title: Probing the Biology of Giardia intestinalis Mitosomes Using In Vivo Enzymatic Tagging.
abstract: 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.
authors.affiliation: Department of Parasitology, Faculty of Science, Charles University in Prague, Prague, Czech Republic.
authors.forename: Eva
authors.initials: E
authors.lastname: Martincová
authors.affiliation: Department of Parasitology, Faculty of Science, Charles University in Prague, Prague, Czech Republic.
authors.forename: Luboš
authors.initials: L
authors.lastname: Voleman
authors.affiliation: Department of Parasitology, Faculty of Science, Charles University in Prague, Prague, Czech Republic.
authors.forename: Jan
authors.initials: J
authors.lastname: Pyrih
authors.affiliation: Department of Parasitology, Faculty of Science, Charles University in Prague, Prague, Czech Republic.
authors.forename: Vojtěch
authors.initials: V
authors.lastname: Žárský
authors.affiliation: Department of Parasitology, Faculty of Science, Charles University in Prague, Prague, Czech Republic.
authors.forename: Pavlína
authors.initials: P
authors.lastname: Vondráčková
authors.affiliation: Centre for Microbial Diversity and Evolution, Department of Botany, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Martin
authors.initials: M
authors.lastname: Kolísko
authors.affiliation: Department of Parasitology, Faculty of Science, Charles University in Prague, Prague, Czech Republic.
authors.forename: Jan
authors.initials: J
authors.lastname: Tachezy
authors.affiliation: Department of Parasitology, Faculty of Science, Charles University in Prague, Prague, Czech Republic pavel.dolezal@natur.cuni.cz.
authors.forename: Pavel
authors.initials: P
authors.lastname: Doležal
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019921:Biotinylation; D019731:Carbon-Nitrogen Ligases / Q000378:metabolism; D002458:Cell Fractionation; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000378:metabolism; D016829:Giardia lamblia / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism*; D005873:Giardiasis / Q000469:parasitology*; D006801:Humans; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015388:Organelles / Q000737:chemistry / Q000378:metabolism; D015800:Protozoan Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D015800:Protozoan Proteins; D012097:Repressor Proteins; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli