Apical Membrane Targeting of Nedd4 Is Mediated by an Association of Its C2 Domain with Annexin XIIIb

• Published in: The Journal of cell biology 2000-06, Vol.149 (7), p.1473-1483
• Main Authors: Plant, Pamela J., Lafont, Frank, Lecat, Sandra, Verkade, Paul, Simons, Kai, Rotin, Daniela
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Annexins; Annexins - metabolism; Binding Sites; Binding Sites - physiology; Calcium; Calcium - metabolism; Calcium-Binding Proteins; Calcium-Binding Proteins - metabolism; Cell Membrane; Cell Membrane - metabolism; Cell Membrane - ultrastructure; Cells, Cultured; Cellular Biology; Endosomal Sorting Complexes Required for Transport; Life Sciences; Ligases; Ligases - metabolism; Molecular Sequence Data; Nedd4 Ubiquitin Protein Ligases; Neurons; Organelles; Organelles - metabolism; Organelles - ultrastructure; Original; Protein Structure, Tertiary; Protein Structure, Tertiary - physiology; Proteins; Subcellular Processes; Ubiquitin-Protein Ligases
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.149.7.1473

Nedd4 is a ubiquitin protein ligase (E3) containing a C2 domain, three or four WW domains, and a ubiquitin ligase HECT domain. We have shown previously that the C2 domain of Nedd4 is responsible for its Ca2+-dependent targeting to the plasma membrane, particularly the apical region of epithelial MDCK cells. To investigate this apical preference, we searched for Nedd4-C2 domain-interacting proteins that might be involved in targeting Nedd4 to the apical surface. Using immobilized Nedd4-C2 domain to trap interacting proteins from MDCK cell lysate, we isolated, in the presence of Ca2+, a ∼35-40-kD protein that we identified as annexin XIII using mass spectrometry. Annexin XIII has two known isoforms, a and b, that are apically localized, although XIIIa is also found in the basolateral compartment. In vitro binding and coprecipitation experiments showed that the Nedd4-C2 domain interacts with both annexin XIIIa and b in the presence of Ca2+and the interaction is direct and optimal at 1 μM Ca2+. Immunofluorescence and immunogold electron microscopy revealed colocalization of Nedd4 and annexin XIIIb in apical carriers and at the apical plasma membrane. Moreover, we show that Nedd4 associates with raft lipid microdomains in a Ca2+-dependent manner, as determined by detergent extraction and floatation assays. These results suggest that the apical membrane localization of Nedd4 is mediated by an association of its C2 domain with the apically targeted annexin XIIIb.