Cechetto J D, Gupta R S
Department of Biochemistry, McMaster University, Hamilton, Ontario, L8N 3Z5, Canada.
Exp Cell Res. 2000 Oct 10;260(1):30-9. doi: 10.1006/excr.2000.4983.
The tumor necrosis factor receptor-associated protein 1 (TRAP-1) interacts with a variety of proteins involved in diverse functions. We have used quantitative immunogold electron microscopy and biochemical analysis to evaluate the subcellular distribution of TRAP-1 in rat tissues. Immunofluorescence employing a polyclonal antibody raised to human recombinant TRAP-1 reveals specific staining of mitochondria and nuclear region in mammalian cells. Western blot analysis of purified rat liver mitochondrial subfractions with the TRAP-1 antibody reveals that the cross-reactive protein (M(r) approximately 80 kDa) is mainly present in the matrix compartment. Immunogold labeling of rat tissue sections embedded in LR Gold resin shows strong labeling of mitochondria in all the tissues examined (viz., liver, heart, pancreas, kidney, spleen, anterior pituitary gland). Additionally, specific and significant labeling with TRAP-1 antibody was also observed in certain tissues in a number of nonmitochondrial locations, including pancreatic zymogen granules, insulin secretory granules, cardiac sarcomeres, and nuclei of pancreatic and heart cells, and on the cell surface of blood vessel endothelial cells. Western blot analysis showed that a cross-reactive protein of similar molecular mass as TRAP-1 is present in purified pancreatic zymogen granules. Immunogold labeling was prevented in all tissues by preadsorption of the TRAP-1 antibody with the purified recombinant TRAP-1 protein. These observations and the fact that TRAP-1 is synthesized with a typical mitochondrial targeting presequence strongly indicate that TRAP-1 is primarily a mitochondrial matrix protein. The localization of this protein at specific extramitochondrial sites raises interesting and fundamental questions regarding the possible mechanisms by which these proteins are translocated to such sites.
肿瘤坏死因子受体相关蛋白1（TRAP-1）与多种参与不同功能的蛋白质相互作用。我们利用定量免疫金电子显微镜和生化分析来评估TRAP-1在大鼠组织中的亚细胞分布。使用针对人重组TRAP-1产生的多克隆抗体进行免疫荧光检测，结果显示在哺乳动物细胞中线粒体和核区域有特异性染色。用TRAP-1抗体对纯化的大鼠肝脏线粒体亚组分进行蛋白质印迹分析，结果表明交叉反应蛋白（分子量约80 kDa）主要存在于基质区室。对包埋在LR Gold树脂中的大鼠组织切片进行免疫金标记，结果显示在所检查的所有组织（即肝脏、心脏、胰腺、肾脏、脾脏、垂体前叶）中线粒体有强烈标记。此外，在一些非线粒体位置的某些组织中也观察到TRAP-1抗体的特异性和显著标记，包括胰腺酶原颗粒、胰岛素分泌颗粒、心肌肌节以及胰腺和心脏细胞的细胞核，还有血管内皮细胞的细胞表面。蛋白质印迹分析表明，纯化的胰腺酶原颗粒中存在一种分子量与TRAP-1相似的交叉反应蛋白。用纯化的重组TRAP-1蛋白预吸附TRAP-1抗体后，所有组织中的免疫金标记均被阻断。这些观察结果以及TRAP-1是带着典型的线粒体靶向前序列合成这一事实，有力地表明TRAP-1主要是一种线粒体基质蛋白。这种蛋白质在特定的线粒体外位点的定位，引发了关于这些蛋白质转运到此类位点的可能机制的有趣且基本的问题。
