The Chloroplast Envelope Protease FTSH11-Interaction With CPN60 and Identification of Potential Substrates
The Chloroplast Envelope Protease FTSH11-Interaction With CPN60 and Identification of Potential Substrates
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DOI：
10.3389/fpls.2019.00428
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发表时间：
2019-04-05
影响因子：
5.6
通讯作者：
Chen, Junping
中科院分区：
文献类型：
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作者：
Adam, Zach;Aviv-Sharon, Elinor;Chen, Junping
FTSH蛋白酶是在细菌、线粒体和叶绿体中发现的膜结合的ATP依赖性金属蛋白酶。拟南芥中编码FTSH蛋白酶的12个基因之一FTSH 11的产物先前已被证明是获得耐热性所必需的。然而，这种蛋白酶的底物以及将其与耐热性联系起来的机制在很大程度上是未知的。为了深入了解这些，在天然启动子的控制下，用HA标签标记的该蛋白酶的蛋白水解活性或无活性变体补充FTSH 11敲除突变体。利用这些植物的耐热性试验表明，蛋白水解活性，而不仅仅是ATP酶的一个，是必不可少的赋予耐热性。叶提取物，分离的细胞器和亚分级的叶绿体膜的免疫印迹分析定位FTSH11主要是叶绿体包膜。亲和纯化，然后质谱分析显示FTSH11和CPN60伴侣蛋白的不同组分之间的相互作用。在亲和富集测定中，发现CPN60以及许多包膜、基质和类囊体蛋白与蛋白水解失活的FTSH 11相关。WT和基因敲除植物，生长在20摄氏度或暴露于30摄氏度6小时的比较蛋白质组学分析，揭示了过量的上调叶绿体蛋白在敲除，其中一些可能是候选底物。其中突出的是TIC40，其在从热应激恢复后稳定在敲除系中，以及在亲和富集测定中发现的三种蛋白质：核苷酸反向转运蛋白PAPST 2、脂肪酸结合蛋白FAP 1和伴侣HSP 70。这四种蛋白在不同测定中的一致行为表明它们是潜在的FTSH11底物。
FTSH proteases are membrane-bound, ATP-dependent metalloproteases found in bacteria, mitochondria and chloroplasts. The product of one of the 12 genes encoding FTSH proteases in Arabidopsis, FTSH11, has been previously shown to be essential for acquired thermotolerance. However, the substrates of this protease, as well as the mechanism linking it to thermotolerance are largely unknown. To get insight into these, the FTSH11 knockout mutant was complemented with proteolytically active or inactive variants of this protease, tagged with HA-tag, under the control of the native promoter. Using these plants in thermotolerance assay demonstrated that the proteolytic activity, and not only the ATPase one, is essential for conferring thermotolerance. Immunoblot analyses of leaf extracts, isolated organelles and sub-fractionated chloroplast membranes localized FTSH11 mostly to chloroplast envelopes. Affinity purification followed by mass spectrometry analysis revealed interaction between FTSH11 and different components of the CPN60 chaperonin. In affinity enrichment assays, CPN60s as well as a number of envelope, stroma and thylakoid proteins were found associated with proteolytically inactive FTSH11. Comparative proteomic analysis of WT and knockout plants, grown at 20 degrees C or exposed to 30 degrees C for 6 h, revealed a plethora of upregulated chloroplast proteins in the knockout, some of them might be candidate substrates. Among these stood out TIC40, which was stabilized in the knockout line after recovery from heat stress, and three proteins that were found trapped in the affinity enrichment assay: the nucleotide antiporter PAPST2, the fatty acid binding protein FAP1 and the chaperone HSP70. The consistent behavior of these four proteins in different assays suggest that they are potential FTSH11 substrates.