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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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Expression and function of m1-HA. (A) Diagram showing the position of the internal HA-tag in μ1 protein (423 amino acids total). (B) Nontransfected LLC-PK1 cells, as well as LLC-PK1::μ1A-HA and LLC-PK1::μ1B-HA transfectants, were lysed in RIPA buffer and 5 and 10 μg of total protein were analyzed by Western blotting. γ-Adaptin, μ1-HA, and μ1 proteins were detected by immunodecoration with anti–γ-adaptin antibodies, anti-HA antibodies, and an antibody that cross-reacts with μ1A and μ1B, respectively. (C) LLC-PK1::μ1A-HA and LLC-PK1::μ1B-HA transfectants were metabolically labeled with [35S]methionine/cysteine overnight and lysed in Triton X-100 buffer. AP-1 complexes were immunoprecipitated using anti–γ-adaptin. The immunoprecipitates were boiled in SDS and one half of the extract was directly subjected to SDS-PAGE analysis. The remaining extract was diluted 20-fold in lysis buffer and μ1A-HA or μ1B-HA proteins were recaptured using anti-HA antibodies or specific anti-μ1B antibodies directed against the COOH terminus of μ1B, respectively. Immunoprecipitates were analyzed by SDS-PAGE and autoradiography. (D) LLC-PK1::μ1A-HA (top) and LLC-PK1::μ1B-HA (bottom) transfectants were grown on Transwell filters and infected with recombinant adenoviruses encoding the FcLR(CT22) chimera (left) or the LDLR (right). 2 d after the infection viable cells were incubated with antibodies directed against the ectodomain of FcLR or LDLR, respectively, fixed, and incubated with Alexa 488–labeled secondary antibodies as described in Material and Methods. Specimens were analyzed by confocal microscopy. Representative X–Z sections are shown.
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Figure 1: Expression and function of m1-HA. (A) Diagram showing the position of the internal HA-tag in μ1 protein (423 amino acids total). (B) Nontransfected LLC-PK1 cells, as well as LLC-PK1::μ1A-HA and LLC-PK1::μ1B-HA transfectants, were lysed in RIPA buffer and 5 and 10 μg of total protein were analyzed by Western blotting. γ-Adaptin, μ1-HA, and μ1 proteins were detected by immunodecoration with anti–γ-adaptin antibodies, anti-HA antibodies, and an antibody that cross-reacts with μ1A and μ1B, respectively. (C) LLC-PK1::μ1A-HA and LLC-PK1::μ1B-HA transfectants were metabolically labeled with [35S]methionine/cysteine overnight and lysed in Triton X-100 buffer. AP-1 complexes were immunoprecipitated using anti–γ-adaptin. The immunoprecipitates were boiled in SDS and one half of the extract was directly subjected to SDS-PAGE analysis. The remaining extract was diluted 20-fold in lysis buffer and μ1A-HA or μ1B-HA proteins were recaptured using anti-HA antibodies or specific anti-μ1B antibodies directed against the COOH terminus of μ1B, respectively. Immunoprecipitates were analyzed by SDS-PAGE and autoradiography. (D) LLC-PK1::μ1A-HA (top) and LLC-PK1::μ1B-HA (bottom) transfectants were grown on Transwell filters and infected with recombinant adenoviruses encoding the FcLR(CT22) chimera (left) or the LDLR (right). 2 d after the infection viable cells were incubated with antibodies directed against the ectodomain of FcLR or LDLR, respectively, fixed, and incubated with Alexa 488–labeled secondary antibodies as described in Material and Methods. Specimens were analyzed by confocal microscopy. Representative X–Z sections are shown.

Mentions: In a previous study, we described two antipeptide antibodies that were specific for μ1B (Fölsch et al. 1999). Unfortunately, neither was suitable for immunofluorescence, a common problem with antibodies raised against the μ chains (Simpson et al. 1996; Dell'Angelica et al. 1999a,Dell'Angelica et al. 1999b). To establish the intracellular localization of μ1B-containing AP-1 complexes, we introduced internal HA tags into μ1A and μ1B between amino acids 230 and 231 (Fig. 1 A). These amino acids lie in a stretch of a weakly conserved region, presumably exposed at the surface of the protein by analogy to the crystal structure of μ2 (Owen and Evans 1998). In addition, the corresponding site in μ2 did not prevent its incorporation into AP-2 complexes (Nesterov et al. 1999).


