Much is known about the transport of arsenite and antimonite into microbes, but the identities of mammalian transport proteins are unknown. The Saccharomyces cerevisiae FPS1 gene encodes a membrane protein homologous to the bacterial aquaglyceroporin GlpF and to mammalian aquaglyceroporins AQP7 and AQP9. Fps1p mediates glycerol uptake and glycerol efflux in response to hypoosmotic shock. Fps1p has been shown to facilitate uptake of the metalloids arsenite and antimonite, and the Escherichia coli homolog, GlpF, facilitates the uptake and sensitivity to metalloid salts. In this study, the ability of mammalian aquaglyceroporins AQP7 and AQP9 to substitute for the yeast Fps1p was examined. The fps1⌬ strain of S. cerevisiae exhibits increased tolerance to arsenite and antimonite compared to a wild-type strain. Introduction of a plasmid containing AQP9 reverses the metalloid tolerance of the deletion strain. AQP7 was not expressed in yeast. The fps1⌬ cells exhibit reduced transport of 73 As(III) or 125 Sb(III), but uptake is enhanced by expression of AQP9. Xenopus laevis oocytes microinjected with either AQP7 or AQP9 cRNA exhibited increased transport of 73 As(III). These results suggest that AQP9 and AQP7 may be a major routes of arsenite uptake into mammalian cells, an observation potentially of large importance for understanding the action of arsenite as a human toxin and carcinogen, as well as its efficacy as a chemotherapeutic agent for acute promyelocytic leukemia.Fps1p ͉ GlpF ͉ acute promyelocytic leukemia

The toxicity of the metalloids arsenic and antimony is related to uptake, whereas detoxification requires efflux. In this report we show that uptake of the trivalent inorganic forms of arsenic and antimony into cells of Escherichia coli is facilitated by the aquaglyceroporin channel GlpF and that transport of Sb(III) is catalyzed by the ArsB carrier protein; everted membrane vesicles accumulated Sb(III) with energy supplied by NADH oxidation, reflecting efflux from intact cells. Dissipation of either the membrane potential or the pH gradient did not prevent Sb(III) uptake, whereas dissipation of both completely uncoupled the carrier protein, suggesting that transport is coupled to either the electrical or the chemical component of the electrochemical proton gradient. Reciprocally, Sb(III) transport via ArsB dissipated both the pH gradient and the membrane potential. These results strongly indicate that ArsB is an antiporter that catalyzes metalloid-proton exchange. Unexpectedly, As(III) inhibited ArsB-mediated Sb(III) uptake, whereas Sb(III) stimulated ArsB-mediated As(III) transport. We propose that the actual substrate of ArsB is a polymer of (AsO) n , (SbO) n , or a co-polymer of the two metalloids.Arsenic, one of the most prevalent toxic metals in the environment, derives primarily from geochemical origins but also from man-made sources. Consequently, nearly every organism has intrinsic or acquired mechanisms for arsenic detoxification (1). The arsenical resistance operon (arsRDABC) of the conjugative R-factor R773 confers resistance to inorganic As(III) and Sb(III) in Escherichia coli. The arsenic transport system exhibits a dual mode of energy coupling depending on the subunit composition (2). When both ArsA and ArsB are present, they form the As(III)-translocating ArsAB ATPase, which is independent of the electrochemical proton gradient (3). In contrast, in the absence of ArsA, ArsB catalyzes As(III) extrusion coupled to electrochemical energy, which suggests that ArsB is a uniporter that extrudes the arsenite anion in response to the positive exterior membrane potential (4). This dual mode of energy coupling led us to propose that the ArsAB pump evolved by association of a secondary carrier with a soluble ATPase (5).Over a decade ago, we proposed that other primary ATP-coupled pumps such as ATP-binding cassette transporters evolved in similar ways (5, 6).ArsB is the most widespread determinant of arsenic resistance in bacteria and archaea, yet its transport properties are not well characterized. It is a member of the ion transporter superfamily (7), with 12 membrane-spanning segments and a membrane topology that is similar to many carrier proteins (8). To date, it has been shown to transport only As(III) (4). Here we report for the first time that ArsB transports inorganic Sb(III) in E. coli, and we describe the relationship with As(III) transport. Considering that the pK a of Sb(III) is 11.8 and As(III) is 9.2, at cytosolic pH the concentration of the oxyanion of either metalloid is negligible. Thus, it...

