Florentin Anat, Stephens Dylon R, Brooks Carrie F, Baptista Rodrigo P, Muralidharan Vasant
Department of Cellular Biology, University of Georgia, Athens, GA 30602.
Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA 30602.
Proc Natl Acad Sci U S A. 2020 Jun 16;117(24):13719-13729. doi: 10.1073/pnas.1919501117. Epub 2020 Jun 1.
The human malaria parasite, , 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 Clp protease (ClpP) has robust enzymatic activity that is essential for apicoplast biogenesis. We developed a CRISPR/Cas9-based system to express catalytically dead ClpP, which showed that ClpP oligomerizes as a zymogen and is matured via transautocatalysis. The expression of both wild-type and mutant Clp chaperone (ClpC) variants revealed a functional chaperone-protease interaction. Conditional mutants of the substrate-adaptor (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.
人类疟原虫含有一种名为顶质体的重要质体。大多数顶质体蛋白由核基因组编码，目前尚不清楚质体蛋白质组是如何调控的。在此，我们研究了一种定位于顶质体的酪蛋白水解蛋白酶（Clp）系统及其如何调节细胞器蛋白质稳态。利用基因敲除和条件性突变体，我们证明Clp蛋白酶（ClpP）具有强大的酶活性，这对顶质体生物发生至关重要。我们开发了一种基于CRISPR/Cas9的系统来表达催化失活的ClpP，结果表明ClpP作为一种酶原寡聚化，并通过自身催化成熟。野生型和突变型Clp伴侣蛋白（ClpC）变体的表达揭示了一种功能性的伴侣蛋白-蛋白酶相互作用。底物衔接蛋白（ClpS）的条件性突变体证明了其在质体生物发生中的重要功能。多种亲和纯化筛选相结合，确定了Clp复合物的组成以及假定的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。
