Functional Analysis of the Hsp93/ClpC Chaperone at the Chloroplast Envelope
Functional Analysis of the Hsp93/ClpC Chaperone at the Chloroplast Envelope
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DOI：
10.1104/pp.15.01538
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发表时间：
2016-01-01
期刊：
影响因子：
7.4
通讯作者：
Jarvis, Paul
中科院分区：
文献类型：
--
作者：
Flores-Perez, Ursula;Bedard, Jocelyn;Jarvis, Paul
Hsp 100型分子伴侣Hsp 93/ClpC在叶绿体生物发生中起着重要作用。除了其在基质中蛋白水解中的作用外，生化和遗传证据还导致了这样的假设，即该分子伴侣与内包膜TIC复合物协作以提供前蛋白输入的动力。最近，有人建议，热休克蛋白93，与Clp蛋白水解核心一起工作，可以赋予在包膜蛋白质质量控制机制。因此，Hsp 93的作用及其参与蛋白质输入的机制尚不清楚。为了分析Hsp 93在蛋白质输入中独立于其ClpP缔合的功能，我们创建了影响其ClpP结合基序（PBM）的Hsp 93突变体（Hsp 93 [P-]），其对于伴侣蛋白与Clp蛋白水解核心的相互作用是必需的。Hsp 93 [P-]构建体在补充Hsp 93拟南芥（Arabidopsis thaliana）突变体的浅黄色表型方面是无效的，表明PBM对于Hsp 93功能是必需的。正如预期的那样，PBM突变负面影响基质Clp蛋白酶的降解活性。该突变还破坏了与Clp蛋白水解核心在信封的Hsp 93的协会，而不影响信封定位的Hsp 93本身或其关联的TIC机械，我们证明是介导的直接相互作用与Tic 110。尽管如此，Hsp 93 [P-]的表达并没有明显提高hsp 93突变体叶绿体的蛋白质输入效率。因此，我们的研究结果不支持建议的功能，热休克蛋白93在蛋白质进口推进，但更符合的概念，热休克蛋白93执行质量控制作用的进口点。
The Hsp100-type chaperone Hsp93/ClpC has crucial roles in chloroplast biogenesis. In addition to its role in proteolysis in the stroma, biochemical and genetic evidence led to the hypothesis that this chaperone collaborates with the inner envelope TIC complex to power preprotein import. Recently, it was suggested that Hsp93, working together with the Clp proteolytic core, can confer a protein quality control mechanism at the envelope. Thus, the role of envelope-localized Hsp93, and the mechanism by which it participates in protein import, remain unclear. To analyze the function of Hsp93 in protein import independently of its ClpP association, we created a mutant of Hsp93 affecting its ClpP-binding motif (PBM) (Hsp93[P-]), which is essential for the chaperone's interaction with the Clp proteolytic core. The Hsp93[P-] construct was ineffective at complementing the pale-yellow phenotype of hsp93 Arabidopsis (Arabidopsis thaliana) mutants, indicating that the PBM is essential for Hsp93 function. As expected, the PBM mutation negatively affected the degradation activity of the stromal Clp protease. The mutation also disrupted association of Hsp93 with the Clp proteolytic core at the envelope, without affecting the envelope localization of Hsp93 itself or its association with the TIC machinery, which we demonstrate to be mediated by a direct interaction with Tic110. Nonetheless, Hsp93[P-] expression did not detectably improve the protein import efficiency of hsp93 mutant chloroplasts. Thus, our results do not support the proposed function of Hsp93 in protein import propulsion, but are more consistent with the notion of Hsp93 performing a quality control role at the point of import.