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Rer1p, a retrieval receptor for endoplasmic reticulum membrane proteins, is dynamically localized to the Golgi apparatus by coatomer.

Sato K, Sato M, Nakano A - J. Cell Biol. (2001)

Bottom Line: Either a lesion of coatomer or deletion of the COOH-terminal tail of Rer1p causes its mislocalization to the vacuole.The COOH-terminal Rer1p tail interacts in vitro with a coatomer complex containing alpha and gamma subunits.These findings not only give the proof that Rer1p is a novel type of retrieval receptor recognizing the TMD in the Golgi but also indicate that coatomer actively regulates the function and localization of Rer1p.

View Article: PubMed Central - PubMed

Affiliation: Molecular Membrane Biology Laboratory, RIKEN (The Institute of Physical and Chemical Research), Saitama 351-0198, Japan. satoken@postman.riken.go.jp

ABSTRACT
Rer1p, a yeast Golgi membrane protein, is required for the retrieval of a set of endoplasmic reticulum (ER) membrane proteins. We present the first evidence that Rer1p directly interacts with the transmembrane domain (TMD) of Sec12p which contains a retrieval signal. A green fluorescent protein (GFP) fusion of Rer1p rapidly cycles between the Golgi and the ER. Either a lesion of coatomer or deletion of the COOH-terminal tail of Rer1p causes its mislocalization to the vacuole. The COOH-terminal Rer1p tail interacts in vitro with a coatomer complex containing alpha and gamma subunits. These findings not only give the proof that Rer1p is a novel type of retrieval receptor recognizing the TMD in the Golgi but also indicate that coatomer actively regulates the function and localization of Rer1p.

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Physical interaction between Rer1p and DSD. Amino acid sequences of the junction regions of DSD and its derivatives are shown in A. Δdap2 cells (SMY22-10B) expressing Rer1-3HAp on a multicopy plasmid and Dap2p, DSD, or DSD mutants (NQ-L, SY-L, Leu × 19, and +2L) on another multicopy plasmid under the TDH3 promoter were spheroplasted, lysed with 25 mM sodium phosphate (pH 7.2), and further incubated with 5 mM DSP at 20°C for 20 min. Reactions were terminated by the addition of 50 mM Tris-HCl (pH 8.0), and then membranes were solubilized with 1% Triton X-100. After the adjustment to 35 mM Tris-HCl (pH 8.0), 120 mM NaCl, and 2% SDS, the samples were heated at 75°C for 10 min and processed for immunoprecipitation with the anti-Dap2p (B) and anti-HA (C) antibodies. The immunoprecipitates were treated with 50 mM DTT to cleave DSP and then analyzed by immunoblotting with anti-Dap2 polyclonal antibody (B and C), and anti-HA monoclonal (B) and polyclonal (C) antibodies. SMY22-10B cells expressing Rer1-3HAp and Dap2p or DSD were also subjected to the same procedures (C, lanes 7 and 8) as controls in the absence of DSP.
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Figure 1: Physical interaction between Rer1p and DSD. Amino acid sequences of the junction regions of DSD and its derivatives are shown in A. Δdap2 cells (SMY22-10B) expressing Rer1-3HAp on a multicopy plasmid and Dap2p, DSD, or DSD mutants (NQ-L, SY-L, Leu × 19, and +2L) on another multicopy plasmid under the TDH3 promoter were spheroplasted, lysed with 25 mM sodium phosphate (pH 7.2), and further incubated with 5 mM DSP at 20°C for 20 min. Reactions were terminated by the addition of 50 mM Tris-HCl (pH 8.0), and then membranes were solubilized with 1% Triton X-100. After the adjustment to 35 mM Tris-HCl (pH 8.0), 120 mM NaCl, and 2% SDS, the samples were heated at 75°C for 10 min and processed for immunoprecipitation with the anti-Dap2p (B) and anti-HA (C) antibodies. The immunoprecipitates were treated with 50 mM DTT to cleave DSP and then analyzed by immunoblotting with anti-Dap2 polyclonal antibody (B and C), and anti-HA monoclonal (B) and polyclonal (C) antibodies. SMY22-10B cells expressing Rer1-3HAp and Dap2p or DSD were also subjected to the same procedures (C, lanes 7 and 8) as controls in the absence of DSP.

