Functional Reassembly of ATP-Dependent Xenobiotic Transport by the N- and C-Terminal Domains of RLIP76 and Identification of ATP Binding Sequences

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• 作者：Sanjay Awasthi ; Ji-Zhong Cheng ; Sharad S. Singhal ; Utpal Pandya ; Rajendra Sharma ; Shivendra V. Singh ; Piotr Zimniak ; and Yogesh C. Awasthi
• 刊名：Biochemistry
• 出版年：2001
• 出版时间：April 3, 2001
• 年：2001
• 卷：40
• 期：13
• 页码：4159 - 4168
• 全文大小：232K
• 年卷期：v.40,no.13(April 3, 2001)
• ISSN：1520-4995

文摘

We have recently shown that RLIP76, a Ral-binding, GTPase-activating protein, is an ATP-dependent transporter of doxorubicin (DOX) as well as glutathione conjugates [Awasthi, S., et al. (2000)Biochemistry 39, 9327-9334]. RLIP76 overexpressed in human cells or transformed E. coli undergoesproteolysis to yield several fragments, including two prominent peptides, N-RLIP76^1-367 and C-RLIP76^410-655,from the N- and C-terminal domains, respectively. To investigate whether the fragmentation of RLIP76has any relevance to its transport function, we have studied the characteristics of these two peptidefragments. Recombinant N-RLIP76^1-367 and C-RLIP76^410-655 were purified from overexpressingtransformed E. coli. While N-RLIP76^1-367 readily underwent proteolysis, showing SDS-gel patterns similarto those of RLIP76, C-RLIP76^410-655 was resistant to such degradation. Both N-RLIP76^1-367 andC-RLIP76^410-655 had ATPase activity (K_m for ATP, 2.5 and 2.0 mM, respectively) which was stimulatedby DNP-SG, DOX, and colchicine (COL). ATP binding to both peptides was confirmed by photoaffinitylabeling with 8-azido-ATP that was increased in the presence of compounds that stimulated their ATPaseactivity. Photoaffinity labeling was also increased in the presence of vanadate, indicating trapping of areaction intermediate in the ATP binding site. The ATP binding sites in N-RLIP76^1-367 and C-RLIP76^410-655were identified to be ⁶⁹GKKKGK⁷⁴ and ⁴¹⁸GGIKDLSK⁴²⁵, respectively. Mutation of K⁷⁴ and K⁴²⁵ to Mresidues, in N-RLIP76^1-367 and C-RLIP76^410-655, respectively, abrogated their ATPase activity as well asazido-ATP labeling. Proteoliposomes reconstituted with either N-RLIP76^1-367 or C-RLIP76^410-655 alonedid not catalyze ATP-dependent transport of DOX or COL. However, proteoliposomes reconstituted witha mixture of N-RLIP76^1-367 and C-RLIP76^410-655 mediated such transport. Proteoliposomes reconstitutedwith the mixture of mutant peptides lacking ATPase activity did not exhibit transport activity. Presentstudies have identified the ATP binding sites in RLIP76, and show that DOX and COL transport can bereconstituted by two fragments of RLIP76.