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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-1–HA is found on clathrin-coated structures. LLC-PK1::μ1A-HA (A) or LLC-PK1::μ1B-HA transfectants (B) were analyzed by immunoelectron microscopy (see Materials and Methods for details). HA-tagged μ1 proteins were visualized using anti-HA antibodies, followed by incubations with anti–mouse IgG antibodies and protein A–gold. Arrowheads highlight gold particles and arrows denote clathrin-coated structures. Bar, 100 nm.
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Figure 4: AP-1–HA is found on clathrin-coated structures. LLC-PK1::μ1A-HA (A) or LLC-PK1::μ1B-HA transfectants (B) were analyzed by immunoelectron microscopy (see Materials and Methods for details). HA-tagged μ1 proteins were visualized using anti-HA antibodies, followed by incubations with anti–mouse IgG antibodies and protein A–gold. Arrowheads highlight gold particles and arrows denote clathrin-coated structures. Bar, 100 nm.

Mentions: To further characterize the intracellular localization of AP-1B, we performed immunoelectron microscopy on frozen sections of LLC-PK1::μ1A-HA or LLC-PK1::μ1B-HA transfectants. Most of the labeling for both AP-1A–HA (Fig. 4 A) and AP-1B–HA (Fig. 4 B) complexes with protein A–gold (arrowheads) was associated with buds, vesicles, and tubules on one side of the Golgi stack. This side of the stack was also characterized by morphologically identifiable clathrin coats, strongly suggesting that it corresponds to the trans side. Indeed, and as expected from previous work for untagged AP-1A complexes, many of the gold particles were associated with clathrin coats (denoted by arrows) (Hirst and Robinson 1998). We conclude that, like AP-1A and AP-1B, complexes can associate with clathrin coats in the TGN, suggesting that their function is associated with clathrin assembly. Often, apparently free-coated vesicles seemed to be more heavily stained for either μ1A or μ1B than were the clathrin-coated tubules that emerged from the TGN. This might reflect either the relative densities or accessibilities of the μ1 or the epitope tags at different stages of coat assembly.


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 is found on clathrin-coated structures. LLC-PK1::μ1A-HA (A) or LLC-PK1::μ1B-HA transfectants (B) were analyzed by immunoelectron microscopy (see Materials and Methods for details). HA-tagged μ1 proteins were visualized using anti-HA antibodies, followed by incubations with anti–mouse IgG antibodies and protein A–gold. Arrowheads highlight gold particles and arrows denote clathrin-coated structures. Bar, 100 nm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: AP-1–HA is found on clathrin-coated structures. LLC-PK1::μ1A-HA (A) or LLC-PK1::μ1B-HA transfectants (B) were analyzed by immunoelectron microscopy (see Materials and Methods for details). HA-tagged μ1 proteins were visualized using anti-HA antibodies, followed by incubations with anti–mouse IgG antibodies and protein A–gold. Arrowheads highlight gold particles and arrows denote clathrin-coated structures. Bar, 100 nm.
Mentions: To further characterize the intracellular localization of AP-1B, we performed immunoelectron microscopy on frozen sections of LLC-PK1::μ1A-HA or LLC-PK1::μ1B-HA transfectants. Most of the labeling for both AP-1A–HA (Fig. 4 A) and AP-1B–HA (Fig. 4 B) complexes with protein A–gold (arrowheads) was associated with buds, vesicles, and tubules on one side of the Golgi stack. This side of the stack was also characterized by morphologically identifiable clathrin coats, strongly suggesting that it corresponds to the trans side. Indeed, and as expected from previous work for untagged AP-1A complexes, many of the gold particles were associated with clathrin coats (denoted by arrows) (Hirst and Robinson 1998). We conclude that, like AP-1A and AP-1B, complexes can associate with clathrin coats in the TGN, suggesting that their function is associated with clathrin assembly. Often, apparently free-coated vesicles seemed to be more heavily stained for either μ1A or μ1B than were the clathrin-coated tubules that emerged from the TGN. This might reflect either the relative densities or accessibilities of the μ1 or the epitope tags at different stages of coat assembly.

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