Multidrug resistance protein 1 (MRP1) and P-glycoprotein, which are ATP-dependent multidrug efflux pumps and involved in multidrug resistance of tumor cells, are members of the ATP binding cassette proteins and contain two nucleotide-binding folds (NBFs). P-glycoprotein hydrolyzes ATP at both NBFs, and vanadateinduced nucleotide trapping occurs at both NBFs. We examined vanadate-induced nucleotide trapping in MRP1 stably expressed in KB cell membrane by using 8-azido-[␣-32 P]ATP. Vanadate-induced nucleotide trapping in MRP1 was found to be stimulated by reduced glutathione, glutathione disulfide, and etoposide and to be synergistically stimulated by the presence of etoposide and either glutathione. These results suggest that glutathione and etoposide interact with MRP1 at different sites and that those bindings cooperatively stimulate the nucleotide trapping. Mild trypsin digestion of MRP1 revealed that vanadate-induced nucleotide trapping mainly occurs at NBF2. Our results suggest that the two NBFs of MRP1 might be functionally nonequivalent.Multidrug resistance of tumor cells is a major obstacle to cancer chemotherapy. This phenomenon is frequently associated with the expression of P-glycoprotein and multidrug resistance protein 1 (MRP1), 1 both of which are ATP binding cassette (ABC) proteins. P-glycoprotein and MRP1 function as ATP-dependent efflux pumps that extrude cytotoxic drugs from the cells before they reach their intracellular targets, thus conferring resistance to many structurally dissimilar anticancer drugs, such as the Vinca alkaloids, colchicine, actinomycin D, etoposide, taxol, and anthracyclines (1-5). However, the mechanism of transport for MRP1 could be different from that for P-glycoprotein, because the depletion of intracellular glutathione (GSH) by buthionine sulfoximine results in a complete reversal of resistance to anticancer drugs of some cell lines expressing MRP1 (6, 7), but buthionine sulfoximine has no effect on P-glycoprotein-mediated multidrug resistance. It has been reported that MRP1 transports GSH-S-conjugates such as leukotriene C 4 , glutathione disulfide (GSSG), and 2,4-dinitrophenyl-S-glutathione (8 -11) and that MRP1 mediates ATP-dependent transport of vincristine, daunorubicin, and etoposide in the presence of . From these findings, it has been postulated that MRP1 can actively cotransport GSH and unmodified xenobiotics as well as GSH-S-conjugates.We have reported that MRP1 in membrane from a human MRP1 cDNA transformant can be specifically photoaffinity labeled with 8-azido-[␣-32 P]ATP by vanadate-induced nucleotide trapping (15). Vanadate and Mg 2ϩ were required for trapping of nucleotide, and photoaffinity labeling was inhibited by the excess ADP as well as ATP. These results have suggested that a stable inhibitory complex MRP1⅐MgADP⅐Vi, an analog of the MRP1⅐MgADP⅐P i transition state complex, is formed in the presence of vanadate, as suggested for P-glycoprotein (16). Vanadate-induced nucleotide trapping in P-glycoprotein has been reported to be stimulated by the t...

