This project will investigate an ABC-type of transport ATPase, DrrAB, that confers resistance to doxorubicin and daunorubicin in the producer organism Streptomyces peucetius. DrrAB consists of two subunits DrrA and DrrB. Interestingly, DrrA bears homology to P-glycoprotein, a multidrug resistance protein, and to other members of the ABC family including CFTR and ABC 1. DrrAB and Pgp are also functionally similar: both confer doxorubicin resistance, DrrAB in the producer organism and Pgp in cancer cells. Because of the sequence, structural and functional similarity between DrrAB and Pgp, it is likely that they share a common ancestor. Hence, elucidation of the function of 'Drr' and the nature of the drug binding sites in 'Drr' will shed light on the mechanism of function of Pgp and on the evolution of multidrug resistance. Furthermore, DrrAB is ideal for understanding interaction between the membrane domain and the catalytic domain of ABC transporters. Preliminary experiments have shown that DrrA and DrrB are biochemically coupled; DrrA is required for the stability and maintenance of DrrB in the membrane and DrrB is required for the activity of DrrA. Experiments will be designed to test the hypothesis that DrrA forms a complex with DrrB and protects it from proteolysis before DrrB is targeted to the membrane, Experiments to study interaction between the two subunits will consist of both biochemical and genetic approaches including isolation of interaction-defective mutants and the second-site suppressors. These studies will have relevance in understanding targeting of membrane proteins and in elucidating the role played by the catalytic domains in stabilizing the membrane domains of multidomain or multisubunit proteins.
