Abstract
Polarity proteins promote the asymmetric organization of cells by orienting intracellular sorting mechanisms, such as protein trafficking and cytoskeletal assembly. The localization of individual polarity proteins in turn is often determined by association with factors that mediate contact with other cells or the substratum. This arrangement for the Par and Crb apical polarity complexes at the tight junction is disrupted by the adaptor protein Amot. Amot directly binds the scaffolding proteins Patj and Mupp1 and redistributes them and their binding partners from the plasma membrane to endosomes. However, the mechanism by which Amot is targeted to endosomes is unknown. Here, a novel lipid binding domain within Amot is shown to selectively bind with high affinity to membranes containing monophosphorylated phosphatidylinositols and cholesterol. With similar lipid specificity, Amot inserts into and tubulates membranes in vitro and enlarges perinuclear endosomal compartments in cells. Based on the similar distribution of Amot with cholesterol, Rab11, and Arf6, such membrane interactions are identified at juxtanuclear endocytic recycling compartments. Taken together, these findings indicate that Amot is targeted along with associated apical polarity proteins to the endocytic recycling compartment via this novel membrane binding domain.
Highlights
Epithelial cells are morphologically plastic and transition between the epithelial and mesenchymal phenotypes
Definition of the Amot coiled-coil homology (ACCH) Domain—The NH2-terminal 240 residues of Amot80 has been predicted to encode a coiled-coil fold with positively charged residues that are positionally conserved with the BAR domain of amphiphysin [22]
Amot encodes the prototypical member of a novel and conserved family of predicted membrane binding functionalities collectively termed ACCH domains
Summary
Reagents—Expression plasmids were constructed using Creator (Clontech)-based vectors described previously [32]. Monolayer Measurements—The monolayer penetration of the ACCH domain into the phospholipid monolayer of different lipid compositions was measured in terms of the change in surface pressure () using a 1-ml circular Teflon trough and wire probe connected to a Kibron MicroTrough X (Kibron, Inc., Helsinki, Finland). Vesicle Dye Leakage Measurements—The release of 5-carboxyfluorescein dye encapsulated into liposomes of different compositions was monitored in the presence of protein using an Aminco Bowman Series 2 spectrofluorometer. Liposomes for these experiments were prepared as described for SPR measurements with the addition of 10 mM 5-carboxyfluorescein in the resuspension buffer. Experiments were performed via the addition of ACCH, epsin N-terminal homology (ENTH), or Triton X-100 to the respective solution of liposomes in a fluorescence cuvette. Control experiments indicate the specificity for membrane-tubulating proteins ACCH and ENTH to disrupt vesicles in accordance with their lipid specificity and affinity
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