Kinetoplastid parasites, including Trypanosoma brucei, Trypanosoma cruzi, and Leishmania, harbor unique organelles known as glycosomes, which are evolutionarily related to peroxisomes. Glycosome/peroxisome biogenesis is mediated by proteins called peroxins that facilitate organelle formation, proliferation, and degradation and import of proteins housed therein. Import of matrix proteins occurs via one of two pathways that are dictated by their peroxisome targeting sequence (PTS). In PTS1 import, a C-terminal tripeptide sequence, most commonly SKL, is recognized by the soluble receptor Pex5. In PTS2 import, a less conserved N-terminal sequence is recognized by Pex7. The soluble receptors deliver their cargo to the import channel consisting minimally of Pex13 and Pex14. While much of the import process is conserved, kinetoplastids are the only organisms to have two Pex13s, Pex13.1 and Pex13.2. It is unclear why trypanosomes require two Pex13s when one is sufficient for most eukaryotes. To interrogate the role of Pex13.2, we have employed biochemical approaches to partially resolve the composition of the Pex13/Pex14 import complexes in T. brucei and characterized glycosome morphology and protein import in Pex13.2-deficient parasites. Here, we show that Pex13.2 is an integral glycosome membrane protein that interacts with Pex13.1 and Pex14. The N terminus of Pex13.2 faces the cytoplasmic side of the membrane, where it can facilitate interactions required for protein import. Two-dimensional gel electrophoresis revealed three glycosome membrane complexes containing combinations of Pex13.1, Pex13.2, and Pex14. The silencing of Pex13.2 resulted in parasites with fewer, larger glycosomes and disrupted glycosome protein import, suggesting the protein is involved in glycosome biogenesis as well as protein import. Furthermore, superresolution microscopy demonstrated that Pex13.2 localizes to discrete foci in the glycosome periphery, indicating that the glycosome periphery is not homogenous.IMPORTANCE Trypanosoma brucei causes human African trypanosomiasis and a wasting disease called Nagana in livestock. Current treatments are expensive, toxic, and difficult to administer. Because of this, the search for new drug targets is essential. T. brucei has glycosomes that are essential to parasite survival; however, our ability to target them in drug development is hindered by our lack of understanding about how these organelles are formed and maintained. This work forwards our understanding of how the parasite-specific protein Pex13.2 functions in glycosome protein import and lays the foundation for future studies focused on blocking Pex13.2 function, which would be lethal to bloodstream-form parasites that reside in the mammalian bloodstream.

中文翻译：

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布氏锥虫 Pex13.2 是一种辅助过氧化物酶，在过氧化物酶体靶向序列 2 型蛋白的导入中发挥作用，并定位于糖体的子结构域。


动质体寄生虫，包括布氏锥虫、克氏锥虫和利什曼原虫，拥有独特的细胞器，称为糖体，其在进化上与过氧化物酶体相关。糖体/过氧化物酶体生物发生是由称为过氧化物酶的蛋白质介导的，其促进细胞器的形成、增殖、降解以及其中蛋白质的输入。基质蛋白的导入通过由其过氧化物酶体靶向序列 (PTS) 决定的两条途径之一进行。在 PTS1 输入中，C 端三肽序列（最常见的是 SKL）被可溶性受体 Pex5 识别。在 PTS2 导入中，Pex7 识别不太保守的 N 端序列。可溶性受体将其货物输送至至少由 Pex13 和 Pex14 组成的输入通道。虽然大部分输入过程是保守的，但动质体是唯一具有两个 Pex13（Pex13.1 和 Pex13.2）的生物体。目前还不清楚为什么锥虫需要两个 Pex13，而 1 个 Pex13 对于大多数真核生物来说就足够了。为了探究 Pex13.2 的作用，我们采用生化方法部分解析了 T. brucei 中 Pex13/Pex14 输入复合物的组成，并表征了 Pex13.2 缺陷寄生虫中的糖体形态和蛋白质输入。在这里，我们证明 Pex13.2 是一种完整的糖体膜蛋白，可与 Pex13.1 和 Pex14 相互作用。 Pex13.2 的 N 末端面向膜的细胞质侧，可以促进蛋白质输入所需的相互作用。二维凝胶电泳揭示了含有 Pex13.1、Pex13.2 和 Pex14 组合的三种糖体膜复合物。 Pex13 的沉默。2导致寄生虫具有更少、更大的糖体，并且糖体蛋白输入被破坏，这表明该蛋白参与糖体生物合成以及蛋白质输入。此外，超分辨率显微镜显示 Pex13.2 定位于糖体外围的离散病灶，表明糖体外围不是同质的。 重要性 布氏锥虫会导致人类非洲锥虫病和牲畜中称为 Nagana 的消耗性疾病。目前的治疗方法昂贵、有毒且难以实施。正因为如此，寻找新的药物靶点至关重要。布氏锥虫具有对寄生虫生存至关重要的糖体；然而，由于我们对这些细胞器如何形成和维持缺乏了解，我们在药物开发中针对它们的能力受到阻碍。这项工作增进了我们对寄生虫特异性蛋白 Pex13.2 如何在糖体蛋白输入中发挥作用的理解，并为未来专注于阻断 Pex13.2 功能的研究奠定了基础，这对于存在于哺乳动物血液中的血流形式寄生虫来说是致命的。