Limits...
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.

Show MeSH

Related in: MedlinePlus

AtRMR1-HA interacts with phaseolin. (A and B) Coimmunoprecipitation of phaseolin with AtRMR1-HA in the presence of 1 mM Ca2+ (A) or 10 mM EDTA (B). Protein extracts were obtained from protoplasts transformed with phaseolin alone or together with AtRMR1-HA and used for immunoprecipitation using anti-HA antibody. Immunoprecipitation was performed at different pH conditions in the presence of 1 mM Ca2+ or 10 mM EDTA. The pellet fractions were then subjected to immunoblot analysis (IB) with anti-HA, antiphaseolin, anti-BiP, or antialeurain antibodies (note that aleurain is the precursor form). (C) Immunoprecipitation of phaseolin with deletion mutants. Phaseolin was expressed in protoplasts together with the indicated AtRMR1 constructs. Protein extracts were prepared from the transformed protoplasts and used for coimmunoprecipitation using anti-HA antibody. The pellet fractions were then subjected to immunoblot analysis (IB) using anti-HA and antiphaseolin antibodies. Tot, total protein extracts; IP, immunoprecipitated fraction; R6, an empty vector; WT, wild-type AtRMR1; ΔLU, AtRMR1ΔLU-HA; ΔCT, AtRMR1ΔCT-HA.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171354&req=5

fig6: AtRMR1-HA interacts with phaseolin. (A and B) Coimmunoprecipitation of phaseolin with AtRMR1-HA in the presence of 1 mM Ca2+ (A) or 10 mM EDTA (B). Protein extracts were obtained from protoplasts transformed with phaseolin alone or together with AtRMR1-HA and used for immunoprecipitation using anti-HA antibody. Immunoprecipitation was performed at different pH conditions in the presence of 1 mM Ca2+ or 10 mM EDTA. The pellet fractions were then subjected to immunoblot analysis (IB) with anti-HA, antiphaseolin, anti-BiP, or antialeurain antibodies (note that aleurain is the precursor form). (C) Immunoprecipitation of phaseolin with deletion mutants. Phaseolin was expressed in protoplasts together with the indicated AtRMR1 constructs. Protein extracts were prepared from the transformed protoplasts and used for coimmunoprecipitation using anti-HA antibody. The pellet fractions were then subjected to immunoblot analysis (IB) using anti-HA and antiphaseolin antibodies. Tot, total protein extracts; IP, immunoprecipitated fraction; R6, an empty vector; WT, wild-type AtRMR1; ΔLU, AtRMR1ΔLU-HA; ΔCT, AtRMR1ΔCT-HA.

Mentions: To obtain more direct evidence that AtRMR1 plays a role in the trafficking of phaseolin to the PSV, we examined whether AtRMR1 interacts with phaseolin in the cell. To do this, we used a coimmunoprecipitation approach. Coimmunoprecipitation was performed in various pH conditions. It is well known that compartments in the endomembrane system are acidic (Taiz, 1992; Sun-Wada et al., 2003), whereas the PSV has neutral or near neutral pH (Swanson et al., 1998). In addition, we examined the effect of Ca2+ on the interaction because, in cases of vacuolar sorting receptors PV72 and AtVSR1, Ca2+ is critical for interaction with their cargo 2S proalbumin (Shimada et al., 2002; 2003). A. thaliana protoplasts were transformed with phaseolin with or without AtRMR1-HA, and protein extracts were prepared. AtRMR1-HA was first immunoprecipitated with anti-HA antibody in the presence of 1 mM Ca2+ at various pH conditions. The pellet fraction was then probed with anti-HA, antiphaseolin, antialeurain, and antibinding protein (BiP) antibodies. Antialeurain and anti-BiP antibodies (Jiang and Rogers, 1998; Lee et al., 2002) were used as controls for nonspecific interactions. AtRMR1-HA was detected in the pellet fractions that were obtained with anti-HA antibody at various pH conditions (Fig. 6 A). Phaseolin was detected in the anti-HA antibody immunoprecipitates that were obtained at pH 4.0 and 6.0 but not in the precipitates that were obtained at pH 7.0. Thus, AtRMR1-HA interacts with phaseolin in protoplasts in acidic conditions. At these conditions, BiP that was present in the ER was not precipitated together with AtRMR1-HA. Furthermore, AtRMR1-HA was not coimmunoprecipitated with aleurain, which is a vacuolar protein that is known to traffic through the Golgi complex to the vacuole (Ahmed et al., 2000). This observation is consistent with results showing that AtRMR1 deletion mutants do not inhibit trafficking of AALP-GFP to the lytic vacuole. Altogether, the results suggest that the interaction between AtRMR1-HA and phaseolin is specific. Next, we performed coimmunoprecipitation experiments in the presence of 10 mM EDTA. The presence of EDTA did not alter the acid-dependent association of phaseolin with AtRMR1-HA (Fig. 6 B). Thus, AtRMR1-HA interacts with phaseolin in vivo in a Ca2+-independent manner. In addition, phaseolin was coimmunoprecipitated with AtRMR1ΔCT-HA but not with AtRMR1ΔLU-HA by anti-HA antibody, which indicates that the LU of AtRMR1 mediates the interaction between AtRMR1 and phaseolin (Fig. 6 C).


