Functional reassembly of ATP-dependent xenobiotic transport by the N- and C-terminal domains of RLIP76 and identification of ATP binding sequences

We have recently shown that RLIP76, a Pal-binding, GTPase-activating protei n, is an ATP-dependent transporter of doxorubicin (DOX) as well as glutathi one conjugates [Awasthi, S., et al. (2000) Biochemistry 39, 9327-9334]. RLI P76 overexpressed in human cells or transformed E. coli undergoes proteolys is to yield several fragments, including two prominent peptides, N-RLIP6(1- 367) and C-RLIP76(410-655), from the N- and C-terminal domains, respectivel y. To investigate whether the fragmentation of RLIP76 has any relevance to its transport function, we have studied the characteristics of these two pe ptide fragments. Recombinant N-RLIP76(1-367) and C-RLIP76(410-655) were pur ified from overexpressing transformed E, coli. While N-RLIp76(1-367) readil y underwent proteolysis, showing SDS-gel patterns similar to those of RLIP7 6, C-RLIP76(410-655) was resistant to such degradation. Both N-RLIp76(1-367 ) and C-RLIP76(410-655) had ATPase activity (K-m for ATP, 2.5 and 2.0 mM, r espectively) which was stimulated by DNP-SG, DOX, and colchicine (COL). ATP binding to both peptides was confirmed by photoaffinity labeling with 8-az ido-ATP that was increased in the presence of compounds that stimulated the ir ATPase activity. Photoaffinity labeling was also increased in the presen ce of vanadate, indicating trapping of a reaction intermediate in the ATP b inding site. The ATP binding sites in N-RLIP76(1-367) and C-RLIP76(410-655) were identified to be (69)GKKKGK(74) and (418)GGIKDLSK(425), respectively. Mutation of K-74 and K-425 to M residues, in N-RLIP76(1-367) and C-RLIP76( 410-655), respectively, abrogated their ATPase activity as well as azido-AT P labeling. Proteoliposomes reconstituted with either N-RLIP76(1-367) or C- RLIP76(410-655) alone did not catalyze ATP-dependent transport of DOX or CO L. However, proteoliposomes reconstituted with a mixture of N-RLIP76(1-367) and C-RLIP76(410-655) mediated such transport. Proteoliposomes reconstitut ed with the mixture of mutant peptides lacking ATPase activity did not exhi bit transport activity. Present studies have identified the ATP binding sit es in RLIP76, and show that DOX and COL transport can be reconstituted by t wo fragments of RLIP76.