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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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Localization of AtRMR1 deletion mutants. (A) Expression of AtRMR1 deletion mutants. Protein extracts were obtained from protoplasts transformed with the indicated constructs and subjected to Western blot analysis using anti-HA and anti-AtRMR1 antibodies. Note that anti-AtRMR1 antibody does not recognize AtRMR1ΔCT. (B) Localization of AtRMR1 deletion mutants. Protoplasts transformed with the indicated constructs were fixed and stained with anti-HA or anti-myc antibodies. The green fluorescent signal of ST-GFP was directly observed from fixed protoplasts. Arrowheads indicate overlaps between the indicated proteins. Bars, 20 μm. (C) Quantification of the overlaps between AtRMR1 deletion mutants and ST-GFP or DIP-myc. The number of punctate stains of AtRMR1 deletion mutants and ST-GFP or DIP-myc that overlapped were counted to determine localization of AtRMR1 deletion mutants. More than 200 punctate stains of AtRMR1 deletion mutants were counted for each comparison in a triplicate experiment. Error bars represent SEM. WT, wild-type AtRMR1; ΔLU, AtRMR1ΔLU-HA; ΔCT, AtRMR1ΔCT-HA.
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fig3: Localization of AtRMR1 deletion mutants. (A) Expression of AtRMR1 deletion mutants. Protein extracts were obtained from protoplasts transformed with the indicated constructs and subjected to Western blot analysis using anti-HA and anti-AtRMR1 antibodies. Note that anti-AtRMR1 antibody does not recognize AtRMR1ΔCT. (B) Localization of AtRMR1 deletion mutants. Protoplasts transformed with the indicated constructs were fixed and stained with anti-HA or anti-myc antibodies. The green fluorescent signal of ST-GFP was directly observed from fixed protoplasts. Arrowheads indicate overlaps between the indicated proteins. Bars, 20 μm. (C) Quantification of the overlaps between AtRMR1 deletion mutants and ST-GFP or DIP-myc. The number of punctate stains of AtRMR1 deletion mutants and ST-GFP or DIP-myc that overlapped were counted to determine localization of AtRMR1 deletion mutants. More than 200 punctate stains of AtRMR1 deletion mutants were counted for each comparison in a triplicate experiment. Error bars represent SEM. WT, wild-type AtRMR1; ΔLU, AtRMR1ΔLU-HA; ΔCT, AtRMR1ΔCT-HA.

Mentions: A previous study suggested that RMR may play a role in protein trafficking to the PSV (Jiang et al., 2000). To investigate this possibility, we examined whether AtRMR1 deletion mutants could inhibit protein trafficking to the PSV through a dominant negative effect. AtRMR1 consists of various domains: the signal peptide (aa 1–27), the lumenal domain (LU; aa 47–149), the transmembrane region (aa 170–190), and the cytosolic ring H2 finger domain (cytosolic tail [CT], aa 222–273; Fig. S6, available at http://www.jcb.org/cgi/content/full/jcb.200504112/DC1). LU may interact with cargo molecules, whereas CT may interact with a cytoplasmic component that is involved in trafficking. We transformed A. thaliana leaf protoplasts with two HA-tagged AtRMR1 deletion mutant constructs, AtRMR1ΔLU-HA and AtRMR1ΔCT-HA, as well as with the corresponding wild-type construct (Fig. S6) and examined their expression by Western blot analysis using anti-HA antibody. AtRMR1ΔLU-HA and AtRMR1ΔCT-HA were detected at the positions of 22 and 30 kD (Fig. 3 A, a), respectively. The expression levels of these transiently expressed wild-type and deletion mutants of AtRMR1-HA were nearly the same as those of endogenous AtRMR1 (Fig. 3 A, b). The polyethylene glycol–mediated transformation efficiency of protoplasts was 30–50% (not depicted; Jin et al., 2001). Thus, the level of transiently expressed AtRMR1 is two- to threefold higher than that of endogenous AtRMR1 in transformed protoplasts. Protoplasts were then transformed with these mutants together with DIP-myc or ST-GFP and were immunostained using anti-HA and anti-myc antibodies. Like wild-type AtRMR1-HA, AtRMR1ΔLU-HA gave a punctate staining pattern, but the majority (87%) of the punctate stains of AtRMR1ΔLU-HA overlapped those of ST-GFP (Fig. 3, B [e–h] and C), and only a minor portion (13%) of AtRMR1ΔLU-HA–positive punctate stains were colocalized with those of DIP-myc (Fig. 3, B [a–d] and C). Furthermore, nearly all of the AtRMR1ΔCT-HA–positive punctate stains colocalized with ST-GFP (Fig. 3, B [m–p] and C). These results suggest that both LU and the cytoplasmic ring H2 region are important for the localization of AtRMR1-HA to the DIP-positive organelle.


