaseolin, ∼70% of protoplasts show the disc pattern that indicates targeting to the PSV, whereas the remaining 30% of protoplasts show network or punctate staining patterns (Park et al., 2004). Protoplasts were cotransformed with phaseolin together with AtRMR1-HA, AtRMR1ΔLU-HA, or AtRMR1ΔCT-HA, and the localization of phaseolin was examined by immunostaining with antiphaseolin antibody. In the presence of AtRMR1-HA, the majority of protoplasts gave the typical disc pattern, as observed when phaseolin alone is expressed, which indicates that phaseolin continues to be targeted to the PSV in the presence of AtRMR1-HA (Fig. 4 A, a). However, in the presence of both AtRMR1ΔLU-HA and AtRMR1ΔCT-HA, the majority of transformed protoplasts produced a punctate staining pattern (Fig. 4 A, b and c). This indicates that the normal trafficking of phaseolin to the PSV is perturbed by the coexpression of AtRMR1 deletion mutants. To estimate the trafficking efficiency of phaseolin to the PSV, we counted protoplasts bearing the disc pattern. The presence of AtRMR1ΔLU-HA and AtRMR1ΔCT-HA reduced the phaseolin trafficking efficiency to 27 and 45%, respectively, from 70% in the presence of AtRMR1-HA (Fig. 4 B). Thus, AtRMR1 deletion mutants inhibit the trafficking of phaseolin to the PSV.


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 deletion mutants inhibit the trafficking of phaseolin to the PSV. (A) Inhibition of phaseolin trafficking to the PSV by AtRMR1 deletion mutants. Protoplasts were transformed with the indicated constructs, and localization of the reporter proteins was examined. GFP signals of AALP-GFP and sporamin-GFP were observed from intact protoplasts, whereas phaseolin was detected from fixed protoplasts by immunostaining with antiphaseolin antibody. Un, untransformed protoplasts; CH, chloroplasts. Insets, bright field images of protoplasts. Bars, 20 μm. (B) Quantification of trafficking efficiency. To estimate the trafficking efficiency, transformed protoplasts were counted based on their GFP or immunostaining patterns. More than 100 protoplasts were counted for each transformation in a triplicate experiment. The numbers and error bars indicate the means and SEM, respectively. (C) Western blot analysis of vacuolar trafficking of AALP-GFP and sporamin-GFP. Protein extracts were prepared from protoplasts transformed with the indicated constructs and used for Western blot analysis using anti-HA or anti-GFP antibodies. WT, wild-type AtRMR1; ΔLU, AtRMR1ΔLU-HA; ΔCT, AtRMR1ΔCT-HA; R6, an empty vector used to balance the amount of plasmid DNA that was introduced into protoplasts. Single arrowhead, precursor; double arrowhead, proteolytically processed form.
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fig4: AtRMR1 deletion mutants inhibit the trafficking of phaseolin to the PSV. (A) Inhibition of phaseolin trafficking to the PSV by AtRMR1 deletion mutants. Protoplasts were transformed with the indicated constructs, and localization of the reporter proteins was examined. GFP signals of AALP-GFP and sporamin-GFP were observed from intact protoplasts, whereas phaseolin was detected from fixed protoplasts by immunostaining with antiphaseolin antibody. Un, untransformed protoplasts; CH, chloroplasts. Insets, bright field images of protoplasts. Bars, 20 μm. (B) Quantification of trafficking efficiency. To estimate the trafficking efficiency, transformed protoplasts were counted based on their GFP or immunostaining patterns. More than 100 protoplasts were counted for each transformation in a triplicate experiment. The numbers and error bars indicate the means and SEM, respectively. (C) Western blot analysis of vacuolar trafficking of AALP-GFP and sporamin-GFP. Protein extracts were prepared from protoplasts transformed with the indicated constructs and used for Western blot analysis using anti-HA or anti-GFP antibodies. WT, wild-type AtRMR1; ΔLU, AtRMR1ΔLU-HA; ΔCT, AtRMR1ΔCT-HA; R6, an empty vector used to balance the amount of plasmid DNA that was introduced into protoplasts. Single arrowhead, precursor; double arrowhead, proteolytically processed form.
Mentions: Next, we examined the effect of these AtRMR1 deletion mutants on the trafficking of phaseolin, which is a storage protein of the common bean (Frigerio et al., 1998). Phaseolin has been shown to be targeted to the PSV when it is transiently expressed in leaf protoplasts (Park et al., 2004). In A. thaliana leaf protoplasts that express phaseolin, ∼70% of protoplasts show the disc pattern that indicates targeting to the PSV, whereas the remaining 30% of protoplasts show network or punctate staining patterns (Park et al., 2004). Protoplasts were cotransformed with phaseolin together with AtRMR1-HA, AtRMR1ΔLU-HA, or AtRMR1ΔCT-HA, and the localization of phaseolin was examined by immunostaining with antiphaseolin antibody. In the presence of AtRMR1-HA, the majority of protoplasts gave the typical disc pattern, as observed when phaseolin alone is expressed, which indicates that phaseolin continues to be targeted to the PSV in the presence of AtRMR1-HA (Fig. 4 A, a). However, in the presence of both AtRMR1ΔLU-HA and AtRMR1ΔCT-HA, the majority of transformed protoplasts produced a punctate staining pattern (Fig. 4 A, b and c). This indicates that the normal trafficking of phaseolin to the PSV is perturbed by the coexpression of AtRMR1 deletion mutants. To estimate the trafficking efficiency of phaseolin to the PSV, we counted protoplasts bearing the disc pattern. The presence of AtRMR1ΔLU-HA and AtRMR1ΔCT-HA reduced the phaseolin trafficking efficiency to 27 and 45%, respectively, from 70% in the presence of AtRMR1-HA (Fig. 4 B). Thus, AtRMR1 deletion mutants inhibit the trafficking of phaseolin to the PSV.

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