Plastid biogenesis in malaria parasites requires the interactions and catalytic activity of the Clp proteolytic system
Plastid biogenesis in malaria parasites requires the interactions and catalytic activity of the Clp proteolytic system
复制标题
DOI：
10.1073/pnas.1919501117
复制
发表时间：
2020-06-16
影响因子：
11.1
通讯作者：
Muralidharan, Vasant
中科院分区：
文献类型：
--
作者：
Florentin, Anat;Stephens, Dylon R.;Muralidharan, Vasant
人类疟疾寄生虫，恶性疟原虫，含有一个基本的质体称为顶质体。大多数顶质体蛋白质由核基因组编码，质体蛋白质组是如何调控的尚不清楚。在这里，我们研究了一个顶质体本地化的酪蛋白水解蛋白酶（CLP）系统，以及它如何调节细胞器蛋白质稳态。使用无效突变体和条件突变体，我们证明了恶性疟原虫Clp蛋白酶（Pf ClpP）具有强大的酶活性，这是顶质体生物发生所必需的。我们开发了一个基于CRISPR/Cas9的系统来表达催化死亡的Pf ClpP，这表明Pf ClpP作为酶原寡聚化并通过transautocatalysis形成。野生型和突变型Clp分子伴侣（Pf ClpC）变体的表达揭示了功能性分子伴侣-蛋白酶相互作用。有条件的突变体的基板适配器（Pf ClpS）证明了其在质体生物发生的重要功能。多个亲和纯化筛选的组合鉴定了Clp复合物的组成以及推定的Clp底物。这项全面的研究揭示了影响顶质体Clp系统蛋白水解功能的分子组成和相互作用，并证明了其在疟原虫质体生物发生中的核心作用。
The human malaria parasite, Plasmodium falciparum , contains an essential plastid called the apicoplast. Most apicoplast proteins are encoded by the nuclear genome and it is unclear how the plastid proteome is regulated. Here, we study an apicoplast-localized caseinolytic-protease (Clp) system and how it regulates organelle proteostasis. Using null and conditional mutants, we demonstrate that the P. falciparum Clp protease (Pf ClpP) has robust enzymatic activity that is essential for apicoplast biogenesis. We developed a CRISPR/Cas9-based system to express catalytically dead Pf ClpP, which showed that Pf ClpP oligomerizes as a zymogen and is ma-tured via transautocatalysis. The expression of both wild-type and mutant Clp chaperone (Pf ClpC) variants revealed a functional chaperone-protease interaction. Conditional mutants of the substrate-adaptor (Pf ClpS) demonstrated its essential function in plastid biogenesis. A combination of multiple affinity purification screens identified the Clp complex composition as well as putative Clp substrates. This comprehensive study reveals the molecular composition and interactions influencing the proteolytic function of the apicoplast Clp system and demonstrates its central role in the biogenesis of the plastid in malaria parasites.