AtCCX3 Is an Arabidopsis Endomembrane H + -Dependent K + Transporter 1[W][OA] Jay Morris, Hui Tian, Sunghun Park, Coimbatore S. Sreevidya, John M. Ward, and Kendal D. Hirschi* Vegetable and Fruit Improvement Center, Texas A&M University, College Station, Texas 77845 (J.M., S.P., K.D.H.); Plant Physiology Group, United States Department of Agriculture/Agriculture Research Service, Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030 (J.M., K.D.H.); Department of Plant Biology, University of Minnesota, St. Paul, Minnesota 55108 (H.T., J.M.W.); and Department of Immunology, M.D. Anderson Cancer Center, Houston, Texas 77030 (C.S.S.) The Arabidopsis (Arabidopsis thaliana) cation calcium exchangers (CCXs) were recently identified as a subfamily of cation transporters; however, no plant CCXs have been functionally characterized. Here, we show that Arabidopsis AtCCX3 (At3g14070) and AtCCX4 (At1g54115) can suppress yeast mutants defective in Na + ,K + , and Mn 2+ transport. We also report high-capacity uptake of 86 Rb + in tonoplast-enriched vesicles from yeast expressing AtCCX3. Cation competition studies showed inhibition of 86 Rb + uptake in AtCCX3 cells by excess Na + ,K + , and Mn 2+ . Functional epitope-tagged AtCCX3 fusion proteins were localized to endomembranes in plants and yeast. In Arabidopsis, AtCCX3 is primarily expressed in flowers, while AtCCX4 is expressed throughout the plant. Quantitative polymerase chain reaction showed that expression of AtCCX3 increased in plants treated with NaCl, KCl, and MnCl 2 . Insertional mutant lines of AtCCX3 and AtCCX4 displayed no apparent growth defects; however, overexpression of AtCCX3 caused increased Na + accumulation and increased 86 Rb + transport. Uptake of 86 Rb + increased in tonoplast-enriched membranes isolated from Arabidopsis lines expressing CCX3 driven by the cauliflower mosaic virus 35S promoter. Overexpression of AtCCX3 in tobacco (Nicotiana tabacum) produced lesions in the leaves, stunted growth, and resulted in the accumulation of higher levels of numerous cations. In summary, these findings suggest that AtCCX3 is an endomembrane-localized H + -dependent K + transporter with apparent Na + and Mn 2+ transport properties distinct from those of previously characterized plant transporters. The plant vacuole and other endomembrane com- partments play an important role in the sequestration of various compounds (Marschner, 1995; Marty, 1999). Concentration gradients of Na + , Ca 2+ , Cd 2+ , NO 3 2 , and Mn 2+ are established across these membranes by cat- ion/H + exchange activities (Schumaker and Sze, 1985; Salt and Wagner, 1993; Barkla and Pantoja, 1996; Gonzalez et al., 1999). Several genes encoding these transport activities have been identified (Shigaki and Hirschi, 2006). However, the biological functions of many of the individual transporters remain for the most part undefined. CCXs (for calcium cation exchangers) were previ- ously identified as CAX (for cation exchanger) homo- logs. Recently CAX7 to CAX11 were reclassified as CCX1 to CCX5 due to higher homology to mammalian K + -dependent Na + /Ca 2+ antiporters (Shigaki et al., 2006). CAXs are cation/H + antiporters that show high- capacity, low-affinity transport and have been charac- terized in a variety of plants (Blumwald and Poole, 1986; Kasai and Muto, 1990; Ettinger et al., 1999; Cheng et al., 2002; Luo et al., 2005). CAXs are energized by the pH gradient established by proton pumps such as H + -ATPase or H + -pyrophosphatase (Kamiya and Maeshima, 2004). Several plant CAXs have been char- acterized as vacuole-localized transporters, which function in H + -coupled antiport of Ca 2+ , Mg 2+ , and Mn 2+ , resulting in the accumulation of these cations in vacuoles (Hirschi, 1999; Pittman and Hirschi, 2001; Pittman et al., 2004a). CCXs have not been studied, and it would be interesting to compare and contrast their activities with those of CAXs and the less closely related Na + (K + )/H + exchangers of the NHX family. CAX proteins have N-terminal regulatory domains (Pittman and Hirschi, 2001), and AtCAX1 and AtCAX2 were originally cloned as functional N-terminal dele- tions (lacking the negative regulatory domain; Hirschi et al., 1996). We refer to these forms as sCAX1 and sCAX2 (Shigaki and Hirschi, 2006). Tobacco (Nicoti- ana tabacum) plants expressing AtsCAX1 exhibit Ca 2+ 1 This work was supported by the U.S. Department of Agricul- ture/Agricultural Research Service (Cooperative Agreement no. 58– 62650–6001), the National Science Foundation (grant nos. NSF 0344350 and NSF 020977), and the U.S. Department of Agriculture Cooperative State Research, Education, and Extension Service (grant no. 2005–34402–17121 to K.D.H.). The National Science Foundation (grant no. 0209792) funded work in the laboratory of J.M.W. * Corresponding author; e-mail kendalh@bcm.tmc.edu. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Kendal D. Hirschi (kendalh@bcm.tmc.edu). [W] The online version of this article contains Web-only data. 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