P-glycoprotein. Associations between domains and between domains and molecular chaperones.

ABSTRACT

P-glycoprotein consists of two homologous halves, each composed of a transmembrane domain and a nucleotide-binding domain. In order to understand how the domains interact in P-glycoprotein, we expressed each domain as a separate polypeptide and tested for associations using coimmunoprecipitation assays. We found that the interactions between the two halves of P-glycoprotein were mediated through associations between the two transmembrane domains as well as through the nucleotide-binding domains. In addition, the nucleotide-binding domain also associated with the transmembrane domain in each half of the molecule. By contrast, we could not detect any association either between the first nucleotide-binding domain and the second transmembrane domain, or between the second nucleotide-binding domain and the first transmembrane domain. We then tested whether individual domains associated with molecular chaperones, since biogenesis of P-glycoprotein appears to involve the chaperones calnexin and Hsc70. We found that calnexin associated only with the transmembrane domains, while Hsc70 associated only with the nucleotide-binding domains. These results suggest that noncovalent interaction between the domains of P-glycoprotein can contribute to structure and function of P-glycoprotein and that chaperones may participate in the folding of each domain.