Data from: A single Tim translocase in the mitosomes of Giardia intestinalis illustrates convergence of protein import machines in anaerobic eukaryotes

Mitochondria have evolved diverse forms across eukaryotic diversity in
adaptation to anaerobiosis. Mitosomes are the simplest and the least
well-studied type of anaerobic mitochondria. Transport of proteins via TIM
complexes, composed of three proteins of the Tim17 protein family
(Tim17/22/23), is one of the key unifying aspects of mitochondrial and
mitochondria-derived organelles. However, multiple experimental and
bioinformatic attempts have so far failed to identify the nature of TIM in
mitosomes of the anaerobic metamonad protist, Giardia intestinalis, one of
the few experimental models for mitosome biology. Here, we present the
identification of a single G. intestinalis Tim17 protein (GiTim17), made
possible only by the implementation of a metamonad-specific hidden Markov
model. While very divergent in primary sequence and in predicted membrane
topology, experimental data suggest that GiTim17 is an inner membrane
mitosomal protein, forming a disulphide-linked dimer. We suggest that the
peculiar GiTim17 sequence reflects adaptation to the unusual, detergent
resistant, inner mitosomal membrane. Specific pull-down experiments
indicate interaction of GiTim17 with mitosomal Tim44, the tethering
component of the import motor complex. Analysis of TIM complexes across
eukaryote diversity indicates that a “single Tim” translocase is a
convergent adaptation of mitosomes in anaerobic protists, with Tim22 and
Tim17 (but not Tim23), providing the protein backbone.

线粒体（Mitochondria）在真核生物的多样类群中演化出多种形态，以适应厌氧环境。纺锤剩体（mitosomes）是结构最简单、研究程度最低的厌氧型线粒体衍生细胞器。由Tim17蛋白家族的3种蛋白（Tim17/22/23）组成的TIM复合体（Translocase of the Inner Membrane，内膜转位酶复合体）介导的蛋白质转运，是线粒体及其衍生细胞器的核心共性特征之一。然而迄今为止，多项实验与生物信息学研究均未能鉴定出厌氧后滴门原生生物——蓝氏贾第鞭毛虫（Giardia intestinalis）——其纺锤剩体中TIM复合体的本质；而蓝氏贾第鞭毛虫是为数不多的纺锤剩体生物学实验模型之一。本研究通过构建后滴门特异性隐马尔可夫模型（Hidden Markov Model, HMM），成功鉴定出蓝氏贾第鞭毛虫中的单拷贝Tim17蛋白（命名为GiTim17）。尽管GiTim17在一级氨基酸序列与预测的膜拓扑结构上均存在显著差异，但实验数据表明其定位于纺锤剩体内膜，可形成二硫键连接的二聚体。我们推测，GiTim17独特的序列特征反映了其对纺锤剩体特殊的、抗去污剂的内膜环境的适应性。特异性亲和纯化下拉实验证实，GiTim17可与纺锤剩体来源的Tim44相互作用，而Tim44是蛋白质输入马达复合体的锚定组分。对不同真核生物类群TIM复合体的分析表明，"单Tim"转位酶是厌氧原生生物纺锤剩体的趋同演化适应，其蛋白骨架由Tim22与Tim17（而非Tim23）构成。