Abstract
BackgroundCalcineurin (CaN) is a Ca2+- and calmodulin (CaM)-dependent serine/threonine phosphatase. Previous studies have found that CaN is involved in the regulation of the stress responses.ResultsIn this study, the growth of Cryptococcus humicola was inhibited by the CaN inhibitor tacrolimus (FK506) under aluminum (Al) stress. The expression of CNA encoding a catalytic subunit A (CNA) and its interaction with CaM were upregulated when the concentration of Al was increased. A CaM-binding domain and key amino acids responsible for interaction with CaM were identified. ∆CNAb with a deletion from S454 to A639 was detected to bind to CaM, while ∆CNAa with a deletion from R436 to A639 showed no binding to CaM. The binding affinities of CNA1 and CNA2, in which I439 or I443 were replaced by Ala, were decreased relative to wild-type CNA. The phosphatase activities of ∆CNAa, CNA1 and CNA2 were lower than the wild-type protein. These results suggest that the region between R436 and S454 is essential for the interaction with CaM and I439, I443 are key amino acids in this region. The ability of the CNA transgenic yeast to develop resistance to Al was significantly higher than that of control yeast. Residual Al in the CNA transgenic yeast culture media was significantly lower than the amount of Al originally added to the media or the residual Al remaining in the control yeast culture media. These findings suggest that CNA confers Al tolerance, and the mechanism of Al tolerance may involve absorption of active Al.ConclusionsAl stress up-regulated the expression of CNA. CaM-binding domain and key amino acids responsible for interaction with CaM were identified and both are required for phosphatase activities. CNA conferred yeast Al resistance indicating that the gene has a potential to improve Al-tolerance through gene engineering.
Highlights
Calcineurin (CaN) is a Ca2+- and calmodulin (CaM)-dependent serine/threonine phosphatase
The role of catalytic subunit A (CNA) in Al stress To examine the impact of CaN on the growth of C. humicola under Al stress, FK506 was added to the culture medium containing 50 mM Al3+
To study the transcription levels of CNA under Al stress, total RNA of cells treated with Al was used as the template for quantitative RT-PCR
Summary
Calcineurin (CaN) is a Ca2+- and calmodulin (CaM)-dependent serine/threonine phosphatase. Previous studies have found that CaN is involved in the regulation of the stress responses. Calmodulin (CaM) is a small, ubiquitous protein that acts as a calcium sensor to mediate several Ca2+ signals [2]. CaM is assumed to be involved in the activation of many enzymes, including CaM-dependent protein kinases and protein phosphatase (calcineurin) [3,4,5]. Calcineurin (CaN) is a heterodimer consisting of a catalytic subunit A (CNA) and a regulatory subunit B (CNB) [6]. It is widely distributed, its primary sequence and structure is conserved among all eukaryotes.
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