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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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The CTPP of phaseolin is critical for its interaction with AtRMR1-HA. (A) Lack of interaction between AtRMR1-HA and phaseolinΔ418. Protein extracts (Tot) were obtained from protoplasts transformed with the indicated constructs. Immunoprecipitation (IP) was performed by using anti-HA antibody at pH 6.0 in the presence of 1 mM Ca2+. The pellet fraction was then subjected to immunoblot analysis (IB) with antiphaseolin antibody. (B) COPII-dependent trafficking of phaseolin and phaseolinΔ418. Protoplasts were transformed with the indicated constructs. Protein extracts were prepared from the transformed protoplasts (C) as well as the incubation medium (M) and were analyzed by Western blotting using antiphaseolin antibody. PhaΔ418, phaseolinΔ418; side bar, the processed forms of phaseolin.
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fig8: The CTPP of phaseolin is critical for its interaction with AtRMR1-HA. (A) Lack of interaction between AtRMR1-HA and phaseolinΔ418. Protein extracts (Tot) were obtained from protoplasts transformed with the indicated constructs. Immunoprecipitation (IP) was performed by using anti-HA antibody at pH 6.0 in the presence of 1 mM Ca2+. The pellet fraction was then subjected to immunoblot analysis (IB) with antiphaseolin antibody. (B) COPII-dependent trafficking of phaseolin and phaseolinΔ418. Protoplasts were transformed with the indicated constructs. Protein extracts were prepared from the transformed protoplasts (C) as well as the incubation medium (M) and were analyzed by Western blotting using antiphaseolin antibody. PhaΔ418, phaseolinΔ418; side bar, the processed forms of phaseolin.

Mentions: To further investigate the relationship between the interaction of phaseolin with AtRMR1 and the trafficking of phaseolin to the PSV, we examined whether AtRMR1-HA can interact with phaseolinΔ418, which lacks CTPP as a result of the deletion of four COOH-terminal amino acids. It is not targeted to the PSV but is, instead, secreted into the extracellular space (Frigerio et al., 1998; Park et al., 2004). Thus, protein extracts were prepared from protoplasts that were cotransformed with phaseolinΔ418 and AtRMR1-HA, and AtRMR1-HA was immunoprecipitated by using anti-HA antibody. PhaseolinΔ418 was not detected from these immunoprecipitates (Fig. 8 A). Thus, AtRMR1-HA does not interact with phaseolinΔ418.


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)

The CTPP of phaseolin is critical for its interaction with AtRMR1-HA. (A) Lack of interaction between AtRMR1-HA and phaseolinΔ418. Protein extracts (Tot) were obtained from protoplasts transformed with the indicated constructs. Immunoprecipitation (IP) was performed by using anti-HA antibody at pH 6.0 in the presence of 1 mM Ca2+. The pellet fraction was then subjected to immunoblot analysis (IB) with antiphaseolin antibody. (B) COPII-dependent trafficking of phaseolin and phaseolinΔ418. Protoplasts were transformed with the indicated constructs. Protein extracts were prepared from the transformed protoplasts (C) as well as the incubation medium (M) and were analyzed by Western blotting using antiphaseolin antibody. PhaΔ418, phaseolinΔ418; side bar, the processed forms of phaseolin.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171354&req=5

fig8: The CTPP of phaseolin is critical for its interaction with AtRMR1-HA. (A) Lack of interaction between AtRMR1-HA and phaseolinΔ418. Protein extracts (Tot) were obtained from protoplasts transformed with the indicated constructs. Immunoprecipitation (IP) was performed by using anti-HA antibody at pH 6.0 in the presence of 1 mM Ca2+. The pellet fraction was then subjected to immunoblot analysis (IB) with antiphaseolin antibody. (B) COPII-dependent trafficking of phaseolin and phaseolinΔ418. Protoplasts were transformed with the indicated constructs. Protein extracts were prepared from the transformed protoplasts (C) as well as the incubation medium (M) and were analyzed by Western blotting using antiphaseolin antibody. PhaΔ418, phaseolinΔ418; side bar, the processed forms of phaseolin.
Mentions: To further investigate the relationship between the interaction of phaseolin with AtRMR1 and the trafficking of phaseolin to the PSV, we examined whether AtRMR1-HA can interact with phaseolinΔ418, which lacks CTPP as a result of the deletion of four COOH-terminal amino acids. It is not targeted to the PSV but is, instead, secreted into the extracellular space (Frigerio et al., 1998; Park et al., 2004). Thus, protein extracts were prepared from protoplasts that were cotransformed with phaseolinΔ418 and AtRMR1-HA, and AtRMR1-HA was immunoprecipitated by using anti-HA antibody. PhaseolinΔ418 was not detected from these immunoprecipitates (Fig. 8 A). Thus, AtRMR1-HA does not interact with phaseolinΔ418.

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