Trafficking of ion transport proteins: The role of new partners

Appropriate intracellular trafficking of proteins is vital to cellular function. In this thesis we investigate the intracellular sorting and interaction partners of the H,K-ATPase. The H,K-ATPase is a heterodimeric ion pump that is principally known for its role in the acidification of the stomach lumen. The beta-subunit of the H,K-ATPase (HKbeta) and its associated proteins are responsible for the pump's intracellular trafficking and its membrane localization in epithelial cells. We find that HKbeta interacts with both the AP-1A and AP-1B subtypes of clathrin adaptor protein-1 (AP-1); however, the expression of AP-1B is not responsible for the pump's basolateral localization in polarized epithelial cells. We also identified the tetraspanin CD63 as a novel interaction partner for the H,K-ATPase beta-subunit. Our data suggest that CD63 may act as a linker between HKbeta and adaptor proteins-2 and -3, enhancing the internalization of the beta-subunit. These results indicate a novel protein trafficking role for tetraspanins. Further characterization of the association between the H,K-ATPase beta-subunit and CD63 using CD63/CD9 chimeras demonstrates that these proteins interact via their transmembrane and extracellular domains. We also find that CHAPS-stable interactions are sufficient for tetraspanins to affect the trafficking of their interaction partners. Additional experiments show that CD63 also interacts with a chloride channel (ClC7) that is found in osteoclasts. We have initiated studies to determine whether CD63 plays a physiologic role in the intracellular trafficking of ClC7. This work contributes new insights into the intracellular trafficking of the H,K-ATPase, and further elucidates the mechanisms of protein sorting within the cell.