N-ethylmaleimide inactivates a nucleotide-free Hsp70 molecular chaperone
N-ethylmaleimide inactivates a nucleotide-free Hsp70 molecular chaperone
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DOI：
10.1074/jbc.271.47.29937
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发表时间：
1996-11-22
影响因子：
4.8
通讯作者：
Chirico, WJ
中科院分区：
文献类型：
--
作者：
Liu, QL;Levy, EJ;Chirico, WJ
Hsp 70分子伴侣是一种ATP酶，它与蛋白质的疏水区域结合，并引导蛋白质的折叠、组装和跨膜转运。纯化的热休克蛋白70的能力uncoat网格蛋白包被的囊泡或刺激前体蛋白的翻译后易位到内质网，线粒体，和细胞核先前被证明是不敏感的巯基修饰试剂N-乙基马来酰亚胺（NEM）。在纯化胞质中蛋白质折叠所需的因子的过程中，我们发现NEM抑制了核糖体后上清液中酵母Hsp 70 Ssa 1 p的ATP-琼脂糖结合活性。我们还发现，从纯化的Ssa 1 p中完全去除核苷酸使其ATP-琼脂糖结合活性、ATP酶活性和翻译后易位刺激活性对NEM敏感。我们用[C-14]NEM修饰无核苷酸的Ssa 1 p，然后用蛋白酶消化。放射性标记的蛋白水解片段的纯化和测序显示，Ssa 1 p的三个半胱氨酸残基（Cys-15，Cys-264和Cys-303）中的每一个都被[C-14]NEM修饰。ADP保护每个半胱氨酸残基的修改和保护Ssa 1 p失活。半胱氨酸残基是三个NEM反应位点（NRS 1 -3）的反应中心。Ssa 1 p的NRSs与其他Hsp 70和肌动蛋白序列的比较显示，NRS 1的Cys-15是高度保守的，对NEM的敏感性可能是许多Hsp 70的特性。基于Hsc 70的三维结构，Ssa 1 p的半胱氨酸残基的预测位置表明，NEM可能会破坏Ssa 1 p的构象或干扰其与核苷酸结合的能力，这些结果表明，Ssa 1 p是一个NEM敏感的因素，从酵母细胞质提取物，刺激蛋白质翻译后易位到细胞器。
Hsp70 molecular chaperones are ATPases that bind to hydrophobic regions of proteins and guide their folding, assembly, and translocation across membranes. The ability of purified Hsp70s to uncoat clathrin-coated vesicles or to stimulate the post-translational translocation of precursor proteins into the endoplasmic reticulum, mitochondria, and the nucleus was previously shown not to be sensitive to the sulfhydryl-modifying reagent N-ethylmaleimide (NEM). During purification of factors required for protein folding in the cytosol, we found that the ATP-agarose binding activity of the yeast Hsp70 Ssa1p in postribosomal supernatants was inhibited by NEM. We also found that completely removing nucleo tides from purified Ssa1p rendered its ATP-agarose binding activity, ATPase activity, and post-translational translocation-stimulating activity sensitive to NEM, We modified nucleotide-free Ssa1p with [C-14]NEM and then digested it with proteases. Purification and sequencing of the radiolabeled proteolytic fragments revealed that each of Ssa1p's three cysteine residues (Cys-15, Cys-264, and Cys-303) was modified with [C-14]NEM. ADP protected each of the cysteine residues from modification and protected Ssa1p from inactivation. The cysteine residues are the reactive centers of three NEM-reactive sites (NRS1-3). A comparison of Ssa1p's NRSs to sequences of other Hsp70s and actin revealed that Cys-15 of NRS1 is highly conserved and that sensitivity to NEM may be a property of many Hsp70s. Based on the three-dimensional structure of Hsc70, the predicted locations of Ssa1p's cysteine residues suggest that NEM may disrupt the conformation of Ssa1p or interfere with its ability to bind nucleotides, Together the results demonstrate that Ssa1p is an NEM-sensitive factor in cytosolic extracts from yeast that stimulates post-translational translocation of proteins into organelles.