Pozos T C, Sekler I, Cyert M S
Department of Biological Sciences, Stanford University, California 94305-5020, USA.
Mol Cell Biol. 1996 Jul;16(7):3730-41. doi: 10.1128/MCB.16.7.3730.
Calcineurin, or PP2B, plays a critical role in mediating Ca2+-dependent signaling in many cell types. In yeast cells, this highly conserved protein phosphatase regulates aspects of ion homeostasis and cell wall synthesis. We show that calcineurin mutants are sensitive to high concentrations of Mn2+ and identify two genes, CCC1 and HUM1, that, at high dosages, increase the Mn2+ tolerance of calcineurin mutants. CCC1 was previously identified by complementation of a Ca2+-sensitive (csg1) mutant. HUM1 (for "high copy number undoes manganese") is a novel gene whose predicted protein product shows similarity to mammalian Na+/Ca2+ exchangers. hum1 mutations confer Mn2+ sensitivity in some genetic backgrounds and exacerbate the Mn2+ sensitivity of calcineurin mutants. Furthermore, disruption of HUM1 in a calcineurin mutant strain results in a Ca2+-sensitive phenotype. We investigated the effect of disrupting HUM1 in other strains with defects in Ca2+ homeostasis. The Ca2+ sensitivity of pmc1 mutants, which lack a P-type ATPase presumed to transport Ca2+ into the vacuole, is exacerbated in a hum1 mutant strain background. Also, the Ca2+ content of hum1 pmc1 cells is less than that of pmc1 cells. In contrast, the Ca2+ sensitivity of vph1 mutants, which are specifically defective in vacuolar acidification, is not significantly altered by disruption of Hum1p function. These genetic interactions suggest that Hum1p may participate in vacuolar Ca2+/H+ exchange. Therefore, we prepared vacuolar membrane vesicles from wild-type and hum1 cells and compared their Ca2+ transport properties. Vacuolar membrane vesicles from hum1 mutants lack all Ca2+/H+ antiport activity, demonstrating that Hum1p catalyzes the exchange of Ca2+ for H+ across the yeast vacuolar membrane.
钙调神经磷酸酶，即PP2B，在介导多种细胞类型中依赖Ca2+的信号传导方面发挥着关键作用。在酵母细胞中，这种高度保守的蛋白磷酸酶调节离子稳态和细胞壁合成的各个方面。我们发现钙调神经磷酸酶突变体对高浓度的Mn2+敏感，并鉴定出两个基因，CCC1和HUM1，高剂量时它们可提高钙调神经磷酸酶突变体对Mn2+的耐受性。CCC1先前是通过对Ca2+敏感（csg1）突变体的互补作用鉴定出来的。HUM1（“高拷贝数消除锰敏感性”之意）是一个新基因，其预测的蛋白质产物与哺乳动物的Na+/Ca2+交换体相似。hum1突变在某些遗传背景下会导致Mn2+敏感性，并加剧钙调神经磷酸酶突变体的Mn2+敏感性。此外，在钙调神经磷酸酶突变菌株中破坏HUM1会导致Ca2+敏感表型。我们研究了在其他Ca2+稳态存在缺陷的菌株中破坏HUM1的影响。在hum1突变菌株背景下，缺乏一种推测可将Ca2+转运到液泡中的P型ATP酶的pmc1突变体的Ca2+敏感性会加剧。而且，hum1 pmc1细胞的Ca2+含量低于pmc1细胞。相反，液泡酸化存在特异性缺陷的vph1突变体的Ca2+敏感性不会因Hum1p功能的破坏而发生显著改变。这些遗传相互作用表明Hum1p可能参与液泡Ca2+/H+交换。因此，我们从野生型和hum1细胞中制备了液泡膜囊泡，并比较了它们的Ca2+转运特性。hum1突变体的液泡膜囊泡缺乏所有Ca2+/H+反向转运活性，这表明Hum1p催化Ca2+与H+跨酵母液泡膜的交换。
