Tissue-specific expression of dominant negative mutant Drosophila HSC70 causes developmental defects and lethality
Tissue-specific expression of dominant negative mutant Drosophila HSC70 causes developmental defects and lethality
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DOI：
10.1091/mbc.10.7.2101
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发表时间：
1999-07-01
影响因子：
3.3
通讯作者：
Palter, KB
中科院分区：
文献类型：
--
作者：
Elefant, F;Palter, KB
果蝇HSC3和HSC4基因分别编码与哺乳动物内质网(ER)蛋白Bip同源的Hsc70蛋白和胞质网状蛋白去包被ATPase。这些蛋白质具有ATP结合/水解活性，通过协调错误折叠蛋白的顺序结合和释放来调节它们帮助蛋白质折叠的能力。为了研究Hsc3(Hsc3p)和Hsc4(Hsc4p)蛋白在果蝇发育过程中的作用，采用GAL4靶向基因表达的方法，分析了果蝇发育过程中特定组织产生显性负性作用的Hsc3p(D231S，K97S)和Hsc4p(D206S，K71S)蛋白的作用。我们表明，每种突变蛋白的产生在一系列发育阶段都会导致致命性，这取决于产生的蛋白质水平和目标组织。我们证明Hsc3p和Hsc4p的功能都是适当的组织建立和维持所必需的。在正常温度下，突变的Hsc4p的产生，而不是Hsc3p的产生，会导致应激诱导的Hsp70的诱导。有证据表明，致命性是由组织特异性缺陷引起的，这些缺陷是由于缺乏功能性Hsc70导致错误折叠蛋白的全球积累所致。我们发现，尽管Hsc3p(D231S)确实经历了ATP诱导的构象变化，但这两个突变的Hsc3p在ATP诱导的底物释放方面都存在缺陷。我们认为，Hsc3p上的氨基酸替换干扰了ATP结合到底物释放的结构偶联，这一缺陷是突变蛋白在体内产生显性负效应的基础。
The Drosophila melanogaster HSC3 and HSC4 genes encode Hsc70 proteins homologous to the mammalian endoplasmic reticulum (ER) protein BiP and the cytoplasmic clathrin uncoating ATPase, respectively. These proteins possess ATP binding/hydrolysis activities that mediate their ability to aid in protein folding by coordinating the sequential binding and release of misfolded proteins. To investigate the roles of HSC3 (Hsc3p) and HSC4 (Hsc4p) proteins during development, GAL4-targeted gene expression was used to analyze the effects of producing dominant negatively acting Hsc3p (D231S, K97S) and Hsc4p (D206S, K71S) proteins, containing single amino acid substitutions in their ATP-binding domains, in specific tissues of Drosophila throughout development. We show that the production of each mutant protein results in lethality over a range of developmental stages, depending on the levels of protein produced and which tissues are targeted. We demonstrate that the functions of both Hsc3p and Hsc4p are required for proper tissue establishment and maintenance. Production of mutant Hsc4p, but not Hsc3p, results in induction of the stress-inducible Hsp70 at normal temperatures. Evidence is presented that lethality is caused by tissue-specific defects that result from a global accumulation of misfolded protein caused by lack of functional Hsc70. We show that both mutant Hsc3ps are defective in ATP-induced substrate release, although Hsc3p(D231S) does undergo an ATP-induced conformational change. We believe that the amino acid substitutions in Hsc3p interfere with the structural coupling of ATP binding to substrate release, and this defect is the basis for the mutant proteins' dominant negative effects in vivo.