AtCAX1 N-terminal can improve Ca2+ and Ba2+ tolerance in yeast  

Hongjun Guo1 , Daisuke Tsugama2 , Tetsuo Takano2 , Yuanyuan Bu1 , Shenkui Liu1
1. Key Laboratory of Saline-alkali Vegetation Ecology Restoration in Oil Field (SAVER), Ministry of Education, Alkali Soil Natural Environmental Science Center (ASNESC), Northeast Forestry University, Harbin, 150040
2. Asian Natural Environmental Science Center (ANESC), The University of Tokyo, Nishi-tokyo-shi, Tokyo, Japan
Author    Correspondence author
Cell Biology and Biophysics, 2015, Vol. 4, No. 3   doi: 10.5376/cbb.2015.04.0003
Received: 08 Jun., 2015    Accepted: 23 Jun., 2015    Published: 29 Jun., 2015
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Hongjun Guo, Daisuke Tsugama, Tetsuo Takano, Yuanyuan Bu and Shenkui Liu, 2015, AtCAX1 N-terminal may enhance Ca2+ and Ba2+ tolerance in yeast, Cell Biology and Biophysics, 4(3): 1-4 (doi: 10.5376/cbb.2015.04.0003)


AtCAX1 is a member of the Ca2+/H+ antiporter family. The N-terminally truncated AtCAX1 (ΔN108AtCAX1) and the C-terminally segment truncated AtCAX1 (ΔC9AtCAX1, ΔC36AtCAX1, and ΔC144AtCAX) and full-length AtCAX1 were created and analyzed their growth in the presence of Ba2+ and Ca2+. The ΔN108AtCAX1 grew well in the presence of 150 mM Ca2+ and 15mM Ba2+. This result indicates that ΔN108AtCAX1 can improve the resistance to Ca2+ and Ba2+. And by atomic absorption spectrophotometer to measure the Ba2+ content of whole yeast cells, the results showed that the Ba2+ content in yeast cell expressing ΔN108AtCAX1 was lower than other yeast transformants. The studies indicate that AtCAX1 may transport Ca2+ and Ba2+.

AtCAX1; Ca2+ and Ba2+ tolerance; Auto-inhibitory domain

In animals and plants, Calcium (Ca2+) is an important second messenger (Bush 1995; Sanders et al. 1999; Qudeimat et al. 2008). Ca2+ signal transduction requires the judicious control of cytosolic Ca2+ levels. This concentration gradient is established across the tonoplast in part by high capacity Ca2+/H+ exchange and via Ca2+ pumping directly energized by ATP hydrolysis (Schumaker, et al. 1986; Sze, et al. 2000). Ca2+ can accumulate to millimolar levels in the vacuole, whereas the concentrations are maintained in the submicromolar range in the cytosol (Marty 1999). Vacuolar transporters may provide an important mechanism for ion sequestration (Salt and Wagner, 1993; Shaul et al. 1999).

