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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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COOH-terminal tail of Rer1p is important for the Golgi localization. (a) Wild-type (ANY21; panels A and B) and Δpep4 (SKY43; panels C and D) cells expressing GFP-Rer1Δ25p were observed by confocal laser microscopy. Nomarski (panels A and C) and fluorescence (panels B and D) images are shown. (b) Immunoblotting analysis of GFP-Rer1p and GFP-Rer1Δ25p. Wild-type (ANY21; lanes 1 and 3) and Δpep4 (SKY43; lanes 2 and 4) cells expressing GFP-Rer1p (lanes 1 and 2) or GFP-Rer1Δ25p (lanes 3 and 4) under the TDH3 promoter were grown at 23°C. Cell extracts (50 μg) were separated by SDS-PAGE and subjected to immunoblotting with the anti-GFP antibody. (c) Immunofluorescence staining of HA-Ste2p and HA–Ste2-Rer1p. Wild-type cells (SEY6211) expressing HA-Ste2p (panels A, C, and E) or HA–Ste2-Rer1p (panels B, D, and F) were grown at 30°C and subjected to immunofluorescence microscopy with the anti-HA monoclonal antibody (16B12). Panels C and D show Alexa 488 fluorescence corresponding to HA-Ste2p and HA–Ste2-Rer1p, respectively. Nomarski images (panels A and B) and DAPI images (panels E and F) are also shown. Panels G and H show double staining of HA–Ste2-Rer1p (G) and myc-Emp47p (H) in the SEY6211 cells expressing these two proteins. (d) Deletion analysis on the COOH-terminal region of GFP-Rer1p. Wild-type cells (ANY21) expressing GFP-Rer1p (Δ0) or its deletion mutants (Δ5, Δ10, Δ15, Δ20, and Δ25) were observed for GFP fluorescence. Bars, 5 μm.
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Figure 4: COOH-terminal tail of Rer1p is important for the Golgi localization. (a) Wild-type (ANY21; panels A and B) and Δpep4 (SKY43; panels C and D) cells expressing GFP-Rer1Δ25p were observed by confocal laser microscopy. Nomarski (panels A and C) and fluorescence (panels B and D) images are shown. (b) Immunoblotting analysis of GFP-Rer1p and GFP-Rer1Δ25p. Wild-type (ANY21; lanes 1 and 3) and Δpep4 (SKY43; lanes 2 and 4) cells expressing GFP-Rer1p (lanes 1 and 2) or GFP-Rer1Δ25p (lanes 3 and 4) under the TDH3 promoter were grown at 23°C. Cell extracts (50 μg) were separated by SDS-PAGE and subjected to immunoblotting with the anti-GFP antibody. (c) Immunofluorescence staining of HA-Ste2p and HA–Ste2-Rer1p. Wild-type cells (SEY6211) expressing HA-Ste2p (panels A, C, and E) or HA–Ste2-Rer1p (panels B, D, and F) were grown at 30°C and subjected to immunofluorescence microscopy with the anti-HA monoclonal antibody (16B12). Panels C and D show Alexa 488 fluorescence corresponding to HA-Ste2p and HA–Ste2-Rer1p, respectively. Nomarski images (panels A and B) and DAPI images (panels E and F) are also shown. Panels G and H show double staining of HA–Ste2-Rer1p (G) and myc-Emp47p (H) in the SEY6211 cells expressing these two proteins. (d) Deletion analysis on the COOH-terminal region of GFP-Rer1p. Wild-type cells (ANY21) expressing GFP-Rer1p (Δ0) or its deletion mutants (Δ5, Δ10, Δ15, Δ20, and Δ25) were observed for GFP fluorescence. Bars, 5 μm.

Mentions: We constructed a mutant version of GFP-Rer1p which lacks the COOH-terminal 25 residues (GFP-Rer1Δ25p). This GFP fusion did not complement the Sec12p-missorting phenotype of rer1. Wild-type and Δpep4 cells expressing GFP-Rer1Δ25p were observed by confocal laser scanning microscopy (Fig. 4 a). Major fluorescent signals were detected in the vacuole, suggesting that the COOH-terminal 25 residues had the information for correct localization to the Golgi. The staining of vacuolar lumen rather than vacuolar membranes was surprising, however, because the GFP moiety of GFP-Rer1Δ25p is expected to face the cytoplasm. This is reminiscent of the behavior of carboxypeptidase S, which is transported to the vacuole via the multivesicular body (MVB)-mediated sorting pathway (Odorizzi et al. 1998). Interestingly, in the Δpep4 cells in which vacuolar proteases are mostly inactive due to the lack of proteinase A (Jones 1984), punctate fluorescent signals of GFP-Rer1Δ25p move around very rapidly in the vacuolar lumen (Fig. 2, D–F; Video 2). Immunoblotting analysis (Fig. 4 b) reveals that GFP-Rer1p remains intact (49 kD) in both wild-type and Δpep4 cells (lanes 1 and 2) but GFP-Rer1Δ25p (46 kD) is processed to the 27-kD species in a PEP4-dependent manner (lanes 3 and 4). Similar results were obtained when GFP fusions were expressed under the authentic RER1 promoter (not shown). These observations suggest that GFP-Rer1Δ25p is targeted to the vacuole via the MVB pathway, and the very mobile structures in the vacuolar lumen of Δpep4 cells are undegraded internal membranes of MVB.


