Subcellular localization of fumarase in mammalian cells and tissues
Subcellular localization of fumarase in mammalian cells and tissues
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DOI：
10.1007/s00418-006-0249-3
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发表时间：
2007-03-01
影响因子：
2.3
通讯作者：
Gupta, Radhey S.
中科院分区：
文献类型：
--
作者：
Bowes, Timothy;Singh, Bhag;Gupta, Radhey S.
富马酸酶是一种线粒体基质蛋白，以前曾指出也大量存在于胞质溶胶中。然而，最近的研究表明，新合成的人胰蛋白酶被有效地输入到线粒体中，在胞质溶胶中没有可检测的量。为了阐明其亚细胞定位，通过许多不同的方法检测了α-淀粉酶在哺乳动物细胞/组织中的亚细胞分布。使用线粒体分级试剂盒或用低浓度毛地黄皂苷提取的细胞分级分离，在100，000 g上清液级分中未检测到脱氢酶。免疫荧光标记的亲和纯化的抗体的线粒体Hsp 60蛋白和抗体的线粒体Hsp 60蛋白显示相同的标记模式，主要在线粒体中看到的标记。使用高分辨率免疫金电子显微镜进行了详细的研究，以确定包埋在LR白色树脂中的大鼠组织中酶的亚细胞定位。在肾脏、肝脏、心脏、肾上腺和垂体前叶的薄切片中，仅在线粒体中检测到由抗人β-半乳糖苷酶抗体引起的强烈和特异性标记。然而，在胰腺腺泡细胞中，除了线粒体外，在酶原颗粒和内质网中也观察到高度显著的标记。在所有情况下观察到的标记在省略一抗后完全消除，表明其具有特异性。在纯化的酶原颗粒的蛋白质印迹中，存在与线粒体中所见分子量相同的酶-抗体交叉反应蛋白。这些结果提供了证据，证明哺乳动物细胞/组织中的线粒体酶主要定位于线粒体中，并且大量的这种蛋白质不存在于胞质溶胶中。然而，这些研究还表明，在某些组织中，除了线粒体外，这种蛋白质也存在于特定的线粒体外位点。尽管线粒体外的这些位置的线粒体酶的细胞功能尚不清楚，但线粒体外的线粒体酶的出现/定位可能有助于解释这种线粒体蛋白的突变如何引起许多不同类型的癌症。
Fumarase, a mitochondrial matrix protein, is previously indicated to be present in substantial amounts in the cytosol as well. However, recent studies show that newly synthesized human fumarase is efficiently imported into mitochondria with no detectable amount in the cytosol. To clarify its subcellular localization, the subcellular distribution of fumarase in mammalian cells/tissues was examined by a number of different methods. Cell fractionation using either a mitochondria fraction kit or extraction with low concentrations of digitonin, detected no fumarase in a 100,000 g supernatant fraction. Immunoflourescence labeling with an affinity-purified antibody to fumarase and an antibody to the mitochondrial Hsp60 protein showed identical labeling pattern with labeling seen mainly in mitochondria. Detailed studies were performed using high-resolution immunogold electron microscopy to determine the subcellular localization of fumarase in rat tissues, embedded in LR White resin. In thin sections from kidney, liver, heart, adrenal gland and anterior pituitary, strong and specific labeling due to fumarase antibody was only detected in mitochondria. However, in the pancreatic acinar cells, in addition to mitochondria, highly significant labeling was also observed in the zymogen granules and endoplasmic reticulum. The observed labeling in all cases was completely abolished upon omission of the primary antibody indicating that it was specific. In a western blot of purified zymogen granules, a fumarase-antibody cross-reactive protein of the same molecular mass as seen in the mitochondria was present. These results provide evidence that fumarase in mammalian cells/tissues is mainly localized in mitochondria and significant amounts of this protein are not present in the cytosol. However, these studies also reveal that in certain tissues, in addition to mitochondria, this protein is also present at specific extramitochondrial sites. Although the cellular function of fumarase at these extramitochondrial locations is not known, the appearance/localization of fumarase outside mitochondria may help explain how mutations in this mitochondrial protein can give rise to a number of different types of cancers.