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Distribution and function of AP-1 clathrin adaptor complexes in polarized epithelial cells.

Fölsch H, Pypaert M, Schu P, Mellman I - J. Cell Biol. (2001)

Bottom Line: Tagged AP-1A and AP-1B complexes were found in the perinuclear region close to the Golgi complex and recycling endosomes, often in clathrin-coated buds and vesicles.Yet, AP-1A and AP-1B localized to different subdomains of the TGN, with only AP-1A colocalizing with furin, a membrane protein that uses AP-1 to recycle between the TGN and endosomes.We conclude that AP-1B functions by interacting with its cargo molecules and clathrin in the TGN, where it acts to sort basolateral proteins from proteins destined for the apical surface and from those selected by AP-1A for transport to endosomes and lysosomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Ludwig Institute for Cancer Research, Yale University School of Medicine, New Haven, Connecticut 06520, USA.

ABSTRACT
Expression of the epithelial cell-specific heterotetrameric adaptor complex AP-1B is required for the polarized distribution of many membrane proteins to the basolateral surface of LLC-PK1 kidney cells. AP-1B is distinguished from the ubiquitously expressed AP-1A by exchange of its single 50-kD mu subunit, mu1A, being replaced by the closely related mu1B. Here we show that this substitution is sufficient to couple basolateral plasma membrane proteins, such as a low-density lipoprotein receptor (LDLR), to the AP-1B complex and to clathrin. The interaction between LDLR and AP-1B is likely to occur in the trans-Golgi network (TGN), as was suggested by the localization of functional, epitope-tagged mu1 by immunofluorescence and immunoelectron microscopy. Tagged AP-1A and AP-1B complexes were found in the perinuclear region close to the Golgi complex and recycling endosomes, often in clathrin-coated buds and vesicles. Yet, AP-1A and AP-1B localized to different subdomains of the TGN, with only AP-1A colocalizing with furin, a membrane protein that uses AP-1 to recycle between the TGN and endosomes. We conclude that AP-1B functions by interacting with its cargo molecules and clathrin in the TGN, where it acts to sort basolateral proteins from proteins destined for the apical surface and from those selected by AP-1A for transport to endosomes and lysosomes.

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AP-1B directly interacts with LDLR. (A) LLC-PK1::μ1A or LLC-PK1::μ1B transfectants were infected with defective adenoviruses encoding the LDLR. 1 d after the infections the cells were lysed and the LDLR was immunoprecipitated (IP) with anti-LDLR antibody (see Materials and Methods for details). Immunoprecipitates were analyzed by SDS-PAGE and Western blotting. LDLR, γ-adaptin, and μ1B were detected by immunodecoration with anti-LDLR antibodies, anti–γ-adaptin antibodies, or specific anti-μ1B antibodies raised against the COOH terminus of μ1B, respectively. Samples representing equivalent amounts of starting cell protein were added to each lane. (B) LLC-PK1 cells transfected with μ1B (lanes 1–4) or μ1B-HA (lanes 5 and 6) were infected with adLDLR. 1 d after infection the cells were incubated with the cross-linker DTSSP (100 μM). After quenching, the cells were lysed and the LDLR was immunoprecipitated as in A (details are described in Materials and Methods). Immunoprecipitates were analyzed as described in A. Clathrin heavy chain was detected by immunodecoration with anticlathrin heavy chain antibodies. (C) LLC-PK1::μ1B transfectants were infected with adLDLR (lanes 1 and 2) or adLDLR(Y18A/G34D) (lanes 3 and 4) and analyzed as described in B.
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Figure 2: AP-1B directly interacts with LDLR. (A) LLC-PK1::μ1A or LLC-PK1::μ1B transfectants were infected with defective adenoviruses encoding the LDLR. 1 d after the infections the cells were lysed and the LDLR was immunoprecipitated (IP) with anti-LDLR antibody (see Materials and Methods for details). Immunoprecipitates were analyzed by SDS-PAGE and Western blotting. LDLR, γ-adaptin, and μ1B were detected by immunodecoration with anti-LDLR antibodies, anti–γ-adaptin antibodies, or specific anti-μ1B antibodies raised against the COOH terminus of μ1B, respectively. Samples representing equivalent amounts of starting cell protein were added to each lane. (B) LLC-PK1 cells transfected with μ1B (lanes 1–4) or μ1B-HA (lanes 5 and 6) were infected with adLDLR. 1 d after infection the cells were incubated with the cross-linker DTSSP (100 μM). After quenching, the cells were lysed and the LDLR was immunoprecipitated as in A (details are described in Materials and Methods). Immunoprecipitates were analyzed as described in A. Clathrin heavy chain was detected by immunodecoration with anticlathrin heavy chain antibodies. (C) LLC-PK1::μ1B transfectants were infected with adLDLR (lanes 1 and 2) or adLDLR(Y18A/G34D) (lanes 3 and 4) and analyzed as described in B.

Mentions: As expected, the LDLR was detected as two bands, the upper band representing the terminally glycosylated mature LDLR (160 kD) and the lower band corresponding to the immature form of the receptor (140 kD) (Fig. 2 A). In LLC-PK1 cells transfected with μ1A, no AP-1 was coprecipitated, as indicated by the absence of staining for γ-adaptin (Fig. 2 A, lane 2); however, dependent on the expression levels in the infected cells, a weak staining for γ-adaptin could be observed (data not shown). Thus, AP-1A interacts only weakly with the LDLR. In LLC-PK1::μ1B transfectants, however, substantially more AP-1 (γ-adaptin) was brought down with anti-LDLR antibodies (Fig. 2 A, compare γ-adaptin bands in lanes 2 and 4). Most importantly, μ1B was also coprecipitated.


