Subcellular localization and oligomeric structure of the yeast putative stretch-activated Ca2+ channel component Mid1.
Subcellular localization and oligomeric structure of the yeast putative stretch-activated Ca2+ channel component Mid1.
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DOI：
10.1016/j.yexcr.2003.09.020
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发表时间：
2004-02
影响因子：
3.7
通讯作者：
H. Yoshimura;T. Tada;H. Iida
中科院分区：
文献类型：
--
作者：
H. Yoshimura;T. Tada;H. Iida
表观分子量为100 kDa的酵母Mid1蛋白是由交配信息素刺激的Ca2+内流所需的，并且通过一种容量性钙进入样机制响应于内质网（ER）的Ca2+耗竭而起作用，并且当在哺乳动物细胞中表达时作为拉伸激活的Ca2+渗透通道起作用。我们以前的工作与蛋白酶保护实验表明，Mid1存在于质膜。在这项研究中，我们研究了一个可能的细胞内定位这种蛋白质的间接荧光显微镜，发现Mid1是目前在ER膜以及质膜。Mid1的细胞内荧光图像与ER标记蛋白Sec71的相同，但与高尔基体蛋白Ypt 1的完全不同。使用Angiografin密度梯度分析通过膜分级法确认结果。我们还研究了Mid1的寡聚体结构和蛋白水平，发现Mid1通过二硫键形成200 kDa的寡聚体。Mid1在质膜和内质网膜中的蛋白水平和修饰不受交配信息素的影响。这些发现提供了新的见解Mid1的功能，有关本地化，修改和激活机制。
The yeast Mid1 protein with an apparent molecular mass of 100 kDa is required for Ca2+influx stimulated by the mating pheromone and by a capacitative calcium entrylike mechanism acting in response to Ca2+depletion from the endoplasmic reticulum (ER) and functions as a stretch-activated Ca2+-permeable channel when expressed in mammalian cells. Our previous work with protease protection experiments has indicated that Mid1 is present in the plasma membrane. In this study, we examined a possible intracellular localization of this protein by indirect fluorescence microscopy and found that Mid1 is present in the ER membrane as well as the plasma membrane. Intracellular fluorescence images for Mid1 were the same as those for the ER marker protein Sec71 but quite different from those of the Golgi protein Ypt1. The results were confirmed by membrane fractionation using Angiografin density gradient analysis. We also investigated the oligomeric structures and protein levels of Mid1 and found that Mid1 forms a 200-kDa oligomer by disulfide bonding. The protein level and modification of Mid1 in the plasma membrane and the ER membrane were unchanged by the mating pheromone. These findings provide new insight into the function of Mid1 in relation to localization, modification, and activation mechanisms.