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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-1A is necessary for furin localization at the TGN. μ1A−/− fibroblasts stably transfected with μ1B alone (top) or with μ1B and μ1A-HA (bottom) were double-labeled with anti–γ-adaptin antibodies (left) and antifurin antibodies (middle) in combination with Alexa 488–labeled (anti-γ staining) and Alexa 594–labeled (antifurin staining) secondary antibodies. Specimens were analyzed by confocal microscopy and representative images are shown. The arrows denote regions in the AP-1B/AP-1A–HA transfectants that are positive for AP-1 but negative for furin.
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Figure 6: AP-1A is necessary for furin localization at the TGN. μ1A−/− fibroblasts stably transfected with μ1B alone (top) or with μ1B and μ1A-HA (bottom) were double-labeled with anti–γ-adaptin antibodies (left) and antifurin antibodies (middle) in combination with Alexa 488–labeled (anti-γ staining) and Alexa 594–labeled (antifurin staining) secondary antibodies. Specimens were analyzed by confocal microscopy and representative images are shown. The arrows denote regions in the AP-1B/AP-1A–HA transfectants that are positive for AP-1 but negative for furin.

Mentions: Recently, Meyer et al. 2000 generated a cell line of mouse embryonic fibroblasts that were deficient in μ1A (μ1A−/− fibroblasts) and exhibited a loss of perinuclear γ-adaptin staining. The diffuse γ-adaptin staining observed in these cells could be rescued by stably transfecting the cells with untagged mouse μ1B cDNA (Schu, P., unpublished observations; Fig. 6), suggesting that μ1B could substitute for μ1A in recruiting the other three AP-1 subunits to the TGN. Therefore, we tested whether μ1B expression in the absence of μ1A would also affect the distribution of furin, which, based on our localization experiments (Fig. 5), would appear to be more likely to interact with AP-1A than with AP-1B.


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-1A is necessary for furin localization at the TGN. μ1A−/− fibroblasts stably transfected with μ1B alone (top) or with μ1B and μ1A-HA (bottom) were double-labeled with anti–γ-adaptin antibodies (left) and antifurin antibodies (middle) in combination with Alexa 488–labeled (anti-γ staining) and Alexa 594–labeled (antifurin staining) secondary antibodies. Specimens were analyzed by confocal microscopy and representative images are shown. The arrows denote regions in the AP-1B/AP-1A–HA transfectants that are positive for AP-1 but negative for furin.
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Related In: Results  -  Collection

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Figure 6: AP-1A is necessary for furin localization at the TGN. μ1A−/− fibroblasts stably transfected with μ1B alone (top) or with μ1B and μ1A-HA (bottom) were double-labeled with anti–γ-adaptin antibodies (left) and antifurin antibodies (middle) in combination with Alexa 488–labeled (anti-γ staining) and Alexa 594–labeled (antifurin staining) secondary antibodies. Specimens were analyzed by confocal microscopy and representative images are shown. The arrows denote regions in the AP-1B/AP-1A–HA transfectants that are positive for AP-1 but negative for furin.
Mentions: Recently, Meyer et al. 2000 generated a cell line of mouse embryonic fibroblasts that were deficient in μ1A (μ1A−/− fibroblasts) and exhibited a loss of perinuclear γ-adaptin staining. The diffuse γ-adaptin staining observed in these cells could be rescued by stably transfecting the cells with untagged mouse μ1B cDNA (Schu, P., unpublished observations; Fig. 6), suggesting that μ1B could substitute for μ1A in recruiting the other three AP-1 subunits to the TGN. Therefore, we tested whether μ1B expression in the absence of μ1A would also affect the distribution of furin, which, based on our localization experiments (Fig. 5), would appear to be more likely to interact with AP-1A than with AP-1B.

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