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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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Related in: MedlinePlus

AP-1–HA localizes to a perinuclear region. LLC-PK1::μ1A-HA transfectants (A–C, top) and LLC-PK1 cells transfected with μ1B-HA (bottom) were fixed and incubated with anti-HA antibodies (A, left) in combination with (A) anti–γ-adaptin antibodies (middle), (B) anti-GM130 antibodies, or (C) Texas red–labeled Tfn. These incubations were followed by an incubation with Alexa 488–labeled (anti-HA staining) and Alexa 594–labeled (anti–γ-adaptin and anti-GM130 staining) secondary antibodies. Specimens were analyzed by confocal microscopy and representative images are shown.
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Figure 3: AP-1–HA localizes to a perinuclear region. LLC-PK1::μ1A-HA transfectants (A–C, top) and LLC-PK1 cells transfected with μ1B-HA (bottom) were fixed and incubated with anti-HA antibodies (A, left) in combination with (A) anti–γ-adaptin antibodies (middle), (B) anti-GM130 antibodies, or (C) Texas red–labeled Tfn. These incubations were followed by an incubation with Alexa 488–labeled (anti-HA staining) and Alexa 594–labeled (anti–γ-adaptin and anti-GM130 staining) secondary antibodies. Specimens were analyzed by confocal microscopy and representative images are shown.

Mentions: Labeling with anti-HA antibodies produced perinuclear staining patterns in both AP-1A–HA– and AP-1B–HA–expressing cell lines (Fig. 3 A, left). As shown in Fig. 3, both μ1A-HA (top) and μ1B-HA (bottom) exhibited good regional colocalization with γ-adaptin. However, the extent of colocalization was not complete, with some μ1 labeling found in the periphery, particularly in the case of μ1B. This might simply reflect different sensitivities of the HA and γ-adaptin antibodies used, relative accessibility of the epitopes, or possibly a somewhat differential distribution of AP-1A and AP-1B complexes.


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-1–HA localizes to a perinuclear region. LLC-PK1::μ1A-HA transfectants (A–C, top) and LLC-PK1 cells transfected with μ1B-HA (bottom) were fixed and incubated with anti-HA antibodies (A, left) in combination with (A) anti–γ-adaptin antibodies (middle), (B) anti-GM130 antibodies, or (C) Texas red–labeled Tfn. These incubations were followed by an incubation with Alexa 488–labeled (anti-HA staining) and Alexa 594–labeled (anti–γ-adaptin and anti-GM130 staining) secondary antibodies. Specimens were analyzed by confocal microscopy and representative images are shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195989&req=5

Figure 3: AP-1–HA localizes to a perinuclear region. LLC-PK1::μ1A-HA transfectants (A–C, top) and LLC-PK1 cells transfected with μ1B-HA (bottom) were fixed and incubated with anti-HA antibodies (A, left) in combination with (A) anti–γ-adaptin antibodies (middle), (B) anti-GM130 antibodies, or (C) Texas red–labeled Tfn. These incubations were followed by an incubation with Alexa 488–labeled (anti-HA staining) and Alexa 594–labeled (anti–γ-adaptin and anti-GM130 staining) secondary antibodies. Specimens were analyzed by confocal microscopy and representative images are shown.
Mentions: Labeling with anti-HA antibodies produced perinuclear staining patterns in both AP-1A–HA– and AP-1B–HA–expressing cell lines (Fig. 3 A, left). As shown in Fig. 3, both μ1A-HA (top) and μ1B-HA (bottom) exhibited good regional colocalization with γ-adaptin. However, the extent of colocalization was not complete, with some μ1 labeling found in the periphery, particularly in the case of μ1B. This might simply reflect different sensitivities of the HA and γ-adaptin antibodies used, relative accessibility of the epitopes, or possibly a somewhat differential distribution of AP-1A and AP-1B complexes.

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