Distribution and function of AP-1 clathrin adaptor complexes in polarized epithelial cells.

Fölsch H, Pypaert M, Schu P, Mellman I - J. Cell Biol. (2001)

AP-1B directly interacts with LDLR. (A) LLC-PK1::μ1A or LLC-PK1::μ1B transfectants were infected with defective adenoviruses encoding the LDLR. 1 d after the infections the cells were lysed and the LDLR was immunoprecipitated (IP) with anti-LDLR antibody (see Materials and Methods for details). Immunoprecipitates were analyzed by SDS-PAGE and Western blotting. LDLR, γ-adaptin, and μ1B were detected by immunodecoration with anti-LDLR antibodies, anti–γ-adaptin antibodies, or specific anti-μ1B antibodies raised against the COOH terminus of μ1B, respectively. Samples representing equivalent amounts of starting cell protein were added to each lane. (B) LLC-PK1 cells transfected with μ1B (lanes 1–4) or μ1B-HA (lanes 5 and 6) were infected with adLDLR. 1 d after infection the cells were incubated with the cross-linker DTSSP (100 μM). After quenching, the cells were lysed and the LDLR was immunoprecipitated as in A (details are described in Materials and Methods). Immunoprecipitates were analyzed as described in A. Clathrin heavy chain was detected by immunodecoration with anticlathrin heavy chain antibodies. (C) LLC-PK1::μ1B transfectants were infected with adLDLR (lanes 1 and 2) or adLDLR(Y18A/G34D) (lanes 3 and 4) and analyzed as described in B.
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Figure 2: AP-1B directly interacts with LDLR. (A) LLC-PK1::μ1A or LLC-PK1::μ1B transfectants were infected with defective adenoviruses encoding the LDLR. 1 d after the infections the cells were lysed and the LDLR was immunoprecipitated (IP) with anti-LDLR antibody (see Materials and Methods for details). Immunoprecipitates were analyzed by SDS-PAGE and Western blotting. LDLR, γ-adaptin, and μ1B were detected by immunodecoration with anti-LDLR antibodies, anti–γ-adaptin antibodies, or specific anti-μ1B antibodies raised against the COOH terminus of μ1B, respectively. Samples representing equivalent amounts of starting cell protein were added to each lane. (B) LLC-PK1 cells transfected with μ1B (lanes 1–4) or μ1B-HA (lanes 5 and 6) were infected with adLDLR. 1 d after infection the cells were incubated with the cross-linker DTSSP (100 μM). After quenching, the cells were lysed and the LDLR was immunoprecipitated as in A (details are described in Materials and Methods). Immunoprecipitates were analyzed as described in A. Clathrin heavy chain was detected by immunodecoration with anticlathrin heavy chain antibodies. (C) LLC-PK1::μ1B transfectants were infected with adLDLR (lanes 1 and 2) or adLDLR(Y18A/G34D) (lanes 3 and 4) and analyzed as described in B.
Mentions: As expected, the LDLR was detected as two bands, the upper band representing the terminally glycosylated mature LDLR (160 kD) and the lower band corresponding to the immature form of the receptor (140 kD) (Fig. 2 A). In LLC-PK1 cells transfected with μ1A, no AP-1 was coprecipitated, as indicated by the absence of staining for γ-adaptin (Fig. 2 A, lane 2); however, dependent on the expression levels in the infected cells, a weak staining for γ-adaptin could be observed (data not shown). Thus, AP-1A interacts only weakly with the LDLR. In LLC-PK1::μ1B transfectants, however, substantially more AP-1 (γ-adaptin) was brought down with anti-LDLR antibodies (Fig. 2 A, compare γ-adaptin bands in lanes 2 and 4). Most importantly, μ1B was also coprecipitated.

Bottom Line: Tagged AP-1A and AP-1B complexes were found in the perinuclear region close to the Golgi complex and recycling endosomes, often in clathrin-coated buds and vesicles.Yet, AP-1A and AP-1B localized to different subdomains of the TGN, with only AP-1A colocalizing with furin, a membrane protein that uses AP-1 to recycle between the TGN and endosomes.We conclude that AP-1B functions by interacting with its cargo molecules and clathrin in the TGN, where it acts to sort basolateral proteins from proteins destined for the apical surface and from those selected by AP-1A for transport to endosomes and lysosomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Ludwig Institute for Cancer Research, Yale University School of Medicine, New Haven, Connecticut 06520, USA.

ABSTRACT
Expression of the epithelial cell-specific heterotetrameric adaptor complex AP-1B is required for the polarized distribution of many membrane proteins to the basolateral surface of LLC-PK1 kidney cells. AP-1B is distinguished from the ubiquitously expressed AP-1A by exchange of its single 50-kD mu subunit, mu1A, being replaced by the closely related mu1B. Here we show that this substitution is sufficient to couple basolateral plasma membrane proteins, such as a low-density lipoprotein receptor (LDLR), to the AP-1B complex and to clathrin. The interaction between LDLR and AP-1B is likely to occur in the trans-Golgi network (TGN), as was suggested by the localization of functional, epitope-tagged mu1 by immunofluorescence and immunoelectron microscopy. Tagged AP-1A and AP-1B complexes were found in the perinuclear region close to the Golgi complex and recycling endosomes, often in clathrin-coated buds and vesicles. Yet, AP-1A and AP-1B localized to different subdomains of the TGN, with only AP-1A colocalizing with furin, a membrane protein that uses AP-1 to recycle between the TGN and endosomes. We conclude that AP-1B functions by interacting with its cargo molecules and clathrin in the TGN, where it acts to sort basolateral proteins from proteins destined for the apical surface and from those selected by AP-1A for transport to endosomes and lysosomes.

Show MeSH
Related in: MedlinePlus