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.

• MeSH
• biotinylace MeSH
• Escherichia coli enzymologie   MeSH
• frakcionace buněk MeSH
• Giardia lamblia chemie   cytologie   metabolismus   MeSH
• giardiáza parazitologie   MeSH
• hmotnostní spektrometrie MeSH
• lidé MeSH
• ligasy tvořící vazby C-N metabolismus   MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• organely chemie   metabolismus   MeSH
• proteiny z Escherichia coli metabolismus   MeSH
• protozoální proteiny analýza   izolace a purifikace   metabolismus   MeSH
• represorové proteiny metabolismus   MeSH
• sekvence aminokyselin MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH