We have identified a detoxifying efflux carrier from Arabidopsis using a functional cloning strategy. A bacterial mutant, KAM3, is deficient in multidrug resistance and does not survive on medium containing norfloxacin. After transformation of KAM3 cells with an Arabidopsis cDNA library, transformants were selected for restored growth on the toxic medium. One cDNA clone that complemented KAM3 encodes a novel protein with twelve putative transmembrane domains and contains limited sequence homology to a multidrug and toxin efflux carrier from bacteria. We named this Arabidopsis protein AtDTX1 (for Arabidopsis thaliana Detoxification 1). A large gene family of at least 56 members encoding related proteins was identified from the Arabidopsis genome. Further functional analysis of AtDTX1 protein in KAM3 mutant demonstrated that AtDTX1 serves as an efflux carrier for plant-derived alkaloids, antibiotics, and other toxic compounds. Interestingly, AtDTX1 was also capable of detoxifying Cd(2+), a heavy metal. Further experiments suggest that AtDTX1 is localized in the plasma membrane in plant cells thereby mediating the efflux of plant-derived or exogenous toxic compounds from the cytoplasm.

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We found that cells of Vibrio parahaemolyticus possess an energy-dependent efflux system for norfloxacin. We cloned a gene for a putative norfloxacin efflux protein from the chromosomal DNA ofV. parahaemolyticus by using an Escherichia coli mutant lacking the major multidrug efflux system AcrAB as the host and sequenced the gene (norM). Cells of E. coli transformed with a plasmid carrying the norMgene showed elevated energy-dependent efflux of norfloxacin. The transformants showed elevated resistance not only to norfloxacin and ciprofloxacin but also to the structurally unrelated compounds ethidium, kanamycin, and streptomycin. These results suggest that this is a multidrug efflux system. The hydropathy pattern of the deduced amino acid sequence of NorM suggested the presence of 12 transmembrane domains. The deduced primary structure of NorM showed 57% identity and 88% similarity with that of a hypothetical E. coli membrane protein, YdhE. No reported drug efflux protein in the sequence databases showed significant sequence similarity with NorM. Thus, NorM seems to be a novel type of multidrug efflux protein. We cloned the ydhE gene from E. coli. Cells ofE. coli transformed with the cloned ydhE gene showed elevated resistance to norfloxacin, ciprofloxacin, acriflavine, and tetraphenylphosphonium ion, but not to ethidium, when MICs were measured. Thus, it seems that NorM and YdhE differ somehow in substrate specificity.

Apoptosis induced by nonsteroidal anti-inflammatory drugs (NSAIDs) is involved not only in the production of NSAIDinduced gastric lesions but also in the antitumor activity of these drugs. The endoplasmic reticulum (ER) stress response is a cellular mechanism that aids in protecting the ER against ER stressors and is involved in ER stressor-induced apoptosis. Here, we examine the relationship between this response and NSAID-induced apoptosis in cultured guineapig gastric mucosal cells. Exposure of cells to indomethacin, a commonly used NSAID, induced GRP78 as well as CHOP, a transcription factor involved in apoptosis. Three factors that positively regulate CHOP expression (ATF6, ATF4 and XBP-1) were activated and/or induced by indomethacin. NSAIDs other than indomethacin (diclofenac, ibuprofen and celecoxib) also induced CHOP. Monitoring of the transcriptional activities of ATF6 and CHOP by luciferase assay revealed that both were stimulated in the presence of indomethacin. Furthermore, indomethacin-induced apoptosis was suppressed in cultured guinea-pig gastric mucosal cells by expression of the dominant-negative form of CHOP, or in peritoneal macrophages from CHOP-deficient mice. These results suggest that ER stress response-related proteins, particularly CHOP, are involved in NSAID-induced apoptosis.

NorM of Vibrio parahaemolyticus apparently is a new type of multidrug efflux protein, with no significant sequence similarity to any known transport proteins. Based on the following experimental results, we conclude that NorM is an Na ؉ -driven Na ؉ /drug antiporter. Drug resistance, especially multidrug resistance, is presently a serious problem in hospitals. Drug efflux from cells is one of the major mechanisms of drug resistance in both prokaryotes and eukaryotes (11,12,15,18,26). Many drug efflux systems are known to exist in the biological world, and these transporters can be divided into four families: the major facilitator (MF) family, the small multidrug resistance (SMR) family, the resistance nodulation cell division (RND) family, and the ATP binding cassette family (4,6,17). Membrane transporters of the MF family possess 12 to 14 transmembrane domains. Transporters of the SMR family are rather small and usually possess four transmembrane domains. Transporters of the RND family require multiple components to function effectively. An electrochemical potential of H ϩ across cell membranes seems to be the driving force for drug efflux by members of the MF, SMR, and RND families of transporters (13,18,28,29). ATP is utilized as the energy donor in members of the ATP binding cassette family of multidrug efflux pumps (3, 26).The electrochemical potential of H ϩ across cell membranes is established mainly by the respiratory chain in aerobic or facultative anaerobic bacteria. The electrochemical potential of H ϩ across the membrane is converted to that of Na ϩ by Na ϩ /H ϩ antiporters (25,27). Both of the electrochemical potentials of H ϩ and Na ϩ across cell membranes can be utilized to drive solute uptake in bacterial cells. Solutes are taken up into cells by an H ϩ /substrate symport mechanism or an Na ϩ / substrate symport mechanism (19). An electrochemical potential of H ϩ is also utilized to drive extrusion of substrate from cells. Most multidrug efflux pumps in bacteria are driven by H ϩ , which is a mechanism for H ϩ /drug antiport (18). However, no Na ϩ -driven extrusion system for drugs, i.e., no Na ϩ / drug antiporter, has been reported for bacterial cell membranes. Although an Na ϩ /Ca 2ϩ exchanger (16) and an Na ϩ / urea antiporter (9) have been reported for animal cells, no Na ϩ /drug antiporter has been reported for animal cells. Vibrio parahaemolyticus, a slightly halophilic marine bacterium, is one of the major causes of food poisoning in Japan and many other countries (14). This microorganism requires Na ϩ for its growth (2). Energy metabolism and energy coupling in membranes of this microorganism are unique (20). Cells of V. parahaemolyticus possess a primary respiratory Na ϩ pump (24) and Na ϩ -coupled membrane processes, such as an Na ϩ / solute symporter (21,22,24) and an Na ϩ -driven flagellar motor (1). We thought that Na ϩ /drug antiporters might exist in this marine organism.If an Na ϩ /drug antiporter were to exist, it would be anticipated that (i) Na ϩ would stimulate drug efflux fro...