Mentions: After a long struggle to prove the physical interaction between Rer1p and the TMD of Sec12p, we decided to use a chimeric protein between Sec12p and Dap2p. Dap2p, a type II vacuolar membrane protein, has been used as a passenger protein to determine the ER localization signals of Sec12p (Sato et al. 1996). DSD, a chimeric protein comprised of the lumenal and cytoplasmic domains from Dap2p and the TMD from Sec12p, is almost completely localized to the ER by the Rer1p-dependent retrieval (Fig. 1 A). Chemical cross-linking experiments using a thiol-cleavable linker dithiobis (succinimidyl propionate) (DSP) were performed with the cell lysate prepared from a yeast strain overexpressing both Rer1-3HAp and DSD. Cells expressing Rer1-3HAp and Dap2p were used as a negative control. Cells were lysed and allowed to react with DSP. After the immunoprecipitation with either anti-Dap2p or anti-HA antibody, DSP was cleaved with 50 mM DTT, and the products were subjected to immunoblotting with anti-Dap2p and anti-HA antibodies. As shown in Fig. 1 (B and C), DSD and Rer1-3HAp were reproducibly coimmunoprecipitated by either anti-Dap2p or anti-HA antibody (lane 2). Such coprecipitation was not observed with the Dap2p control (Fig. 1B and Fig. C, lane 1). Mutants of DSD, which have amino acid replacements in the TMD, and thus show less Rer1p dependency (NQ-L, SY-L, +2L, and LeuX19; Fig. 1 A) (Sato et al. 1996; Sato, M., unpublished data), were not efficiently coimmunoprecipitated with Rer1-3HAp by the anti-Dap2p antibody and vice versa (Fig. 1B and Fig. C, lanes 3–6), indicating that Rer1p indeed recognizes the polar residues in the TMD of Sec12p.


Rer1p, a retrieval receptor for endoplasmic reticulum membrane proteins, is dynamically localized to the Golgi apparatus by coatomer.

Sato K, Sato M, Nakano A - J. Cell Biol. (2001)

Physical interaction between Rer1p and DSD. Amino acid sequences of the junction regions of DSD and its derivatives are shown in A. Δdap2 cells (SMY22-10B) expressing Rer1-3HAp on a multicopy plasmid and Dap2p, DSD, or DSD mutants (NQ-L, SY-L, Leu × 19, and +2L) on another multicopy plasmid under the TDH3 promoter were spheroplasted, lysed with 25 mM sodium phosphate (pH 7.2), and further incubated with 5 mM DSP at 20°C for 20 min. Reactions were terminated by the addition of 50 mM Tris-HCl (pH 8.0), and then membranes were solubilized with 1% Triton X-100. After the adjustment to 35 mM Tris-HCl (pH 8.0), 120 mM NaCl, and 2% SDS, the samples were heated at 75°C for 10 min and processed for immunoprecipitation with the anti-Dap2p (B) and anti-HA (C) antibodies. The immunoprecipitates were treated with 50 mM DTT to cleave DSP and then analyzed by immunoblotting with anti-Dap2 polyclonal antibody (B and C), and anti-HA monoclonal (B) and polyclonal (C) antibodies. SMY22-10B cells expressing Rer1-3HAp and Dap2p or DSD were also subjected to the same procedures (C, lanes 7 and 8) as controls in the absence of DSP.
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Related In: Results  -  Collection

