Cyanide-resistant alternative oxidase (AOX) is a nuclear-encoded quinol oxidase located in the inner mitochondrial membrane. Although the quality control of AOX proteins is expected to have a role in elevated respiration in mitochondria, it remains unclear whether thermogenic plants possess molecular mechanisms for the mitochondrial degradation of AOX. To better understand the mechanism of AOX turnover in mitochondria, we performed a series of in organello AOX degradation assays using mitochondria from various stages of the appendices of Arum maculatum. Our analyses clearly indicated that AOX proteins at certain stages in the appendices are degraded at 30°C, which is close to the maximum appendix temperature observed during thermogenesis. Interestingly, such temperature-dependent protease activities were specifically inhibited by E-64, a cysteine protease inhibitor. Moreover, purification and subsequent nano LC–MS/MS analyses of E-64-sensitive and DCG-04-labeled active mitochondrial protease revealed an ∼30 kDa protein with an identical partial peptide sequence to the cysteine protease 1-like protein from Phoenix dactylifera. Our data collectively suggest that AOX is a potential target for temperature-dependent E-64-sensitive cysteine protease in the appendices of A. maculatum. A possible retrograde signalling cascade mediated by specific degradation of AOX proteins and its physiological significance are discussed.

中文翻译：

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斑果附睾中线粒体替代氧化酶的降解

抗氰化物的替代氧化酶（AOX）是位于线粒体内膜上的核编码喹诺酮氧化酶。尽管预期AOX蛋白的质量控制在线粒体呼吸增加中起作用，但尚不清楚致热植物是否具有AOX线粒体降解的分子机制。为了更好地了解线粒体中AOX转换的机制，我们使用了来自Arum maculatum阑尾各个阶段的线粒体的一系列器官内AOX降解测定。我们的分析清楚地表明，附录中某些阶段的AOX蛋白在30°C时降解，这与生热过程中观察到的最高附录温度接近。有趣的是，这种温度依赖性蛋白酶活性被E-64特异性抑制，半胱氨酸蛋白酶抑制剂。此外，对E-64敏感和DCG-04-标记的活性线粒体蛋白酶的纯化和随后的纳米LC-MS / MS分析显示，约30 kDa的蛋白具有与Phoenix dactylifera的半胱氨酸蛋白酶1样蛋白相同的部分肽序列。 。我们的数据共同表明，AOX是斑纹拟南芥附录中对温度依赖性E-64敏感的半胱氨酸蛋白酶的潜在靶标。讨论了由AOX蛋白的特异性降解介导的可能的逆行信号级联反应及其生理意义。纯化和随后的对E-64敏感和DCG-04-标记的活性线粒体蛋白酶的纳米LC-MS / MS分析显示，一个约30 kDa的蛋白具有与Phoenix dactylifera的半胱氨酸蛋白酶1样蛋白相同的部分肽序列。我们的数据共同表明，AOX是斑纹拟南芥附录中温度依赖性E-64敏感的半胱氨酸蛋白酶的潜在靶标。讨论了由AOX蛋白的特异性降解介导的可能的逆行信号级联反应及其生理意义。纯化和随后的对E-64敏感和DCG-04-标记的活性线粒体蛋白酶的纳米LC-MS / MS分析显示，一个约30 kDa的蛋白具有与Phoenix dactylifera的半胱氨酸蛋白酶1样蛋白相同的部分肽序列。我们的数据共同表明，AOX是斑纹拟南芥附录中温度依赖性E-64敏感的半胱氨酸蛋白酶的潜在靶标。讨论了由AOX蛋白的特异性降解介导的可能的逆行信号级联反应及其生理意义。