The generation of high-density lipoprotein (HDL), one of the most critical events for preventing atherosclerosis, is mediated by ATPbinding cassette protein A1 (ABCA1). ABCA1 is known to transfer cellular cholesterol and phospholipids to apolipoprotein A-I (apoA-I) for generating discoidal HDL (dHDL) particles, composed of 100-200 lipid molecules surrounded by two apoA-I molecules; however, the regulatory mechanisms are still poorly understood. Here we observed ABCA1-GFP and apoA-I at the level of single molecules on the plasma membrane via a total internal reflection fluorescence microscope. We found that about 70% of total ABCA1-GFP spots are immobilized on the plasma membrane and estimated that about 89% of immobile ABCA1 molecules are in dimers. Furthermore, an ATPase-deficient ABCA1 mutant failed to be immobilized or form a dimer. We found that the lipid acceptor apoA-I interacts with the ABCA1 dimer to generate dHDL and is followed by ABCA1 dimermonomer interconversion. This indicates that the formation of the ABCA1 dimer is the key for apoA-I binding and nascent HDL generation. Our findings suggest the physiological significance of conversion of the ABCA1 monomer to a dimer: The dimer serves as a receptor for two apoA-I molecules for dHDL particle generation.membrane protein | transporter P lasma high-density lipoprotein (HDL) is critical for preventing coronary artery disease (1). A member of the ATPdependent transporter family of ABC proteins, ATP-binding cassette protein A1 (ABCA1) initiates the generation of discoidal HDL (dHDL), a bilayer fragment consisting of 100-200 lipids wrapped by two molecules of apolipoprotein A-I (apoA-I) (2-4), by exporting cholesterol and phospholipids to lipid-free apoA-I in serum (5). More than 70 mutations have been identified in the ABCA1 gene. Indeed, mutations in ABCA1 lead to Tangier disease, which is characterized by plasma HDL deficiency (6-10). ABCA1 has two large extracellular domains (ECDs), and the two intramolecular disulfide bonds between them are necessary for apoA-I binding and HDL formation (11-13) (Fig. 1A). Two pieces of evidence suggest that apoA-I interacts with a specific conformation of the ECDs in an ATP-dependent manner: Chemical cross-linkers can cross-link apoA-I with ABCA1, and ATPasedeficient ABCA1 mutants fail to mediate apoA-I binding and crosslinking. However, the importance of direct binding of ABCA1-apoA-I in HDL formation is still controversial and, furthermore, how a dHDL particle containing two molecules of apoA-I is formed from lipid-free apoA-I monomers and membrane lipids is unknown.To address these issues, we performed single-molecule fluorescence imaging (14, 15) of ABCA1 and apoA-I on the plasma membrane (PM) via a total internal reflection fluorescence (TIRF) microscope in living cells. We examined the dynamic behaviors of ABCA1 as well as the interaction of ABCA1 with apoA-I, and found that ABCA1 forms an immobile dimer on the PM; the ABCA1 dimer then dissociates into diffusing monomers upon interaction with apoA-I, finally gen...

No abstract

No abstract

A small-molecule fluorescent probe specific for human pluripotent stem cells would serve as a useful tool for basic cell biology research and stem cell therapy. Screening of fluorescent chemical libraries with human induced pluripotent stem cells (iPSCs) and subsequent evaluation of hit molecules identified a fluorescent compound (Kyoto probe 1 [KP-1]) that selectively labels human pluripotent stem cells. Our analyses indicated that the selectivity results primarily from a distinct expression pattern of ABC transporters in human pluripotent stem cells and from the transporter selectivity of KP-1. Expression of ABCB1 (MDR1) and ABCG2 (BCRP), both of which cause the efflux of KP-1, is repressed in human pluripotent stem cells. Although KP-1, like other pluripotent markers, is not absolutely specific for pluripotent stem cells, the identified chemical probe may be used in conjunction with other reagents.

Forty-nine ABC protein genes exist on human chromosomes. Eukaryotic ABC proteins were originally recognized as drug efflux pumps involved in the multidrug resistance of cancer cells. However, it is now realized that one of their major physiological roles is cellular lipid transport and homeostasis, and their dysfunction is often associated with human diseases. ABCA1 and ABCA7 mediate the apolipoprotein-dependent formation of a high-density lipoprotein-cholesterol complex. ABCA3 is indispensable for pulmonary surfactant secretion. ABCG5 and ABCG8 are involved in the secretion of plant sterols and cholesterol into bile. However, the primary substrates and mechanism of action of these ABC proteins have not been precisely defined. In this review article, we first describe the general structure and functions of eukaryotic ABC proteins. The current model of ABCA1 functionality is then explained based on studies on a topological model, subcellular localization, apoA-I dependence of HDL formation, functional defects of Tangier disease mutants, and ATP hydrolysis of purified ABCA1. ABCA1 is supposed to function as a transporter of lipids as well as a receptor for apoA-I. ABCA3 is likely involved in accumulating phospholipids and cholesterol in lamellar bodies and in generating multivesicular structures.

Rings with platinum: The first platinum disulfur and diselenium complexes have been synthesized by taking advantage of new phosphane ligands bearing an extremely bulky substituent (see structure). The molecular structures of the disulfur and diselenium complexes are very similar, and these complexes, as well as an analogous dioxygen complex [(PPh3)2PtO2], have a square‐planar geometry and a three‐membered PtE2 (E=S, Se) ring.

Novel platinum complexes having an oxochalcogenido ligand such as S2O, Se2O, S2O3, and SeO3 were synthesized by the oxidation reactions of the overcrowded dichalcogenido platinum complexes [(BbtMe2P)2PtX2] (Bbt = 2,6-bis[bis(trimethylsilyl)methyl]-4-[tris(trimethylsilyl)methyl]phenyl; X = S, Se).