Ca2+/H+ exchange helps to establish the concentration gradient of Ca2+ across the tonoplast (vacuolar membrane; Schumaker and Sze, 1985; Blumwald and Poole, 1986). Plant CAXs are characterized by 11 TM helices (Shigaki and Hirschi, 2006). TMs of CAXs are generally divided into three components, namely TM1, TM2-6, and TM7-11. TM2-6 and TM7-11 are weakly homologous elements connected by a loop with acidic motif, and TM1 appears to be a dispensable element for cation transport function in some CAXs (Schaaf et al. 2002). Plant Ca2+/H+ antiporter genes were identified by their ability to suppress the Ca2+-hypersensitive phenotype of a yeast mutant lacking the vacuolar Ca2+-ATPase PMC1 and VCX1 (Hirschi et al. 1996; Ueoka-Nakanishi et al. 2000). Research indicated that the scale of the border substance of CAXs family is very extensive by means of suppressing Ca2+ sensitive yeast (Shigaki & Hirschi 2006).
Using yeast as an experimental tool, two domains have been identified that modulate CAX1 activity (Pittman and Hirschi, 2001; Shigaki et al. 2001). The second domain that regulates CAX function has been termed the regulatory or autoinhibitory domain (Pittman and Hirschi, 2001). The full-length open reading frame of CAX1 contains an extended N-terminal tail of 36 amino acids, termed the N-terminal regulatory region (NRR), which prevents Ca2+ transport activity (Pittman and Hirschi, 2001). Using a series of N-terminal-truncated CAX (sCAX) chimeric constructs, a second domain, the Ca2+ domain (CaD), has been identified that appears to modulate Ca2+ transport (Shigaki, et al. 2001). Early studies showed N-terminally truncated AtCAX1 and AtCAX2 could suppress the Ca2+-and Mn2+-sensitivity of a yeast mutants (Hirschi et al. 1996; Pittman and Hirschi 2001; Pittman et al. 2002a; Pittman et al. 2004). In this study, we will demonstrate the relationship between the five transformants and Ba2+ or Ca2+.
1 Materials and Methods
1.1 Plasmids Construction and Yeast strain
The coding region of AtCAX1 was amplified by PCR. The primer F: 5′-TCAGTAGAGAAATGGCGGGA-3′, the primer R: 5′-TCCATGTCTCTCGCTTTGG-3′ Four truncated forms of AtCAX1 were constructed: N-terminally truncated AtCAX1 (ΔN108AtCAX1), C-terminally truncated AtCAX1 (ΔC9AtCAX1, ΔC36AtCAX1, ΔC144AtCAX1). The primer N108-F: 5′-ATGTCTTCTTCTTCTTTGAGG-3′. The primer C9-F: 5′-TTAGAAAACTCCTCCTCCTGTTG-3′, C36-R: 5′-TTAGACATTGTTCATCGCTTGATGTC-3′, C144-R: 5′-TTATCCCTTCATGTAGTGAGAACTCCC-3′ was designed to place a stop codon. ΔN108AtCAX1, ΔC9AtCAX1, ΔC36AtCAX1 andΔC144AtCAX1 were amplified from the AtCAX1 cDNA using the primer pairs of N108-F and R, F and C9-R, F and C36-R, F and C144-R, respectively. The amplified fragment was ligated into BamHI/XbaI sites of the pYES2 vector (Invitrogen, CA, USA), respectively. The expression plasmids were selected and propagated in Escherichia coli strain JM109 with BamHI/XbaI sites, and then to Saccharomyces cerevisiae strain K667 (cnb1::LEU2 pmc1::TRP1 vcx1). The K667 yeast strain lacks of vacuolar Ca2+-ATPase (PMC1) and mutated vacuolar Ca2+/H+ exchanger (VCX1) (Cunningham and Fink, 1996). Positive CAX yeast transformants were selected on SD-ura medium (pH=5.8), which contains 0.67 % yeast nitrogen base (without amino acids), 0.0776 % -ura clontech, 2 % D-glucose, and 1.5 % Agar. The yeast strains transformed with the empty pYES2 vector were used as a control.
1.2 Yeast Growth Conditions
Transformed cells were tested for their capability to suppress the K667. Growth assays using K667 cells were conducted using YPD medium supplemented with or without CaCl2 (50, 100, or 150 mM), or BaCl2 (5, 8, 10, or 15 mM). The BaCl2 and CaCl2 stock solution was filter-sterilized and added to the autoclaved YPD agar medium to final concentrations. K667 cells expressing various constructs were grown in SD-ura selection liquid medium overnight at 30 °C until an OD≈1 was reached, then diluted 10-1, 10-2, 10-3, and 10-4-fold using distilled water. After a series of 5-fold dilutions, 5 µl cell suspensions were spotted on the indicated media, and the cells were grown for 3-8 days.
1.3 Ba2+ content in transformed yeast
The yeast transforments pyes2-AtCAX1, pYES2- ΔN108AtCAX1, pyes2-ΔC9AtCAX, pyes2-ΔC36AtCAX1, pyes2-ΔC144AtCAX1 were cultured in the SD-ura liquid culture medium until they reach the level of OD600≈1, and then they will be processed for 8 hours in uptake buffer (2% galactose and 10mM MES; the pH6.0 of different levels of Ba2+, and the yeast cells will be bleached twice with distilled water, after which they will be dissolved by 0.6% HNO3, then the sample were collected by centrifugation. Then we use atomic absorption spectrophotometer (AA800, Perkin Elmer, USA) method to measure the content of Ba2+.
2 Results and Discussion
2.1 Hyper-resistance of AtCAX1-overexpressing cells to Ca2+ and Ba2+
To confirm the function of AtCAX1, six transformed yeast lines were constructed. One was transformed with empty vector pYES2 as a control (Figure 1), and five were transformed with a vector containing AtCAX1, ΔN108AtCAX1, ΔC9AtCAX, ΔC36AtCAX, and ΔC144AtCAX. In the presence of Ca2+, the growth of the AtCAX1 transformant was weaker but it was still better than the growth of the vector transformant (Figure 1). In the presence of Ba2+ (8, 10, 15mM), the ΔN108AtCAX1 transformant grew well in the presence of 15 mM Ba2+, while the vector transformant did not grow at media (Figure 1). This means that ΔN108AtCAX1 transformant yeast can improve the resistance to Ca2+ can greatly improve the resistance to Ba2+, even with 15 mM Ba2+ under the conditions of normal growth (Figure 1), the experimental results show that ΔN108AtCAX1 improves the resistance of yeast to Ca2+ and Ba2+. Therefore, future studies should be performed to determine whether this was mediated directly by AtCAX1. In Figure 1B, the ΔN108AtCAX1 transformants were able to grow in the presence of 150 mM Ca2+ as well as in the presence 15 mM Ba2+. Collectively, these findings suggest that the NRR of these CAXs acts as an auto-inhibitory domain for cation transport. We demonstrated tolerance of yeast significantly, whereas the absence of the N-terminal region of PutCAX1 decreased the Ca2+ and Ba2+ tolerance of yeast significantly (Liu et al. 2009). These results show that the absence of the N-terminal region of AtCAX1 significantly decreased the Ca2+ and Ba2+ tolerance of yeast.

