Ca2+ transport in mitochondria from yeast expressing recombinant aequorin.
Ca2+ transport in mitochondria from yeast expressing recombinant aequorin.
复制标题
来自表达重组水母发光蛋白的酵母的线粒体中的 Ca2 转运。
DOI：
10.1016/j.ab.2003.10.029
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发表时间：
2004
影响因子：
2.9
通讯作者：
Pfeiffer,DouglasR
中科院分区：
文献类型：
--
作者：
Jung,DennisW;Bradshaw,PatrickC;Litsky,Monica;Pfeiffer,DouglasR
我们已经表达了水母发光蛋白的酵母酿酒酵母的线粒体和其特征在于所得到的菌株相对于线粒体Ca 2+转运在体内和体外。当将完整细胞悬浮在含有1.4mM乙醇和14 mM CaCl 2的水中时，基质游离Ca 2+浓度为200 nM，与细胞质中预期的值相似。添加离子载体ETH 129可使Ca 2+主动蓄积，并迅速将值增加至1.2μM。在这些条件下，升高的Ca 2+浓度维持6分钟或更长的时间。当ETH 129存在时，分离的酵母线粒体氧化乙醇也积累Ca 2+，但阳离子不保留，这取决于培养基条件。这一发现证实了如前所述需要游离脂肪酸的Ca 2+释放机制的存在[P.C. Bradshaw等人（2001）J.Biol.Chem.276，40502-40509]。当呼吸底物不存在时，Ca 2+缓慢地进入和离开酵母线粒体，比活度接近0.2nmol/min/mg蛋白质。在这些条件下的运输平衡的内部和外部浓度的Ca 2+，并不受钌红，解偶联剂，或离子载体，扰乱跨膜梯度的电荷和pH值。这种活动显示S形动力学和K1/2值的Ca 2+是接近900 nM，在没有乙醇或当它存在。此外，它表明，在酵母线粒体中的Ca ~（2+）的活性系数是一个函数的矩阵Ca ~（2+）的含量，并大大大于在哺乳动物线粒体。水母发光蛋白表达菌株的特征似乎适合于其在针对从哺乳动物线粒体克隆Ca 2+转运蛋白的基于表达的方法中的使用，并用于进一步研究酵母中线粒体和细胞质Ca 2+之间的相互关系。
We have expressed aequorin in mitochondria of the yeast Saccharomyces cerevisiae and characterized the resulting strain with respect to mitochondrial Ca2+transport in vivo and in vitro. When intact cells are suspended in water containing 1.4mM ethanol and 14mM CaCl2, the matrix free Ca2+concentration is 200nM, similar to the values expected in cytoplasm. Addition of ionophore ETH 129 allows an active accumulation of Ca2+and promptly increases the value to 1.2μM. Elevated Ca2+concentrations are maintained for periods of 6 min or longer under these conditions. Isolated yeast mitochondria oxidizing ethanol also accumulate Ca2+when ETH 129 is present, but the cation is not retained depending on the medium conditions. This finding confirms the presence of a Ca2+release mechanism that requires free fatty acids as previously described [P.C. Bradshaw et al. (2001) J. Biol. Chem. 276, 40502–40509]. When a respiratory substrate is not present, Ca2+enters and leaves yeast mitochondria slowly, at a specific activity near 0.2nmol/min/mg protein. Transport under these conditions equilibrates the internal and external concentrations of Ca2+and is not affected by ruthenium red, uncouplers, or ionophores that perturb transmembrane gradients of charge and pH. This activity displays sigmoid kinetics and a K1/2value for Ca2+that is near to 900nM, in the absence of ethanol or when it is present. It is furthermore shown that the activity coefficient of Ca2+in yeast mitochondria is a function of the matrix Ca2+content and is substantially larger than that in mammalian mitochondria. Characteristics of the aequorin-expressing strain appear suitable for its use in expression-based methods directed at cloning Ca2+transporters from mammalian mitochondria and for further examining the interrelationships between mitochondrial and cytoplasmic Ca2+in yeast.