Date of Award

11-30-2008

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Dr. Parjit Kaur - Chair

Second Advisor

Dr. Chung-Dar Lu

Third Advisor

Dr. P.C Tai

Fourth Advisor

Dr. Zehava Eichenbaum

Abstract

ATP binding cassette (ABC) transporters constitute one of the largest families of transport proteins. The occurrence of multidrug resistance (MDR) in human cancer cells has been correlated with the over expression of human ABC, P-glycoprotein (Pgp). Streptomyces peucetius produces two anticancer agents, doxorubicin and daunorubicin, that belong to the anthracycline family of antibiotics. The organism is self-resistant to the potent effects of the antibiotics it produces due to the action of an efflux pump, DrrAB. Both Pgp and DrrAB carry out similar functions, but in two different cell types. An understanding of the bacterial drug transporter DrrAB is thus expected to help in obtaining a better understanding of the function and evolution of the multidrug transporter P-glycoprotein. In DrrAB, the catalytic and membrane domains are present on separate subunits, DrrA and DrrB respectively. How the catalytic ATP-binding domains and the membrane domains in transporters interact with each other, or how energy is transduced between them, is not well understood. We introduced several single cysteine substitutions in DrrB and then by using a cysteine to amine hetero-bifunctional cross-linker showed that DrrA interacts predominantly with the N-terminal cytoplasmic tail of DrrB. Within this region of DrrB, we also identified a sequence with similarities to the EAA motif found in importers of the ABC family of proteins, thus leading to the proposal that the EAA or the EAA-like motif may be involved in forming a generalized interface between the ABC and the TMD of both uptake and export systems. By using a combination of approaches, including point mutations and disulfide cross-linking analysis, we show here that the Q-loop region of DrrA plays an important role in dimerization of DrrA as well as in interactions with DrrB. Furthermore, we also show that the interaction of the Q-loop with the N-terminus of DrrB is involved in transmitting conformational changes between DrrA and DrrB. The scope of the present study further extends into identifying the factors involved in the biogenesis of the DrrAB pump. We have identified two accessory proteins namely, FtsH and GroEL that may be involved in proper folding and assembly of the transporter.

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Biology Commons

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