Novel Function of Human RLIP76: ATP-Dependent Transport of Glutathione Conjugates and Doxorubicin

详细信息    查看全文

• 作者：Sanjay Awasthi ; Jizhong Cheng ; Sharad S. Singhal ; Manjit K. Saini ; Utpal Pandya ; Slawomir Pikula ; Joanna Bandorowicz-Pikula ; Shivendra V. Singh ; Piotr Zimniak ; and Yogesh C. Awasthi
• 刊名：Biochemistry
• 出版年：2000
• 出版时间：August 8, 2000
• 年：2000
• 卷：39
• 期：31
• 页码：9327 - 9334
• 全文大小：140K
• 年卷期：v.39,no.31(August 8, 2000)
• ISSN：1520-4995

文摘

Active transport of conjugated and unconjugated electrophiles out of cells is essential for cellularhomeostasis. We have previously identified in human tissues a transporter, DNP-SG [S-(2,4-dinitrophenyl)glutathione] ATPase, capable of carrying out this function [Awasthi et al. (1998) Biochemistry 37, 5231-5238, 5239-5248]. We now report the cloning of DNP-SG ATPase. The sequence of the cDNA clonewas identical to that of human RLIP76, a known Ral-binding protein. RLIP76 expressed in E. coli waspurified by DNP-SG affinity chromatography. Purified recombinant RLIP76: (1) had ATPase activitystimulated by DNP-SG or doxorubicin (DOX), and the K_m values of RLIP76 for ATP, DOX, and DNP-SG were similar to those reported for DNP-SG ATPase; (2) upon reconstitution with asolectin as well aswith defined lipids, catalyzed ATP-dependent transport of DNP-SG and DOX with kinetic parameterssimilar to those of DNP-SG ATPase; (3) when transfected into K562 cells, resulted in increased resistanceto DOX, and increased ATP-dependent transport of DNP-SG and DOX by inside-out membrane vesiclesfrom transfected cells; (4) direct uptake of purified RLIP76 protein into mammalian cells from donorproteoliposomes confers DOX resistance. These results indicate that RLIP76, in addition to its role insignal transduction, can catalyze transport of glutathione conjugates and xenobiotics, and may contributeto the multidrug resistance phenomenon.