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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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Colocalization of phaseolin with AtRMR1-HA. Protoplasts were transformed with phaseolin together with AtRMR1-HA, and localization of both of these proteins was examined by immunostaining with antiphaseolin and anti-HA antibodies. To quantify the localization patterns of AtRMR1-HA and phaseolin, the protoplasts were counted based on their immunostaining pattern. More than 100 protoplasts were counted for each protein. Three independent experiments were performed to obtain means and SEM. PS, small punctate staining pattern; PSV, disc pattern. Bars, 20 μm.
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fig7: Colocalization of phaseolin with AtRMR1-HA. Protoplasts were transformed with phaseolin together with AtRMR1-HA, and localization of both of these proteins was examined by immunostaining with antiphaseolin and anti-HA antibodies. To quantify the localization patterns of AtRMR1-HA and phaseolin, the protoplasts were counted based on their immunostaining pattern. More than 100 protoplasts were counted for each protein. Three independent experiments were performed to obtain means and SEM. PS, small punctate staining pattern; PSV, disc pattern. Bars, 20 μm.

Mentions: To confirm the interaction between AtRMR1-HA and phaseolin, we examined whether and where these two proteins colocalize with each other. To address these questions, protoplasts were cotransformed with AtRMR1-HA and phaseolin, and the localization of both of these proteins was examined by immunostaining using anti-HA and antiphaseolin antibodies, respectively. When A. thaliana leaf protoplasts are transformed with phaseolin alone, ∼70% show a disc pattern that indicates targeting to the PSV, whereas the remaining protoplasts show either network or small punctate staining patterns (Park et al., 2004). When phaseolin and AtRMR1-HA were coexpressed, we found that phaseolin was again distributed in a disc pattern in ∼70% of the protoplasts (Fig. 7), whereas it was located in the small punctate staining pattern in the remaining 30% of protoplasts. AtRMR1-HA showed a disc pattern to which phaseolin colocalized in the remaining 5% of protoplasts, whereas AtRMR1-HA was in a small punctate staining pattern in 95% of transformed protoplasts (Fig. 7, e–g). Thus, 5% of transformed protoplasts had AtRMR1-HA at the PSV, which is unlike the situation when AtRMR1-HA was expressed on its own. This indicates that coexpressing phaseolin may induce the localization of AtRMR1-HA at the PSV. In addition, in 30% of coexpressing protoplasts, both phaseolin and AtRMR1-HA gave punctate staining patterns that closely overlapped (Fig. 7, i–k), which indicates that phaseolin may localize at the PVC for the PSV. These results strongly suggest that phaseolin traffics through the AtRMR1-positive compartment on its way to the PSV. Furthermore, the presence of AtRMR1-HA in the PSV when AtRMR1-HA and phaseolin were coexpressed suggests that AtRMR1-HA may traffic to the PSV together with phaseolin.


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)

Colocalization of phaseolin with AtRMR1-HA. Protoplasts were transformed with phaseolin together with AtRMR1-HA, and localization of both of these proteins was examined by immunostaining with antiphaseolin and anti-HA antibodies. To quantify the localization patterns of AtRMR1-HA and phaseolin, the protoplasts were counted based on their immunostaining pattern. More than 100 protoplasts were counted for each protein. Three independent experiments were performed to obtain means and SEM. PS, small punctate staining pattern; PSV, disc pattern. Bars, 20 μm.
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Related In: Results  -  Collection

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fig7: Colocalization of phaseolin with AtRMR1-HA. Protoplasts were transformed with phaseolin together with AtRMR1-HA, and localization of both of these proteins was examined by immunostaining with antiphaseolin and anti-HA antibodies. To quantify the localization patterns of AtRMR1-HA and phaseolin, the protoplasts were counted based on their immunostaining pattern. More than 100 protoplasts were counted for each protein. Three independent experiments were performed to obtain means and SEM. PS, small punctate staining pattern; PSV, disc pattern. Bars, 20 μm.
Mentions: To confirm the interaction between AtRMR1-HA and phaseolin, we examined whether and where these two proteins colocalize with each other. To address these questions, protoplasts were cotransformed with AtRMR1-HA and phaseolin, and the localization of both of these proteins was examined by immunostaining using anti-HA and antiphaseolin antibodies, respectively. When A. thaliana leaf protoplasts are transformed with phaseolin alone, ∼70% show a disc pattern that indicates targeting to the PSV, whereas the remaining protoplasts show either network or small punctate staining patterns (Park et al., 2004). When phaseolin and AtRMR1-HA were coexpressed, we found that phaseolin was again distributed in a disc pattern in ∼70% of the protoplasts (Fig. 7), whereas it was located in the small punctate staining pattern in the remaining 30% of protoplasts. AtRMR1-HA showed a disc pattern to which phaseolin colocalized in the remaining 5% of protoplasts, whereas AtRMR1-HA was in a small punctate staining pattern in 95% of transformed protoplasts (Fig. 7, e–g). Thus, 5% of transformed protoplasts had AtRMR1-HA at the PSV, which is unlike the situation when AtRMR1-HA was expressed on its own. This indicates that coexpressing phaseolin may induce the localization of AtRMR1-HA at the PSV. In addition, in 30% of coexpressing protoplasts, both phaseolin and AtRMR1-HA gave punctate staining patterns that closely overlapped (Fig. 7, i–k), which indicates that phaseolin may localize at the PVC for the PSV. These results strongly suggest that phaseolin traffics through the AtRMR1-positive compartment on its way to the PSV. Furthermore, the presence of AtRMR1-HA in the PSV when AtRMR1-HA and phaseolin were coexpressed suggests that AtRMR1-HA may traffic to the PSV together with phaseolin.

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