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-HA interacts with phaseolin. (A and B) Coimmunoprecipitation of phaseolin with AtRMR1-HA in the presence of 1 mM Ca2+ (A) or 10 mM EDTA (B). Protein extracts were obtained from protoplasts transformed with phaseolin alone or together with AtRMR1-HA and used for immunoprecipitation using anti-HA antibody. Immunoprecipitation was performed at different pH conditions in the presence of 1 mM Ca2+ or 10 mM EDTA. The pellet fractions were then subjected to immunoblot analysis (IB) with anti-HA, antiphaseolin, anti-BiP, or antialeurain antibodies (note that aleurain is the precursor form). (C) Immunoprecipitation of phaseolin with deletion mutants. Phaseolin was expressed in protoplasts together with the indicated AtRMR1 constructs. Protein extracts were prepared from the transformed protoplasts and used for coimmunoprecipitation using anti-HA antibody. The pellet fractions were then subjected to immunoblot analysis (IB) using anti-HA and antiphaseolin antibodies. Tot, total protein extracts; IP, immunoprecipitated fraction; R6, an empty vector; WT, wild-type AtRMR1; ΔLU, AtRMR1ΔLU-HA; ΔCT, AtRMR1ΔCT-HA.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: AtRMR1-HA interacts with phaseolin. (A and B) Coimmunoprecipitation of phaseolin with AtRMR1-HA in the presence of 1 mM Ca2+ (A) or 10 mM EDTA (B). Protein extracts were obtained from protoplasts transformed with phaseolin alone or together with AtRMR1-HA and used for immunoprecipitation using anti-HA antibody. Immunoprecipitation was performed at different pH conditions in the presence of 1 mM Ca2+ or 10 mM EDTA. The pellet fractions were then subjected to immunoblot analysis (IB) with anti-HA, antiphaseolin, anti-BiP, or antialeurain antibodies (note that aleurain is the precursor form). (C) Immunoprecipitation of phaseolin with deletion mutants. Phaseolin was expressed in protoplasts together with the indicated AtRMR1 constructs. Protein extracts were prepared from the transformed protoplasts and used for coimmunoprecipitation using anti-HA antibody. The pellet fractions were then subjected to immunoblot analysis (IB) using anti-HA and antiphaseolin antibodies. Tot, total protein extracts; IP, immunoprecipitated fraction; R6, an empty vector; WT, wild-type AtRMR1; ΔLU, AtRMR1ΔLU-HA; ΔCT, AtRMR1ΔCT-HA.
Mentions: To obtain more direct evidence that AtRMR1 plays a role in the trafficking of phaseolin to the PSV, we examined whether AtRMR1 interacts with phaseolin in the cell. To do this, we used a coimmunoprecipitation approach. Coimmunoprecipitation was performed in various pH conditions. It is well known that compartments in the endomembrane system are acidic (Taiz, 1992; Sun-Wada et al., 2003), whereas the PSV has neutral or near neutral pH (Swanson et al., 1998). In addition, we examined the effect of Ca2+ on the interaction because, in cases of vacuolar sorting receptors PV72 and AtVSR1, Ca2+ is critical for interaction with their cargo 2S proalbumin (Shimada et al., 2002; 2003). A. thaliana protoplasts were transformed with phaseolin with or without AtRMR1-HA, and protein extracts were prepared. AtRMR1-HA was first immunoprecipitated with anti-HA antibody in the presence of 1 mM Ca2+ at various pH conditions. The pellet fraction was then probed with anti-HA, antiphaseolin, antialeurain, and antibinding protein (BiP) antibodies. Antialeurain and anti-BiP antibodies (Jiang and Rogers, 1998; Lee et al., 2002) were used as controls for nonspecific interactions. AtRMR1-HA was detected in the pellet fractions that were obtained with anti-HA antibody at various pH conditions (Fig. 6 A). Phaseolin was detected in the anti-HA antibody immunoprecipitates that were obtained at pH 4.0 and 6.0 but not in the precipitates that were obtained at pH 7.0. Thus, AtRMR1-HA interacts with phaseolin in protoplasts in acidic conditions. At these conditions, BiP that was present in the ER was not precipitated together with AtRMR1-HA. Furthermore, AtRMR1-HA was not coimmunoprecipitated with aleurain, which is a vacuolar protein that is known to traffic through the Golgi complex to the vacuole (Ahmed et al., 2000). This observation is consistent with results showing that AtRMR1 deletion mutants do not inhibit trafficking of AALP-GFP to the lytic vacuole. Altogether, the results suggest that the interaction between AtRMR1-HA and phaseolin is specific. Next, we performed coimmunoprecipitation experiments in the presence of 10 mM EDTA. The presence of EDTA did not alter the acid-dependent association of phaseolin with AtRMR1-HA (Fig. 6 B). Thus, AtRMR1-HA interacts with phaseolin in vivo in a Ca2+-independent manner. In addition, phaseolin was coimmunoprecipitated with AtRMR1ΔCT-HA but not with AtRMR1ΔLU-HA by anti-HA antibody, which indicates that the LU of AtRMR1 mediates the interaction between AtRMR1 and phaseolin (Fig. 6 C).

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