Figure 1 Hyper-resistance of AtCAX1 and the N-terminal, C-terminal truncated AtCAX1 cells to Ca2+ and Ba2+. In (A) and (B), the yeast cell incubated as described in ‘‘Materials and methods’’. Serial dilutions were spotted onto YPD agar plates (control) supplemented with CaCl2 and BaCl2 at the concentrations indicated. Growth was monitored for 5 days at 30°C

(A) Structure of AtCAX1, and the N-terminal
, C-terminal segment truncated AtCAX1. a, Full-length AtCAX1. b-e, N-terminal truncated AtCAX1 (ΔN108AtCAX1), C-terminal segment truncated AtCAX1 (ΔC9AtCAX1, ΔC36AtCAX1 and ΔC144AtCAX).
(B) The resistance of cells overexpressing full-length AtCAX1 and truncated AtCAX1 to Ca2+ and Ba2+.
To confirm Ca2+ and Ba2+ between the transfer relationship, in different concentration of Ca2+ medium supplemented with different concentrations of exogenous Ba2+, the results showed that transformants of AtCAX1, ΔN108AtCAX1, ΔC9AtCAX1, ΔC36AtCAX1 and ΔC144AtCAX1 yeast cells grow weakly (Figure 2), which indicated that there might be some kind of interaction between Ca2+ and Ba2+.

Figure 2 The resistance of cells overexpressing full-length AtCAX1 and truncated AtCAX1 to Ca2+ and Ba2+ together.Yeast cells were incubated as described in Materials and methods, and serial dilutions were spotted onto solid YPG media supplemented with or without additional cations. Growth was monitored for 3–8 days at 30°C

To further confirm this speculation, the cultivation of the same concentration of exogenous Ca2+ medium added in different concentrations of Ba2+, Ba2+ (3, 5 mM) conditions, the over expression of ΔC36AtCAX1, ΔC144AtCAX, and the control group with yeast grows weaker in the presence of 50 mM Ca2+ (10, 20 mM Ba2+) conditions, the overexpression of ΔC9AtCAX1, AtCAX1 and ΔN108AtCAX1, the full-length yeast growth was weak in the presence of 50 mM Ca2+ (Figure 2).
2.2 Ba2+ content in transformed yeast
To further investigate whether the Ba2+-resistance of yeast mutants expressing ΔN108AtCAX1 was due to their ability to transport Ba2+, the Ba2+ content in whole yeast cells was measured. As shown in Figure 3, the Ba2+ accumulation in yeast cells expressing ΔN108AtCAX1 was lower than in those expressing AtCAX1 and ΔCAtCAX1 (Figure 3). This is supported by the better growth of theΔN108AtCAX1 transformants in Ba2+-containing medium (Figure 1B). Raghu reported that Ba was present at high concentrations in plants around barite mining areas, and that high concentrations of Ba2+ inhibited normal plant growth (Raghu 2001). High concentrations of Ba2+ (5 mM) were found to prevent stomatal opening, perturb carbon fixation metabolism and translocation, disturb intracellular metal homeostasis, and decrease overall plant growth (M. Llugany, et al. 2000).

Figure 3 The Ba2+ content of whole yeast cells expressing full-length AtCAX1 and four truncated AtCAX1 in the presence of 0, 5, 10, 15, 20 mM BaCl2. The yeast strain K667 was transformed with empty vector pYES2, vector containing AtCAX1, ΔN108 AtCAX1, ΔC9AtCAX1, ΔC36AtCAX1, or ΔC144AtCAX1. Results are expressed as means ± SE (n = 3)

3 Conclusions
The growth of yeasts expressing the full-length and four truncated forms of AtCAX1 in media containing Ca2+ and Ba2+ was examined. Moreover, the Ba2+ content in whole yeast cells expressing ΔN108AtCAX1 was lower than other yeast transformants. These results suggests thatΔN108AtCAX1 can enhance the tolerance of Ba2+ and Ca2+ in yeast, even transports the Ba2+ and Ca2+.
This work was supported by specific fund for forest scientific research in the public welfare (201404220) and Program for Changjiang Scholars and Innovative Research Team in University of China (PCSIRT) (IRT13053).
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