CLONING AND SUBCELLULAR-LOCALIZATION OF HUMAN MITOCHONDRIAL HSP70
CLONING AND SUBCELLULAR-LOCALIZATION OF HUMAN MITOCHONDRIAL HSP70
复制标题
DOI：
10.1074/jbc.270.4.1705
复制
发表时间：
1995-01-27
影响因子：
4.8
通讯作者：
MORIMOTO, RI
中科院分区：
文献类型：
--
作者：
BHATTACHARYYA, T;KARNEZIS, AN;MORIMOTO, RI
我们报道了人类HSP70多基因家族成员线粒体HSP70的克隆、核苷酸序列和定位。通过使用识别人HSP70家族三个成员(HSP70、hsc70和一个75 kDa蛋白，其特征与先前建立的线粒体HSP70相同)的单抗3A3筛选表达文库，克隆了人mthsp75基因。通过对HeLa细胞的亚细胞分离和3A3反应性75-kDa蛋白与线粒体定位蛋白的共分离，证实了75 kDa蛋白的同源性。各克隆的核苷酸序列显示了一个由679个氨基酸组成的开放阅读框，与人HSP70家族成员具有广泛的序列同源性，推导出的氨基末端前序列具有其他线粒体靶向序列的共同特征，通过在克隆基因的羧基末端引入表位标签明确了该cDNA的同源性，并通过免疫荧光分析，标记的75 kDa蛋白定位于线粒体，从而提供了确凿的证据，证明它对应于人线粒体HSP70，这里指的是mthsp75。
We report the cloning, nucleotide sequence, and localization of mitochondrial hsp70, a member of the human hsp70 multi-gene family, The human mthsp75 gene was cloned by screening an expression library with monoclonal antibody 3A3 that recognizes three members of the human hsp70 family (hsp70, hsc70, and a 75-kDa protein with characteristics identical to that previously established for mitochondrial hsp70), The identity of the 75-kDa protein was confirmed by subcellular fractionation of HeLa cells and the demonstration that the 3A3-reactive 75-kDa protein co-fractionates with mitochondrial localized proteins, The nucleotide sequence of the respective cDNA clone revealed an open reading frame of 679 amino acids with extensive sequence identity with members of the human hsp70 family, The derived amino-terminal pre-sequence shares features common to other mitochondrial targeting sequences, The identity of the cDNA was unequivocally established by introduction of an epitope-tag at the carboxyl terminus of the cloned gene, transfection and analysis by immunofluorescence, The tagged 75-kDa protein localizes to mito chondria, thus providing conclusive evidence that it corresponds to the human mitochondrial hsp70, referred to here as mthsp75.