Bowman Barry J, Abreu Stephen, Margolles-Clark Emilio, Draskovic Marija, Bowman Emma Jean
Department of Molecular, Cell, and Developmental Biology, University of California, Santa Cruz, CA 95064, USA.
Eukaryot Cell. 2011 May;10(5):654-61. doi: 10.1128/EC.00239-10. Epub 2011 Feb 18.
We have examined the distribution of calcium in Neurospora crassa and investigated the role of four predicted calcium transport proteins. The results of cell fractionation experiments showed 4% of cellular calcium in mitochondria, approximately 11% in a dense vacuolar fraction, 40% in an insoluble form that copurifies with microsomes, and 40% in a high-speed supernatant, presumably from large vacuoles that had broken. Strains lacking NCA-1, a SERCA-type Ca(2+)-ATPase, or NCA-3, a PMC-type Ca(2+)-ATPase, had no obvious defects in growth or distribution of calcium. A strain lacking NCA-2, which is also a PMC-type Ca(2+)-ATPase, grew slowly in normal medium and was unable to grow in high concentrations of calcium tolerated by the wild type. Furthermore, when grown in normal concentrations of calcium (0.68 mM), this strain accumulated 4- to 10-fold more calcium than other strains, elevated in all cell fractions. The data suggest that NCA-2 functions in the plasma membrane to pump calcium out of the cell. In this way, it resembles the PMC-type enzymes of animal cells, not the Pmc1p enzyme in Saccharomyces cerevisiae that resides in the vacuole. Strains lacking the cax gene, which encodes a Ca(2+)/H(+) exchange protein in vacuolar membranes, accumulate very little calcium in the dense vacuolar fraction but have normal levels of calcium in other fractions. The cax knockout strain has no other observable phenotypes. These data suggest that "the vacuole" is heterogeneous and that the dense vacuolar fraction contains an organelle that is dependent upon the CAX transporter for accumulation of calcium, while other components of the vacuolar system have multiple calcium transporters.
我们研究了粗糙脉孢菌中钙的分布，并探究了四种预测的钙转运蛋白的作用。细胞分级分离实验结果表明，线粒体中细胞钙含量为4%，致密液泡部分约为11%，与微粒体共纯化的不溶性部分为40%，高速上清液中为40%，可能来自破裂的大液泡。缺乏NCA - 1（一种SERCA型Ca(2 +)-ATP酶）或NCA - 3（一种PMC型Ca(2 +)-ATP酶）的菌株在生长或钙分布方面没有明显缺陷。缺乏同样是PMC型Ca(2 +)-ATP酶的NCA - 2的菌株在正常培养基中生长缓慢，并且无法在野生型能够耐受的高浓度钙环境中生长。此外，当在正常钙浓度（0.68 mM）下生长时，该菌株积累的钙比其他菌株多4至10倍，且在所有细胞组分中均升高。数据表明，NCA - 2在质膜中发挥作用，将钙泵出细胞。这样，它类似于动物细胞的PMC型酶，而不像酿酒酵母中位于液泡中的Pmc1p酶。缺乏cax基因（该基因编码液泡膜中的Ca(2 +)/H(+)交换蛋白）的菌株在致密液泡部分积累的钙很少，但其他部分的钙水平正常。cax基因敲除菌株没有其他可观察到的表型。这些数据表明“液泡”是异质的，致密液泡部分含有一种依赖于CAX转运蛋白积累钙的细胞器，而液泡系统的其他组分具有多种钙转运蛋白。
