Loo T W, Clarke D M
MRC Group in Membrane Biology, Departments of Medicine, University of Toronto, Ontario, Canada.
J Biol Chem. 1995 Sep 15;270(37):21839-44. doi: 10.1074/jbc.270.37.21839.
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.
P-糖蛋白由两个同源部分组成，每个部分都由一个跨膜结构域和一个核苷酸结合结构域构成。为了了解这些结构域在P-糖蛋白中是如何相互作用的，我们将每个结构域作为一个单独的多肽进行表达，并使用免疫共沉淀分析来检测其相互作用。我们发现，P-糖蛋白两个部分之间的相互作用是通过两个跨膜结构域之间的相互作用以及核苷酸结合结构域之间的相互作用介导的。此外，核苷酸结合结构域还与分子每一半中的跨膜结构域相关联。相比之下，我们没有检测到第一个核苷酸结合结构域与第二个跨膜结构域之间或第二个核苷酸结合结构域与第一个跨膜结构域之间存在任何相互作用。由于P-糖蛋白的生物发生似乎涉及伴侣蛋白钙连蛋白和热休克蛋白70（Hsc70），我们随后测试了各个结构域是否与分子伴侣相关联。我们发现钙连蛋白仅与跨膜结构域相关联，而Hsc70仅与核苷酸结合结构域相关联。这些结果表明，P-糖蛋白各结构域之间的非共价相互作用可能有助于P-糖蛋白的结构和功能，并且分子伴侣可能参与每个结构域的折叠。
