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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 LU of AtRMR1 binds to the CTPP peptide of phaseolin. (A) Peptide sequences. (B) In vitro binding assay. The in vitro binding assay was performed as described in Materials and methods. Peptides immobilized on Sepharose beads were incubated with purified GST-LU or GST alone under different pH conditions at 4°C. Sepharose beads were washed three times with binding buffer, and bound proteins were eluted three times with elution buffer. The unbound (Un), wash (Wa), and eluted (El) fractions were analyzed by Western blot analysis using anti-GST antibody. In, input GST-LU.
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fig9: The LU of AtRMR1 binds to the CTPP peptide of phaseolin. (A) Peptide sequences. (B) In vitro binding assay. The in vitro binding assay was performed as described in Materials and methods. Peptides immobilized on Sepharose beads were incubated with purified GST-LU or GST alone under different pH conditions at 4°C. Sepharose beads were washed three times with binding buffer, and bound proteins were eluted three times with elution buffer. The unbound (Un), wash (Wa), and eluted (El) fractions were analyzed by Western blot analysis using anti-GST antibody. In, input GST-LU.

Mentions: To further examine the interaction between AtRMR1 and phaseolin, we synthesized the peptide pCTPP, which consists of 14 amino acid residues from the phaseolin COOH-terminal region (Fig. 9 A). Two control peptides, pCTPP(rev) and pCTPPΔ(AFVY), were synthesized; pCTPP(rev) had identical amino acid residues but had a reverse sequence to that of pCTPP, and pCTPPΔ(AFVY) had a four–amino acid (AFVY) deletion in its COOH terminus. Two additional peptides, pNTPP and pNTPP(rev), were also synthesized. pNTPP consisted of 21 amino acid residues that included the NH2-terminal sorting signal of AALP, and pNTPP(rev) had identical amino acid residues but had a reverse sequence to that of pNTPP. The LU of AtRMR1 was expressed as a GST fusion protein (GST-LU) in Escherichia coli and was used in the binding assay. GST-LU bound to pCTPP at pH 4.0 and 6.0 but not at pH 7.0 (Fig. 9 B, lane El). However, GST-LU did not bind to any of the other peptides — pCTPP(rev), pCTPPΔ(AFVY), pNTPP, or pNTPP(rev) — at any pH. GST alone did not bind to any of these peptides. These results strongly suggest that the LU of AtRMR1 binds specifically to the CTPP of 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)

The LU of AtRMR1 binds to the CTPP peptide of phaseolin. (A) Peptide sequences. (B) In vitro binding assay. The in vitro binding assay was performed as described in Materials and methods. Peptides immobilized on Sepharose beads were incubated with purified GST-LU or GST alone under different pH conditions at 4°C. Sepharose beads were washed three times with binding buffer, and bound proteins were eluted three times with elution buffer. The unbound (Un), wash (Wa), and eluted (El) fractions were analyzed by Western blot analysis using anti-GST antibody. In, input GST-LU.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171354&req=5

fig9: The LU of AtRMR1 binds to the CTPP peptide of phaseolin. (A) Peptide sequences. (B) In vitro binding assay. The in vitro binding assay was performed as described in Materials and methods. Peptides immobilized on Sepharose beads were incubated with purified GST-LU or GST alone under different pH conditions at 4°C. Sepharose beads were washed three times with binding buffer, and bound proteins were eluted three times with elution buffer. The unbound (Un), wash (Wa), and eluted (El) fractions were analyzed by Western blot analysis using anti-GST antibody. In, input GST-LU.
Mentions: To further examine the interaction between AtRMR1 and phaseolin, we synthesized the peptide pCTPP, which consists of 14 amino acid residues from the phaseolin COOH-terminal region (Fig. 9 A). Two control peptides, pCTPP(rev) and pCTPPΔ(AFVY), were synthesized; pCTPP(rev) had identical amino acid residues but had a reverse sequence to that of pCTPP, and pCTPPΔ(AFVY) had a four–amino acid (AFVY) deletion in its COOH terminus. Two additional peptides, pNTPP and pNTPP(rev), were also synthesized. pNTPP consisted of 21 amino acid residues that included the NH2-terminal sorting signal of AALP, and pNTPP(rev) had identical amino acid residues but had a reverse sequence to that of pNTPP. The LU of AtRMR1 was expressed as a GST fusion protein (GST-LU) in Escherichia coli and was used in the binding assay. GST-LU bound to pCTPP at pH 4.0 and 6.0 but not at pH 7.0 (Fig. 9 B, lane El). However, GST-LU did not bind to any of the other peptides — pCTPP(rev), pCTPPΔ(AFVY), pNTPP, or pNTPP(rev) — at any pH. GST alone did not bind to any of these peptides. These results strongly suggest that the LU of AtRMR1 binds specifically to the CTPP of 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