Arsenic exposure is associated with hypertension, diabetes and cancer. Some mammals methylate arsenic. Saccharomyces cerevisiae hexose permeases catalyze As(OH) 3 uptake. Here we report that mammalian glucose transporter GLUT1 catalyzes As(OH) 3 and CH 3 As(OH) 2 uptake in yeast or in Xenopus laevis öocytes. Expression of GLUT1 in a yeast lacking other glucose transporters allows for growth on glucose. Yeast expressing yeast HXT1 or rat GLUT1 transport As(OH) 3 and CH 3 As (OH) 2 . The K m of GLUT1 is to 1.2 mM for CH 3 As(OH) 2 , compared to a K m of 3 mM for glucose. Inhibition between glucose and CH 3 As(OH) 2 is noncompetitive, suggesting differences between the translocation pathways of hexoses and arsenicals. Both human and rat GLUT1 catalyze uptake of both As(OH) 3 and CH 3 As(OH) 2 in öocytes. Thus GLUT1 may be a major pathway uptake of both inorganic and methylated arsenicals in erythrocytes or the epithelial cells of the blood-brain barrier, contributing to arsenic-related cardiovascular problems and neurotoxicity.Arsenic ranks first on the United States Government's Comprehensive Environmental Response, Compensation, and Liability (Superfund) Act Priority List of Hazardous Substances . In response to the Safe Water Drinking Act, the United States Environmental Protection Agency has set the allowable level of arsenic in drinking water at 10 ppb , which is less than the level in the water supplies of many U.S. municipalities . In addition, the Environmental Protection Agency's Office of Pesticide Programs is concerned with exposure to the organic arsenicals methylarsenic acid and dimethylarsenic acid, pentavalent arsenicals used as pesticides and herbicides .Health effects associated with arsenic exposure include cardiovascular and peripheral vascular disease, neurological disorders, diabetes mellitus and various cancers, including liver, bladder, kidney and skin [1][2][3]. Arsenic trioxide, which is As 2 O 3 in the solid, anhydrous form and As (OH) 3 in solution at physiological pH [4], is used clinically as a chemotherapeutic drug for treatment of acute promyelocytic leukemia [5]. Although epidemiological studies demonstrated that arsenic exposure is associated with a high frequency of circulatory and neurological problems [2,6,7], how arsenic causes non-cancer-related diseases is far from clear. Here we report that GLUT1 facilitates uptake of As(OH) 3 and CH 3 As(OH) 2 using heterologous expression in S. cerevisiae and X. laevis öocytes. S. cerevisiae is a valuable model system for structure-function studies of mammalian membrane proteins such as GLUT1 [22,23]. A yeast strain with low uptake of As(OH) 3 and CH 3 As(OH) 2 was constructed by deletion of all 18 hexose transporters, FPS1, which encodes an aquaglyceroporin that facilitates As(OH) 3 influx, and ACR3, the gene for an...

Arsenic trioxide is a toxic metalloid and carcinogen that is also used as an anticancer drug, and for this reason it is important to identify the routes of arsenite uptake by cells. In this study the ability of hexose transporters to facilitate arsenic trioxide uptake in Saccharomyces cerevisiae was examined. In the absence of glucose, strains with disruption of the arsenite efflux gene ACR3 accumulated high levels of 73 As(OH) 3 . The addition of glucose inhibited uptake by ϳ80%. Disruption of FPS1, the aquaglyceroporin gene, reduced glucose-independent uptake by only about 25%, and the residual uptake was nearly completely inhibited by hexoses, including glucose, galactose, mannose, and fructose but not pentoses or disaccharides. A strain lacking FPS1, ACR3, and all genes for hexose permeases except for HXT3, HXT6, HXT7, and GAL2 exhibited hexoseinhibitable 73 As(OH) 3 uptake, whereas a strain lacking all 18 hexose transport-related genes (HXT1 to HXT17 and GAL2), FPS1 and ACR3, exhibited <10% of wild type 73 As(OH) 3 transport. When HXT1, HXT3, HXT4, HXT5, HXT7, or HXT9 was individually expressed in that strain, hexose-inhibitable 73 As(OH) 3 uptake was restored. In addition, the transport of [ 14 C]glucose was inhibited by As(OH) 3 . These results clearly demonstrate that hexose permeases catalyze the majority of the transport of the trivalent metalloid arsenic trioxide.Arsenic trioxide is a carcinogen (1) and is also the active ingredient in the chemotherapeutic drug Trisenox, which is used for the treatment of acute promyelocytic leukemia (2). For trivalent arsenic to be a carcinogen or for Trisenox to be a useful drug, they must first be taken up into cells. Because it is unlikely that transport systems evolved for the uptake of arsenic trioxide, it is most likely taken up by transporters for biological molecules. To predict the routes of uptake, it is necessary to have a clear understanding of the species present in solution at neutral pH. Arsenic trioxide is in equilibrium with the oxyanion arsenite, but, with a pK a of 9.2, it should be fully protonated and uncharged at neutral pH. We have shown by extended x-ray absorption fine structure spectroscopy that in solution at neutral pH, the predominate species is As(OH) 3 .1 Thus, physiologically, As(III) appears to be a polyhydroxylated molecule; therefore, polyol transporters would be reasonable candidates for uptake systems. One class of polyol transporters are the aquaglyceroporins, which are channels for small uncharged solutes, such as glycerol (3). Members of the aquaglyceroporin family from bacteria (GlpF), yeast (Fps1p), and mammals (Aqp9) are As(OH) 3 channels (4 -6). Disruption of the yeast FPS1 gene resulted in substantial loss of uptake of As(OH) 3 when glucose was present in the assay medium (4).In this study, we report that hexose permeases are responsible for the majority of As(OH) 3 accumulation in Saccharomyces cerevisiae. In glucose-free medium, the fps1⌬ strain exhibits only a 25% reduction in 73 As(OH) 3 uptake compared with its parent...