作为植物中的钙传感器继电器,钙调磷酸酶B样(CBL)蛋白为解码各种非生物胁迫引起的Ca2信号提供了重要贡献。目前,众所周知,CBL感知并将Ca2+信号主要传递给一组称为CBL-相互作用蛋白激酶(CIPK)的丝氨酸/苏氨酸蛋白激酶。在这项研究中,我们报道,该家族的CBL10成员除了CIPK蛋白外,还有一个新的相互作用伴侣。以CBL10为诱饵的酵母双杂交筛选鉴定了编码TOC34蛋白的拟南芥cDNA克隆,它是TOC(叶绿体外膜转位)复合物的成员,具有GTP酶活性。进一步的分析显示,除了CBL10之外,CBL7还在酵母双杂交系统中以低得多的强度与TOC34相互作用。然而,其余的CBL家庭成员未能与TOC34互动。双分子荧光互补(BiFC)分析证实,CBL10-TOC34相互作用发生在体内叶绿体的外膜。此外,我们还证明了CBL10在体外与TOC34物理相关,导致TOC34蛋白的GTP酶活性显著降低。一起来看,我们的发现清楚地表明,CBL家族的一员,CBL10不仅可以调节CIPK成员,还可以调节TOC34,从而使CBL家族以比目前已知的更多方式中继Ca2信号。
As calcium sensor relays in plants, calcineurin B-like (CBL) proteins provide an important contribution to decoding Ca2+ signatures elicited by a variety of abiotic stresses. Currently, it is well known that CBLs perceive and transmit the Ca2+ signals mainly to a group of serine/threonine protein kinases called CBL-interacting protein kinases (CIPKs). In this study, we report that the CBL10 member of this family has a novel interaction partner besides the CIPK proteins. Yeast two-hybrid screening with CBL10 as bait identified an Arabidopsis cDNA clone encoding a TOC34 protein, which is a member of the TOC (Translocon of the Outer membrane of the Chloroplasts) complex and possesses the GTPase activity. Further analyses showed that in addition to CBL10, CBL7 also interacts with TOC34 at much lower strength in the yeast two-hybrid system. However, the rest of the CBL family members failed to interact with TOC34. Bimolecular fluorescence complementation (BiFC) analysis verified that the CBL10-TOC34 interaction occurs at the outer membrane of chloroplasts in vivo. In addition, we also demonstrated that CBL10 physically associates with TOC34 in vitro, resulting in a significant decrease in the GTPase activity of the TOC34 protein. Taken together, our findings clearly indicate that a member of the CBL family, CBL10, can modulate not only the CIPK members but also TOC34, allowing the CBL family to relay the Ca2+ signals in more diverse ways than currently known.