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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. Anin vivo enzymatic tagging technique based on theEscherichia coli biotin ligase (BirA) has been introduced toG. intestinalis ; this method allows for the compartment-specific biotinylation of a protein of interest. Known proteins involved in the mitosomal protein import werein 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 (Gi Tim44), 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 toG. intestinalis and even absent from the related diplomonad parasiteSpironucleus salmonicida , thus reflecting the unique character of the mitosomal metabolism. Thein vivo enzymatic tagging also showed that proteins enter the mitosome posttranslationally in an unfolded state and without vesicular transport.

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