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Probing the Biology of Giardia intestinalis Mitosomes Using In Vivo Enzymatic Tagging

机译：使用体内酶标记法探查贾第鞭毛虫肠线粒体的生物学

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Giardia intestinalis parasites contain mitosomes, one of the simplest mitochondrion-related organelles. Strategies to identify the functions of mitosomes have been limited mainly to homology detection, which is not suitable for identifying species-specific proteins and their functions. An in vivo enzymatic tagging technique based on the Escherichia coli biotin ligase (BirA) has been introduced to G. intestinalis; this method allows for the compartment-specific biotinylation of a protein of interest. Known proteins involved in the mitosomal protein import were in vivo tagged, cross-linked, and used to copurify complexes from the outer and inner mitosomal membranes in a single step. New proteins were then identified by mass spectrometry. This approach enabled the identification of highly diverged mitosomal Tim44 (GiTim44), the first known component of the mitosomal inner membrane translocase (TIM). In addition, our subsequent bioinformatics searches returned novel diverged Tim44 paralogs, which mediate the translation and mitosomal insertion of mitochondrially encoded proteins in other eukaryotes. However, most of the identified proteins are specific to G. intestinalis and even absent from the related diplomonad parasite Spironucleus salmonicida, thus reflecting the unique character of the mitosomal metabolism. The in vivo enzymatic tagging also showed that proteins enter the mitosome posttranslationally in an unfolded state and without vesicular transport.
机译：贾第鞭毛虫肠道寄生虫含有线粒体，它是最简单的线粒体相关细胞器之一。鉴定线粒体功能的策略主要限于同源性检测，这不适用于鉴定物种特异性蛋白质及其功能。一种基于大肠杆菌生物素连接酶（BirA）的体内酶标记技术已被引入到肠小肠结肠菌中。该方法允许目的蛋白的区室特异性生物素化。体内已对参与线粒体蛋白质导入的已知蛋白质进行了标记，进行了交联，并用于一步纯化共纯化线粒体外膜和内膜的复合物。然后通过质谱鉴定新蛋白。这种方法能够鉴定出高度分离的线粒体Tim44（ Gi Tim44），这是线粒体内膜转运蛋白（TIM）的第一个已知成分。此外，我们随后的生物信息学搜索返回了新的发散的Tim44旁系同源物，其介导其他真核生物中线粒体编码蛋白的翻译和微粒体插入。然而，大多数鉴定出的蛋白质对肠球菌具有特异性，甚至在相关的双文凭寄生虫螺旋体鲑鱼中也没有，因此反映了微粒体代谢的独特性。体内酶标还显示蛋白质在翻译后以未折叠状态且无水泡转运进入线粒体。

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