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)

COOH-terminal tail of Rer1p is important for the Golgi localization. (a) Wild-type (ANY21; panels A and B) and Δpep4 (SKY43; panels C and D) cells expressing GFP-Rer1Δ25p were observed by confocal laser microscopy. Nomarski (panels A and C) and fluorescence (panels B and D) images are shown. (b) Immunoblotting analysis of GFP-Rer1p and GFP-Rer1Δ25p. Wild-type (ANY21; lanes 1 and 3) and Δpep4 (SKY43; lanes 2 and 4) cells expressing GFP-Rer1p (lanes 1 and 2) or GFP-Rer1Δ25p (lanes 3 and 4) under the TDH3 promoter were grown at 23°C. Cell extracts (50 μg) were separated by SDS-PAGE and subjected to immunoblotting with the anti-GFP antibody. (c) Immunofluorescence staining of HA-Ste2p and HA–Ste2-Rer1p. Wild-type cells (SEY6211) expressing HA-Ste2p (panels A, C, and E) or HA–Ste2-Rer1p (panels B, D, and F) were grown at 30°C and subjected to immunofluorescence microscopy with the anti-HA monoclonal antibody (16B12). Panels C and D show Alexa 488 fluorescence corresponding to HA-Ste2p and HA–Ste2-Rer1p, respectively. Nomarski images (panels A and B) and DAPI images (panels E and F) are also shown. Panels G and H show double staining of HA–Ste2-Rer1p (G) and myc-Emp47p (H) in the SEY6211 cells expressing these two proteins. (d) Deletion analysis on the COOH-terminal region of GFP-Rer1p. Wild-type cells (ANY21) expressing GFP-Rer1p (Δ0) or its deletion mutants (Δ5, Δ10, Δ15, Δ20, and Δ25) were observed for GFP fluorescence. Bars, 5 μm.
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Figure 4: COOH-terminal tail of Rer1p is important for the Golgi localization. (a) Wild-type (ANY21; panels A and B) and Δpep4 (SKY43; panels C and D) cells expressing GFP-Rer1Δ25p were observed by confocal laser microscopy. Nomarski (panels A and C) and fluorescence (panels B and D) images are shown. (b) Immunoblotting analysis of GFP-Rer1p and GFP-Rer1Δ25p. Wild-type (ANY21; lanes 1 and 3) and Δpep4 (SKY43; lanes 2 and 4) cells expressing GFP-Rer1p (lanes 1 and 2) or GFP-Rer1Δ25p (lanes 3 and 4) under the TDH3 promoter were grown at 23°C. Cell extracts (50 μg) were separated by SDS-PAGE and subjected to immunoblotting with the anti-GFP antibody. (c) Immunofluorescence staining of HA-Ste2p and HA–Ste2-Rer1p. Wild-type cells (SEY6211) expressing HA-Ste2p (panels A, C, and E) or HA–Ste2-Rer1p (panels B, D, and F) were grown at 30°C and subjected to immunofluorescence microscopy with the anti-HA monoclonal antibody (16B12). Panels C and D show Alexa 488 fluorescence corresponding to HA-Ste2p and HA–Ste2-Rer1p, respectively. Nomarski images (panels A and B) and DAPI images (panels E and F) are also shown. Panels G and H show double staining of HA–Ste2-Rer1p (G) and myc-Emp47p (H) in the SEY6211 cells expressing these two proteins. (d) Deletion analysis on the COOH-terminal region of GFP-Rer1p. Wild-type cells (ANY21) expressing GFP-Rer1p (Δ0) or its deletion mutants (Δ5, Δ10, Δ15, Δ20, and Δ25) were observed for GFP fluorescence. Bars, 5 μm.
Mentions: We constructed a mutant version of GFP-Rer1p which lacks the COOH-terminal 25 residues (GFP-Rer1Δ25p). This GFP fusion did not complement the Sec12p-missorting phenotype of rer1. Wild-type and Δpep4 cells expressing GFP-Rer1Δ25p were observed by confocal laser scanning microscopy (Fig. 4 a). Major fluorescent signals were detected in the vacuole, suggesting that the COOH-terminal 25 residues had the information for correct localization to the Golgi. The staining of vacuolar lumen rather than vacuolar membranes was surprising, however, because the GFP moiety of GFP-Rer1Δ25p is expected to face the cytoplasm. This is reminiscent of the behavior of carboxypeptidase S, which is transported to the vacuole via the multivesicular body (MVB)-mediated sorting pathway (Odorizzi et al. 1998). Interestingly, in the Δpep4 cells in which vacuolar proteases are mostly inactive due to the lack of proteinase A (Jones 1984), punctate fluorescent signals of GFP-Rer1Δ25p move around very rapidly in the vacuolar lumen (Fig. 2, D–F; Video 2). Immunoblotting analysis (Fig. 4 b) reveals that GFP-Rer1p remains intact (49 kD) in both wild-type and Δpep4 cells (lanes 1 and 2) but GFP-Rer1Δ25p (46 kD) is processed to the 27-kD species in a PEP4-dependent manner (lanes 3 and 4). Similar results were obtained when GFP fusions were expressed under the authentic RER1 promoter (not shown). These observations suggest that GFP-Rer1Δ25p is targeted to the vacuole via the MVB pathway, and the very mobile structures in the vacuolar lumen of Δpep4 cells are undegraded internal membranes of MVB.

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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