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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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Dilysine-like motif and two tyrosines are required for the correct localization of Rer1p. (a) Mutational analysis of the COOH-terminal tail of GFP-Rer1p. The ability of each fusion to complement rer1-2 in terms of missorting of Sec12-Mfα1p (Sato et al. 1995) is shown on the right. (b) Localization of a series of point mutants of GFP-Rer1p. Wild-type cells (ANY21) expressing each of GFP-Rer1p point mutants were harvested and subjected to fluorescence microscopy. Nomarski images (panels A–C and G–I) and GFP images (panels D–F and J–L) are shown. (c) Pulse–chase analyses of GFP-Rer1p mutants. Wild-type cells (ANY21) expressing each GFP-Rer1p derivative were labeled with Tran35S-label (ICN Biochemicals) at 30°C for 10 min and chased for the indicated times. GFP fusions were immunoprecipitated with the anti-GFP antibody and analyzed by SDS-PAGE and radioimaging. I and P indicate intact and processed forms, respectively. Bar, 5 μm.
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Figure 5: Dilysine-like motif and two tyrosines are required for the correct localization of Rer1p. (a) Mutational analysis of the COOH-terminal tail of GFP-Rer1p. The ability of each fusion to complement rer1-2 in terms of missorting of Sec12-Mfα1p (Sato et al. 1995) is shown on the right. (b) Localization of a series of point mutants of GFP-Rer1p. Wild-type cells (ANY21) expressing each of GFP-Rer1p point mutants were harvested and subjected to fluorescence microscopy. Nomarski images (panels A–C and G–I) and GFP images (panels D–F and J–L) are shown. (c) Pulse–chase analyses of GFP-Rer1p mutants. Wild-type cells (ANY21) expressing each GFP-Rer1p derivative were labeled with Tran35S-label (ICN Biochemicals) at 30°C for 10 min and chased for the indicated times. GFP fusions were immunoprecipitated with the anti-GFP antibody and analyzed by SDS-PAGE and radioimaging. I and P indicate intact and processed forms, respectively. Bar, 5 μm.

Mentions: To define the localization signal(s) in the COOH-terminal tail of Rer1p more precisely, we generated a series of mutants from GFP-Rer1p. Deletion of the COOH-terminal 10 residues (Δ10) led to the mislocalization to the vacuole and the failure to complement rer1, but the Δ5 construct was normal (Fig. 4 d). We noticed that the amino acid sequence GKKKY (179–183) contains a dilysine-type motif and performed a mutational analysis on this. Single mutations affected the ability to complement rer1 in different degrees (Fig. 5 a). As shown in Fig. 5 b, K180S and Y183A mutations caused clear mislocalization of GFP-Rer1p to the vacuole, whereas G179A and K182S had a marginal effect on the Golgi localization. K181S mutant was partially missorted to the vacuole. The KKSS double mutant (K180S K181S) completely lost the function as Rer1p (Fig. 5 a) and was severely mislocalized to the vacuole (data not shown). Interestingly, Y183A showed clear mislocalization to the vacuole but retained the Rer1p function to sort 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)

Dilysine-like motif and two tyrosines are required for the correct localization of Rer1p. (a) Mutational analysis of the COOH-terminal tail of GFP-Rer1p. The ability of each fusion to complement rer1-2 in terms of missorting of Sec12-Mfα1p (Sato et al. 1995) is shown on the right. (b) Localization of a series of point mutants of GFP-Rer1p. Wild-type cells (ANY21) expressing each of GFP-Rer1p point mutants were harvested and subjected to fluorescence microscopy. Nomarski images (panels A–C and G–I) and GFP images (panels D–F and J–L) are shown. (c) Pulse–chase analyses of GFP-Rer1p mutants. Wild-type cells (ANY21) expressing each GFP-Rer1p derivative were labeled with Tran35S-label (ICN Biochemicals) at 30°C for 10 min and chased for the indicated times. GFP fusions were immunoprecipitated with the anti-GFP antibody and analyzed by SDS-PAGE and radioimaging. I and P indicate intact and processed forms, respectively. Bar, 5 μm.
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Related In: Results  -  Collection

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Figure 5: Dilysine-like motif and two tyrosines are required for the correct localization of Rer1p. (a) Mutational analysis of the COOH-terminal tail of GFP-Rer1p. The ability of each fusion to complement rer1-2 in terms of missorting of Sec12-Mfα1p (Sato et al. 1995) is shown on the right. (b) Localization of a series of point mutants of GFP-Rer1p. Wild-type cells (ANY21) expressing each of GFP-Rer1p point mutants were harvested and subjected to fluorescence microscopy. Nomarski images (panels A–C and G–I) and GFP images (panels D–F and J–L) are shown. (c) Pulse–chase analyses of GFP-Rer1p mutants. Wild-type cells (ANY21) expressing each GFP-Rer1p derivative were labeled with Tran35S-label (ICN Biochemicals) at 30°C for 10 min and chased for the indicated times. GFP fusions were immunoprecipitated with the anti-GFP antibody and analyzed by SDS-PAGE and radioimaging. I and P indicate intact and processed forms, respectively. Bar, 5 μm.
Mentions: To define the localization signal(s) in the COOH-terminal tail of Rer1p more precisely, we generated a series of mutants from GFP-Rer1p. Deletion of the COOH-terminal 10 residues (Δ10) led to the mislocalization to the vacuole and the failure to complement rer1, but the Δ5 construct was normal (Fig. 4 d). We noticed that the amino acid sequence GKKKY (179–183) contains a dilysine-type motif and performed a mutational analysis on this. Single mutations affected the ability to complement rer1 in different degrees (Fig. 5 a). As shown in Fig. 5 b, K180S and Y183A mutations caused clear mislocalization of GFP-Rer1p to the vacuole, whereas G179A and K182S had a marginal effect on the Golgi localization. K181S mutant was partially missorted to the vacuole. The KKSS double mutant (K180S K181S) completely lost the function as Rer1p (Fig. 5 a) and was severely mislocalized to the vacuole (data not shown). Interestingly, Y183A showed clear mislocalization to the vacuole but retained the Rer1p function to sort 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.

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