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

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.

Giardia intestinalis incorporates heme into cytosolic cytochrome b5.

The anaerobic intestinal pathogen Giardia intestinalis does not possess enzymes for heme synthesis, and it also lacks the typical set of hemoproteins that are involved in mitochondrial respiration and cellular oxygen stress management. Nevertheless, G. intestinalis may require heme for the function of particular hemoproteins, such as cytochrome b5 (cytb5). We have analyzed the sequences of eukaryotic cytb5 proteins and identified three distinct cytb5 groups: group I, which consists of C-tail membrane-anchored cytb5 proteins; group II, which includes soluble cytb5 proteins; and group III, which comprises the fungal cytb5 proteins. The majority of eukaryotes possess both group I and II cytb5 proteins, whereas three Giardia paralogs belong to group II. We have identified a fourth Giardia cytb5 paralog (gCYTb5-IV) that is rather divergent and possesses an unusual 134-residue N-terminal extension. Recombinant Giardia cytb5 proteins, including gCYTb5-IV, were expressed in Escherichia coli and exhibited characteristic UV-visible spectra that corresponded to heme-loaded cytb5 proteins. The expression of the recombinant gCYTb5-IV in G. intestinalis resulted in the increased import of extracellular heme and its incorporation into the protein, whereas this effect was not observed when gCYTb5-IV containing a mutated heme-binding site was expressed. The electrons for Giardia cytb5 proteins may be provided by the NADPH-dependent Tah18-like oxidoreductase GiOR-1. Therefore, GiOR-1 and cytb5 may constitute a novel redox system in G. intestinalis. To our knowledge, G. intestinalis is the first anaerobic eukaryote in which the presence of heme has been directly demonstrated.

Live imaging of mitosomes and hydrogenosomes by HaloTag technology.

Hydrogenosomes and mitosomes represent remarkable mitochondrial adaptations in the anaerobic parasitic protists such as Trichomonas vaginalis and Giardia intestinalis, respectively. In order to provide a tool to study these organelles in the live cells, the HaloTag was fused to G. intestinalis IscU and T. vaginalis frataxin and expressed in the mitosomes and hydrogenosomes, respectively. The incubation of the parasites with the fluorescent Halo-ligand resulted in highly specific organellar labeling, allowing live imaging of the organelles. With the array of available ligands the HaloTag technology offers a new tool to study the dynamics of mitochondria-related compartments as well as other cellular components in these intriguing unicellular eukaryotes.