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)

Localization of AtRMR1 deletion mutants. (A) Expression of AtRMR1 deletion mutants. Protein extracts were obtained from protoplasts transformed with the indicated constructs and subjected to Western blot analysis using anti-HA and anti-AtRMR1 antibodies. Note that anti-AtRMR1 antibody does not recognize AtRMR1ΔCT. (B) Localization of AtRMR1 deletion mutants. Protoplasts transformed with the indicated constructs were fixed and stained with anti-HA or anti-myc antibodies. The green fluorescent signal of ST-GFP was directly observed from fixed protoplasts. Arrowheads indicate overlaps between the indicated proteins. Bars, 20 μm. (C) Quantification of the overlaps between AtRMR1 deletion mutants and ST-GFP or DIP-myc. The number of punctate stains of AtRMR1 deletion mutants and ST-GFP or DIP-myc that overlapped were counted to determine localization of AtRMR1 deletion mutants. More than 200 punctate stains of AtRMR1 deletion mutants were counted for each comparison in a triplicate experiment. Error bars represent SEM. WT, wild-type AtRMR1; ΔLU, AtRMR1ΔLU-HA; ΔCT, AtRMR1ΔCT-HA.
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fig3: Localization of AtRMR1 deletion mutants. (A) Expression of AtRMR1 deletion mutants. Protein extracts were obtained from protoplasts transformed with the indicated constructs and subjected to Western blot analysis using anti-HA and anti-AtRMR1 antibodies. Note that anti-AtRMR1 antibody does not recognize AtRMR1ΔCT. (B) Localization of AtRMR1 deletion mutants. Protoplasts transformed with the indicated constructs were fixed and stained with anti-HA or anti-myc antibodies. The green fluorescent signal of ST-GFP was directly observed from fixed protoplasts. Arrowheads indicate overlaps between the indicated proteins. Bars, 20 μm. (C) Quantification of the overlaps between AtRMR1 deletion mutants and ST-GFP or DIP-myc. The number of punctate stains of AtRMR1 deletion mutants and ST-GFP or DIP-myc that overlapped were counted to determine localization of AtRMR1 deletion mutants. More than 200 punctate stains of AtRMR1 deletion mutants were counted for each comparison in a triplicate experiment. Error bars represent SEM. WT, wild-type AtRMR1; ΔLU, AtRMR1ΔLU-HA; ΔCT, AtRMR1ΔCT-HA.
Mentions: A previous study suggested that RMR may play a role in protein trafficking to the PSV (Jiang et al., 2000). To investigate this possibility, we examined whether AtRMR1 deletion mutants could inhibit protein trafficking to the PSV through a dominant negative effect. AtRMR1 consists of various domains: the signal peptide (aa 1–27), the lumenal domain (LU; aa 47–149), the transmembrane region (aa 170–190), and the cytosolic ring H2 finger domain (cytosolic tail [CT], aa 222–273; Fig. S6, available at http://www.jcb.org/cgi/content/full/jcb.200504112/DC1). LU may interact with cargo molecules, whereas CT may interact with a cytoplasmic component that is involved in trafficking. We transformed A. thaliana leaf protoplasts with two HA-tagged AtRMR1 deletion mutant constructs, AtRMR1ΔLU-HA and AtRMR1ΔCT-HA, as well as with the corresponding wild-type construct (Fig. S6) and examined their expression by Western blot analysis using anti-HA antibody. AtRMR1ΔLU-HA and AtRMR1ΔCT-HA were detected at the positions of 22 and 30 kD (Fig. 3 A, a), respectively. The expression levels of these transiently expressed wild-type and deletion mutants of AtRMR1-HA were nearly the same as those of endogenous AtRMR1 (Fig. 3 A, b). The polyethylene glycol–mediated transformation efficiency of protoplasts was 30–50% (not depicted; Jin et al., 2001). Thus, the level of transiently expressed AtRMR1 is two- to threefold higher than that of endogenous AtRMR1 in transformed protoplasts. Protoplasts were then transformed with these mutants together with DIP-myc or ST-GFP and were immunostained using anti-HA and anti-myc antibodies. Like wild-type AtRMR1-HA, AtRMR1ΔLU-HA gave a punctate staining pattern, but the majority (87%) of the punctate stains of AtRMR1ΔLU-HA overlapped those of ST-GFP (Fig. 3, B [e–h] and C), and only a minor portion (13%) of AtRMR1ΔLU-HA–positive punctate stains were colocalized with those of DIP-myc (Fig. 3, B [a–d] and C). Furthermore, nearly all of the AtRMR1ΔCT-HA–positive punctate stains colocalized with ST-GFP (Fig. 3, B [m–p] and C). These results suggest that both LU and the cytoplasmic ring H2 region are important for the localization of AtRMR1-HA to the DIP-positive organelle.

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