Protein features for SVM from A mitochondrion-free eukaryote contains proteins capable of import into an exogenous mitochondrion-related organelle

The endobiotic flagellate Monocercomonoides exilis is the only known eukaryote to have lost mitochondria and all its associated proteins in its evolutionary past. This final stage of the mitochondrial evolutionary pathway may serve as a model to explain events at their very beginning such as the initiation of protein import. We have assessed the capability of proteins from this eukaryote to enter emerging mitochondria using a specifically designed in vitro assay. Hydrogenosomes (reduced mitochondria) of Trichomonas vaginalis were incubated with a soluble protein pool derived from a cytosolic fraction of M. exilis, and proteins entering hydrogenosomes were subsequently detected by mass spectrometry. The assay detected 19 specifically and reproducibly imported proteins, and in 14 cases the import was confirmed by the overexpression of their tagged version in T. vaginalis. In most cases, only a small portion of the signal reached the hydrogenosomes, suggesting specific but inefficient transport. Most of these proteins represent enzymes of carbon metabolism, and none exhibited clear signatures of proteins targeted to hydrogenosomes or mitochondria, which is consistent with their inefficient import. The observed phenomenon may resemble a primaeval type of protein import which might play a role in the establishment of the organelle and shaping of its proteome in the initial stages of endosymbiosis.

内生鞭毛虫单尾鞭滴虫（Monocercomonoides exilis）是目前已知唯一一种在演化历程中丢失了线粒体（mitochondria）及其所有关联蛋白的真核生物（eukaryote）。该线粒体演化通路的最终阶段，可作为模型阐释其起始阶段的相关事件，例如蛋白质导入的启动过程。本研究采用定制化设计的体外实验体系（in vitro assay），评估了该真核生物的蛋白质进入新生线粒体的能力。研究人员将阴道毛滴虫（Trichomonas vaginalis）的氢化酶体（hydrogenosomes，即退化型线粒体）与源自单尾鞭滴虫胞质组分的可溶性蛋白池共同孵育，随后通过质谱法（mass spectrometry）检测进入氢化酶体的蛋白质。该实验体系共检测到19种可特异性且可重复地被导入的蛋白质，其中14种的导入过程通过在阴道毛滴虫中过表达（overexpression）其带标签的重组版本得到了验证。多数情况下，仅少量靶蛋白能抵达氢化酶体，提示该转运过程具备特异性但效率偏低。这类蛋白质大多属于碳代谢（carbon metabolism）相关酶类，且均未表现出靶向氢化酶体或线粒体的蛋白质典型特征，这与其导入效率偏低的现象相符。本研究观测到的这一现象，可能类似于一种原始的蛋白质导入机制，该机制或许在内共生（endosymbiosis）初期参与了细胞器的建立及其蛋白质组（proteome）的塑造过程。