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首页> 外文期刊>Biochemistry >Self-processing of FtsH and its implication for the cleavage specificity of this protease.
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Self-processing of FtsH and its implication for the cleavage specificity of this protease.

机译：FtsH的自我加工及其对这种蛋白酶切割特异性的影响。

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摘要

FtsH, a membrane-bound and ATP-dependent protease of Escherichia coli, is involved in degradation of some of uncomplexed integral membrane proteins and short-lived cytoplasmic proteins. It is composed of an N-terminal membrane-spanning region and a following large cytoplasmic domain that contains ATPase and protease active sites. In the present study, it was found that FtsH undergoes C-terminal processing in vivo. The processing was blocked by loss of function mutations of FtsH. Purified FtsH-His(6)-Myc, a C-terminally tagged derivative of FtsH, was self-processed in vitro. This in vitro processing was observed only in the presence of ATP and not in the presence of adenosine 5'-(beta,gamma-imino)triphosphate (AMP-PNP). Moreover, such processing did not occur in the case of the ATPase motif mutant protein. These results indicated that this processing is a self-catalyzed reaction that needs ATP hydrolysis. Mutations in the hflKC genes that encode a possible modulator of FtsH, and the growth phase of the cells as well, affected the processing. Complementation experiments with genetically constructed variants suggested that both the processed and the unprocessed forms of FtsH are functional. The cleavage was found to occur between Met-640 and Ser-641, removing a heptapeptide from the C-terminus of FtsH. Systematic mutational analyses of Met-640 and Ser-641 revealed preferences for positively charged and hydrophobic amino acid residues at these positions for processing. This cleavage specificity may be shared by the self-cleavage and the substrate-cleavage reactions of this protease.
机译：FtsH是大肠杆菌的一种与膜结合且依赖ATP的蛋白酶，它参与一些未复杂的整合膜蛋白和短寿命细胞质蛋白的降解。它由一个N端跨膜区域和一个包含ATPase和蛋白酶活性位点的大细胞质结构域组成。在本研究中，发现FtsH在体内经历C末端加工。 FtsH功能突变的丧失阻止了该过程。纯化的FtsH-His（6）-Myc，FtsH的C末端标记衍生物，在体外进行了自我加工。仅在ATP存在下观察到这种体外加工，而在5'-β-γ-亚氨基三磷酸腺苷（AMP-PNP）存在下则未观察到。而且，在ATPase基序突变蛋白的情况下，这种处理没有发生。这些结果表明该过程是需要ATP水解的自催化反应。编码可能的FtsH调节剂的hflKC基因突变以及细胞的生长期也影响了加工过程。具有遗传构建的变体的互补实验表明，FtsH的加工形式和未加工形式均具有功能。发现该切割发生在Met-640和Ser-641之间，从FtsH的C末端去除了七肽。对Met-640和Ser-641进行的系统突变分析显示，在这些位置进行加工时，偏爱带正电和疏水性氨基酸残基。该切割特异性可以由该蛋白酶的自切割和底物切割反应共享。

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