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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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A minor portion of AtRMR1 localizes to the Golgi complex. Protoplasts were transformed with the indicated constructs. ST-GFP was directly observed with green fluorescent signals from the fixed protoplasts. AtRMR1-HA was detected with anti-HA antibody. Endogenous γ-COP and AtPEP12p were detected with anti–γ-COP and anti-AtPEP12p antibodies, respectively. (a and b) Insets show bright field images. Arrowheads indicate overlaps between the indicated proteins. Bars, 20 μm.
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fig2: A minor portion of AtRMR1 localizes to the Golgi complex. Protoplasts were transformed with the indicated constructs. ST-GFP was directly observed with green fluorescent signals from the fixed protoplasts. AtRMR1-HA was detected with anti-HA antibody. Endogenous γ-COP and AtPEP12p were detected with anti–γ-COP and anti-AtPEP12p antibodies, respectively. (a and b) Insets show bright field images. Arrowheads indicate overlaps between the indicated proteins. Bars, 20 μm.

Mentions: To further define the organelle to which AtRMR1 localizes, we compared its localization with that of other organellar marker proteins. When protoplasts were cotransformed with AtRMR1-HA and sialyltransferase (ST)-GFP, which is a marker of the Golgi complex (Lee et al., 2002), both proteins gave punctate staining patterns. As expected, the majority of AtRMR1-HA did not colocalize with ST-GFP. However, a small fraction of the punctate stains consisted of overlapping AtRMR1-HA and ST-GFP signals (Fig. 2, c–f). To confirm this, we examined the colocalization of AtRMR1-HA with endogenous γ–coat protein complex (COP), which is a component of the COPI vesicle that localizes to the Golgi complex (Fig. 2, g–j; Pimpl et al., 2000). Again, a minor portion of AtRMR1-HA–positive punctate stains overlapped with γ-COP stains. To estimate the relative distribution of AtRMR1-HA between these two different organelles, we counted the punctate stains of AtRMR1-HA that overlapped with those of DIP-myc, ST-GFP, or γ-COP. AtRMR1-HA stains overlapped with these proteins 77, 23, and 24% of the time, respectively (Fig. S5, available at http://www.jcb.org/cgi/content/full/jcb.200504112/DC1). This shows that AtRMR1-HA in A. thaliana leaf protoplasts mainly localizes to the DIP-positive organelle but is also found, at lower levels, in the Golgi complex.


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)

A minor portion of AtRMR1 localizes to the Golgi complex. Protoplasts were transformed with the indicated constructs. ST-GFP was directly observed with green fluorescent signals from the fixed protoplasts. AtRMR1-HA was detected with anti-HA antibody. Endogenous γ-COP and AtPEP12p were detected with anti–γ-COP and anti-AtPEP12p antibodies, respectively. (a and b) Insets show bright field images. Arrowheads indicate overlaps between the indicated proteins. Bars, 20 μm.
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Related In: Results  -  Collection

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fig2: A minor portion of AtRMR1 localizes to the Golgi complex. Protoplasts were transformed with the indicated constructs. ST-GFP was directly observed with green fluorescent signals from the fixed protoplasts. AtRMR1-HA was detected with anti-HA antibody. Endogenous γ-COP and AtPEP12p were detected with anti–γ-COP and anti-AtPEP12p antibodies, respectively. (a and b) Insets show bright field images. Arrowheads indicate overlaps between the indicated proteins. Bars, 20 μm.
Mentions: To further define the organelle to which AtRMR1 localizes, we compared its localization with that of other organellar marker proteins. When protoplasts were cotransformed with AtRMR1-HA and sialyltransferase (ST)-GFP, which is a marker of the Golgi complex (Lee et al., 2002), both proteins gave punctate staining patterns. As expected, the majority of AtRMR1-HA did not colocalize with ST-GFP. However, a small fraction of the punctate stains consisted of overlapping AtRMR1-HA and ST-GFP signals (Fig. 2, c–f). To confirm this, we examined the colocalization of AtRMR1-HA with endogenous γ–coat protein complex (COP), which is a component of the COPI vesicle that localizes to the Golgi complex (Fig. 2, g–j; Pimpl et al., 2000). Again, a minor portion of AtRMR1-HA–positive punctate stains overlapped with γ-COP stains. To estimate the relative distribution of AtRMR1-HA between these two different organelles, we counted the punctate stains of AtRMR1-HA that overlapped with those of DIP-myc, ST-GFP, or γ-COP. AtRMR1-HA stains overlapped with these proteins 77, 23, and 24% of the time, respectively (Fig. S5, available at http://www.jcb.org/cgi/content/full/jcb.200504112/DC1). This shows that AtRMR1-HA in A. thaliana leaf protoplasts mainly localizes to the DIP-positive organelle but is also found, at lower levels, in the Golgi complex.

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