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)

Expression and function of m1-HA. (A) Diagram showing the position of the internal HA-tag in μ1 protein (423 amino acids total). (B) Nontransfected LLC-PK1 cells, as well as LLC-PK1::μ1A-HA and LLC-PK1::μ1B-HA transfectants, were lysed in RIPA buffer and 5 and 10 μg of total protein were analyzed by Western blotting. γ-Adaptin, μ1-HA, and μ1 proteins were detected by immunodecoration with anti–γ-adaptin antibodies, anti-HA antibodies, and an antibody that cross-reacts with μ1A and μ1B, respectively. (C) LLC-PK1::μ1A-HA and LLC-PK1::μ1B-HA transfectants were metabolically labeled with [35S]methionine/cysteine overnight and lysed in Triton X-100 buffer. AP-1 complexes were immunoprecipitated using anti–γ-adaptin. The immunoprecipitates were boiled in SDS and one half of the extract was directly subjected to SDS-PAGE analysis. The remaining extract was diluted 20-fold in lysis buffer and μ1A-HA or μ1B-HA proteins were recaptured using anti-HA antibodies or specific anti-μ1B antibodies directed against the COOH terminus of μ1B, respectively. Immunoprecipitates were analyzed by SDS-PAGE and autoradiography. (D) LLC-PK1::μ1A-HA (top) and LLC-PK1::μ1B-HA (bottom) transfectants were grown on Transwell filters and infected with recombinant adenoviruses encoding the FcLR(CT22) chimera (left) or the LDLR (right). 2 d after the infection viable cells were incubated with antibodies directed against the ectodomain of FcLR or LDLR, respectively, fixed, and incubated with Alexa 488–labeled secondary antibodies as described in Material and Methods. Specimens were analyzed by confocal microscopy. Representative X–Z sections are shown.
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Related In: Results  -  Collection

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Figure 1: Expression and function of m1-HA. (A) Diagram showing the position of the internal HA-tag in μ1 protein (423 amino acids total). (B) Nontransfected LLC-PK1 cells, as well as LLC-PK1::μ1A-HA and LLC-PK1::μ1B-HA transfectants, were lysed in RIPA buffer and 5 and 10 μg of total protein were analyzed by Western blotting. γ-Adaptin, μ1-HA, and μ1 proteins were detected by immunodecoration with anti–γ-adaptin antibodies, anti-HA antibodies, and an antibody that cross-reacts with μ1A and μ1B, respectively. (C) LLC-PK1::μ1A-HA and LLC-PK1::μ1B-HA transfectants were metabolically labeled with [35S]methionine/cysteine overnight and lysed in Triton X-100 buffer. AP-1 complexes were immunoprecipitated using anti–γ-adaptin. The immunoprecipitates were boiled in SDS and one half of the extract was directly subjected to SDS-PAGE analysis. The remaining extract was diluted 20-fold in lysis buffer and μ1A-HA or μ1B-HA proteins were recaptured using anti-HA antibodies or specific anti-μ1B antibodies directed against the COOH terminus of μ1B, respectively. Immunoprecipitates were analyzed by SDS-PAGE and autoradiography. (D) LLC-PK1::μ1A-HA (top) and LLC-PK1::μ1B-HA (bottom) transfectants were grown on Transwell filters and infected with recombinant adenoviruses encoding the FcLR(CT22) chimera (left) or the LDLR (right). 2 d after the infection viable cells were incubated with antibodies directed against the ectodomain of FcLR or LDLR, respectively, fixed, and incubated with Alexa 488–labeled secondary antibodies as described in Material and Methods. Specimens were analyzed by confocal microscopy. Representative X–Z sections are shown.
Mentions: In a previous study, we described two antipeptide antibodies that were specific for μ1B (Fölsch et al. 1999). Unfortunately, neither was suitable for immunofluorescence, a common problem with antibodies raised against the μ chains (Simpson et al. 1996; Dell'Angelica et al. 1999a,Dell'Angelica et al. 1999b). To establish the intracellular localization of μ1B-containing AP-1 complexes, we introduced internal HA tags into μ1A and μ1B between amino acids 230 and 231 (Fig. 1 A). These amino acids lie in a stretch of a weakly conserved region, presumably exposed at the surface of the protein by analogy to the crystal structure of μ2 (Owen and Evans 1998). In addition, the corresponding site in μ2 did not prevent its incorporation into AP-2 complexes (Nesterov et al. 1999).

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