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Figure 1: Physical interaction between Rer1p and DSD. Amino acid sequences of the junction regions of DSD and its derivatives are shown in A. Δdap2 cells (SMY22-10B) expressing Rer1-3HAp on a multicopy plasmid and Dap2p, DSD, or DSD mutants (NQ-L, SY-L, Leu × 19, and +2L) on another multicopy plasmid under the TDH3 promoter were spheroplasted, lysed with 25 mM sodium phosphate (pH 7.2), and further incubated with 5 mM DSP at 20°C for 20 min. Reactions were terminated by the addition of 50 mM Tris-HCl (pH 8.0), and then membranes were solubilized with 1% Triton X-100. After the adjustment to 35 mM Tris-HCl (pH 8.0), 120 mM NaCl, and 2% SDS, the samples were heated at 75°C for 10 min and processed for immunoprecipitation with the anti-Dap2p (B) and anti-HA (C) antibodies. The immunoprecipitates were treated with 50 mM DTT to cleave DSP and then analyzed by immunoblotting with anti-Dap2 polyclonal antibody (B and C), and anti-HA monoclonal (B) and polyclonal (C) antibodies. SMY22-10B cells expressing Rer1-3HAp and Dap2p or DSD were also subjected to the same procedures (C, lanes 7 and 8) as controls in the absence of DSP.
Mentions: After a long struggle to prove the physical interaction between Rer1p and the TMD of Sec12p, we decided to use a chimeric protein between Sec12p and Dap2p. Dap2p, a type II vacuolar membrane protein, has been used as a passenger protein to determine the ER localization signals of Sec12p (Sato et al. 1996). DSD, a chimeric protein comprised of the lumenal and cytoplasmic domains from Dap2p and the TMD from Sec12p, is almost completely localized to the ER by the Rer1p-dependent retrieval (Fig. 1 A). Chemical cross-linking experiments using a thiol-cleavable linker dithiobis (succinimidyl propionate) (DSP) were performed with the cell lysate prepared from a yeast strain overexpressing both Rer1-3HAp and DSD. Cells expressing Rer1-3HAp and Dap2p were used as a negative control. Cells were lysed and allowed to react with DSP. After the immunoprecipitation with either anti-Dap2p or anti-HA antibody, DSP was cleaved with 50 mM DTT, and the products were subjected to immunoblotting with anti-Dap2p and anti-HA antibodies. As shown in Fig. 1 (B and C), DSD and Rer1-3HAp were reproducibly coimmunoprecipitated by either anti-Dap2p or anti-HA antibody (lane 2). Such coprecipitation was not observed with the Dap2p control (Fig. 1B and Fig. C, lane 1). Mutants of DSD, which have amino acid replacements in the TMD, and thus show less Rer1p dependency (NQ-L, SY-L, +2L, and LeuX19; Fig. 1 A) (Sato et al. 1996; Sato, M., unpublished data), were not efficiently coimmunoprecipitated with Rer1-3HAp by the anti-Dap2p antibody and vice versa (Fig. 1B and Fig. C, lanes 3–6), indicating that Rer1p indeed recognizes the polar residues in the TMD of Sec12p.

Bottom Line: Either a lesion of coatomer or deletion of the COOH-terminal tail of Rer1p causes its mislocalization to the vacuole.The COOH-terminal Rer1p tail interacts in vitro with a coatomer complex containing alpha and gamma subunits.These findings not only give the proof that Rer1p is a novel type of retrieval receptor recognizing the TMD in the Golgi but also indicate that coatomer actively regulates the function and localization of Rer1p.

View Article: PubMed Central - PubMed

Affiliation: Molecular Membrane Biology Laboratory, RIKEN (The Institute of Physical and Chemical Research), Saitama 351-0198, Japan. satoken@postman.riken.go.jp

ABSTRACT
Rer1p, a yeast Golgi membrane protein, is required for the retrieval of a set of endoplasmic reticulum (ER) membrane proteins. We present the first evidence that Rer1p directly interacts with the transmembrane domain (TMD) of Sec12p which contains a retrieval signal. A green fluorescent protein (GFP) fusion of Rer1p rapidly cycles between the Golgi and the ER. Either a lesion of coatomer or deletion of the COOH-terminal tail of Rer1p causes its mislocalization to the vacuole. The COOH-terminal Rer1p tail interacts in vitro with a coatomer complex containing alpha and gamma subunits. These findings not only give the proof that Rer1p is a novel type of retrieval receptor recognizing the TMD in the Golgi but also indicate that coatomer actively regulates the function and localization of Rer1p.

Show MeSH
Related in: MedlinePlus