Structure and Distribution of Organelles and Cellular Location of Calcium Transporters in Neurospora crassa
Structure and Distribution of Organelles and Cellular Location of Calcium Transporters in Neurospora crassa
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DOI：
10.1128/ec.00174-09
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发表时间：
2009-12-01
期刊：
影响因子：
--
通讯作者：
Bowman, Emma Jean
中科院分区：
文献类型：
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作者：
Bowman, Barry J.;Draskovic, Marija;Bowman, Emma Jean
我们想要检查四种运输钙的粗糙脉孢菌蛋白的细胞位置。然而，粗糙脉孢菌活菌丝中细胞器的结构和分布尚未得到全面描述。因此，我们制作了重组基因，可产生假定的细胞器标记蛋白与绿色或红色荧光蛋白的翻译融合。我们在核膜和相关膜中观察到由 grp-78 和 dpm 编码的推定内质网蛋白。由 vam-3 和 vma-1 编码的液泡膜蛋白质位于菌丝尖端附近的小管和囊泡的互连网络中，而在更远端的区域，它们位于大大小小的球形液泡中。用标记的 ARG-4 可视化的线粒体在菌丝的所有区域都丰富。同样，我们用绿色或红色荧光蛋白标记了四种粗糙脉孢菌蛋白，以检查它们的细胞位置。 NCA-1蛋白是动物细胞SERCA型Ca(2+)-ATP酶的同源物，与内质网标记物共定位。 NCA-2 和 NCA-3 蛋白是酵母液泡膜或动物细胞质膜中 Ca(2+)-ATP 酶的同源物。它们与液泡膜中的标记共定位，并且它们也出现在距尖端超过 1 毫米的菌丝区域的质膜中。 cax 基因编码液泡中的 Ca2(+)/H(+) 交换蛋白。正如预期的那样，CAX 蛋白定位于液泡区室。我们观察到，距尖端约 50 至 100 μm 处，有一些球形细胞器，其中含有大量标记的 CAX 蛋白和标记的液泡 ATP 酶亚基（VMA-1 和 VMA-5）。我们认为，这种细胞器（以前在粗糙链孢菌中没有描述过）可能具有螯合钙的作用。
We wanted to examine the cellular locations of four Neurospora crassa proteins that transport calcium. However, the structure and distribution of organelles in live hyphae of N. crassa have not been comprehensively described. Therefore, we made recombinant genes that generate translational fusions of putative organellar marker proteins with green or red fluorescent protein. We observed putative endoplasmic reticulum proteins, encoded by grp-78 and dpm, in the nuclear envelope and associated membranes. Proteins of the vacuolar membrane, encoded by vam-3 and vma-1, were in an interconnected network of small tubules and vesicles near the hyphal tip, while in more distal regions they were in large and small spherical vacuoles. Mitochondria, visualized with tagged ARG-4, were abundant in all regions of the hyphae. Similarly, we tagged the four N. crassa proteins that transport calcium with green or red fluorescent protein to examine their cellular locations. NCA-1 protein, a homolog of the SERCA-type Ca(2+)-ATPase of animal cells, colocalized with the endoplasmic reticulum markers. The NCA-2 and NCA-3 proteins are homologs of Ca(2+)-ATPases in the vacuolar membrane in yeast or in the plasma membrane in animal cells. They colocalized with markers in the vacuolar membrane, and they also occurred in the plasma membrane in regions of the hyphae more than 1 mm from the tip. The cax gene encodes a Ca2(+)/H(+) exchange protein found in vacuoles. As expected, the CAX protein localized to the vacuolar compartment. We observed, approximately 50 to 100 mu m from the tip, a few spherical organelles that had high amounts of tagged CAX protein and tagged subunits of the vacuolar ATPase (VMA-1 and VMA-5). We suggest that this organelle, not described previously in N. crassa, may have a role in sequestering calcium.