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.
Highlights
Epithelial cells generate and maintain at least two biochemically distinct plasma membrane domains that reside within a continuous lipid bilayer (Rodriguez-Boulan and Powell, 1992; Drubin and Nelson, 1996; Yeaman et al, 1999)
Neither AP-1B nor clathrin could be cross-linked to the mutant low-density lipoprotein receptor (LDLR), indicating that AP-1B interacts with its basolateral sorting signals (Fig. 2 C, lane 4). These results suggest that AP-1B mediates basolateral targeting by directly interacting with the basolateral targeting motifs of cargo molecules, and that clathrin is involved in the formation of transport vesicles targeted to the basolateral plasma membrane
We have yet to achieve furin staining at the electron microscopic level, these results suggest that AP1A and AP-1B may define at least partially distinct subdomains of the trans-Golgi network (TGN) and/or endosomes
Summary
Epithelial cells generate and maintain at least two biochemically distinct plasma membrane domains that reside within a continuous lipid bilayer (Rodriguez-Boulan and Powell, 1992; Drubin and Nelson, 1996; Yeaman et al, 1999). The polarized distribution of membrane proteins and lipids to the apical or basolateral domain depends on several factors. Most epithelial cells have the ability to sort newly synthesized plasma membrane components as they exit the TGN and package them into transport vesicles that are delivered to the correct target membrane. To ensure that this polarized distribution is maintained despite rapid endocytosis at both the apical and basolateral surfaces, endosomes in epithelial cells are likewise capable of polarized sorting and transport to allow for recycling of internalized proteins and lipids to their correct domains. Some membrane proteins may achieve polarity by selective retention at the apical or basolateral surface. This may reflect interactions with extracellular ligands or with intracellular
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