|
pubmed:abstractText |
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 Ca(2+)-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 Ca(2+), a approximately 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 Ca(2+), and the interaction is direct and optimal at 1 microM Ca(2+). 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 Ca(2+)-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.
|
|
pubmed:affiliation |
Program in Cell Biology, The Hospital for Sick Children and Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada, M5G 1X8.
|
