The highest activity, 15 ± 3% dequenching, was detected at pH 7.5 (Fig. 3a). The KM value for Ca2+ was 0.5 ± 0.1 mM at pH 7.5. The Ca2+/H+ antiport activity was inhibited by lanthanum, which has been reported as the inhibitor of Ca2+/H+ antiporters (Fig. 3b) (Matsushita et al., 1986). The Li+/H+ and K+/H+ antiport activities of Tr-Mrp were measured, but no activities were detected with the dequenching assay at pH 7.5 (Fig. 3b) or at other pH values tested (data not shown). Antiport activities were measured in inside-out membrane Dinaciclib vesicles derived directly from T. roseum cells (called Tr-vesicles). As shown in Fig. 4a, Tr-vesicles exhibited significant Na+/H+

antiport activity, consistent with annotation of antiporters in the CPA2 family, which could account for this activity (Waser et al., 1992; Mesbah et al., 2009). In addition, lower but reproducible Ca2+/H+ antiport activity was also observed

(Fig. 4b). see more This is the first example of a Ca2+-translocating Mrp antiporter. All of the Mrp antiporters characterized previously were monovalent CPAs which utilized Na+, Li+, and/or K+ as counter ions for H+. Other members of the CPA superfamily have been shown to possess a wide spectrum of cations as the transported substrate (Southworth et al., 2001; Waditee et al., 2001; Wei et al., 2003; Fujisawa et al., 2005). Our finding indicates that there is a Mrp antiporter family member that possesses a capacity to transport divalent cations. It will be of interest to learn, as this capacity is assessed for other Mrp antiporters, how widespread it is and whether some Mrp antiporters can use both divalent and monovalent cations as efflux substrates. The dissolved calcium concentration has been reported to be low (0.28 mg L−1) in the alkaline

hot spring, Mushroom Spring, where T. roseum was isolated (Ball et al., 1998). Possibly, this suggests that Tr-Mrp contributes primarily to the cytoplasmic calcium homeostasis, rather than to adaptation to problem of pH homeostasis at both alkaline pH and high temperature. The monovalent CPAs detected in the T. roseum vesicles might play the major role in pH homeostasis, but a role for the Mrp antiporter cannot be ruled out. Calcium ions are known to play diverse physiological roles, including regulatory and signaling roles Ribonucleotide reductase in prokaryotic processes such as cell morphogenesis, motility and sporulation (Seto-Young & Ellar, 1981; Smith, 1995; Michiels et al., 2002; Dominguez, 2004), but they might also make some contribution to pH homeostasis in partnership with other antiporters in this multi-extremophile. We are grateful to Dr. Terry A. Krulwich and Dr. Arthur A. Guffanti (Mount Sinai School of Medicine, New York, NY, USA) for critical reading of the manuscript. This work was also supported by a special grant-in-aid from Toyo University, a grant from a High-Tech Research Center program of the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to M.I.