Trichomonas vaginalis is a parasitic protist of the Excavata group. It contains an anaerobic form of mitochondria called hydrogenosomes, which produce hydrogen and ATP; the majority of mitochondrial pathways and the organellar genome were lost during the mitochondrion-to-hydrogenosome transition. Consequently, all hydrogenosomal proteins are encoded in the nucleus and imported into the organelles. However, little is known about the membrane machineries required for biogenesis of the organelle and metabolite exchange. Using a combination of mass spectrometry, immunofluorescence microscopy, in vitro import assays and reverse genetics, we characterized the membrane proteins of the hydrogenosome. We identified components of the outer membrane (TOM) and inner membrane (TIM) protein translocases include multiple paralogs of the core Tom40-type porins and Tim17/22/23 channel proteins, respectively, and uniquely modified small Tim chaperones. The inner membrane proteins TvTim17/22/23-1 and Pam18 were shown to possess conserved information for targeting to mitochondrial inner membranes, but too divergent in sequence to support the growth of yeast strains lacking Tim17, Tim22, Tim23 or Pam18. Full complementation was seen only when the J-domain of hydrogenosomal Pam18 was fused with N-terminal region and transmembrane segment of the yeast homolog. Candidates for metabolite exchange across the outer membrane were identified including multiple isoforms of the β-barrel proteins, Hmp35 and Hmp36; inner membrane MCF-type metabolite carriers were limited to five homologs of the ATP/ADP carrier, Hmp31. Lastly, hydrogenosomes possess a pathway for the assembly of C-tail-anchored proteins into their outer membrane with several new tail-anchored proteins being identified. These results show that hydrogenosomes and mitochondria share common core membrane components required for protein import and metabolite exchange; however, they also reveal remarkable differences that reflect the functional adaptation of hydrogenosomes to anaerobic conditions and the peculiar evolutionary history of the Excavata group.

Department of Parasitology Charles University Prague Faculty of Science Prague Czech Republic

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A hybrid TIM complex mediates protein import into hydrogenosomes of Trichomonas vaginalis

The divergent ER-mitochondria encounter structures (ERMES) are conserved in parabasalids but lost in several anaerobic lineages with hydrogenosomes

Comparative analysis of mitochondrion-related organelles in anaerobic amoebozoans

A mitochondrion-free eukaryote contains proteins capable of import into an exogenous mitochondrion-related organelle

Reduced mitochondria provide an essential function for the cytosolic methionine cycle

Triplet-pore structure of a highly divergent TOM complex of hydrogenosomes in Trichomonas vaginalis

N-Terminal Presequence-Independent Import of Phosphofructokinase into Hydrogenosomes of Trichomonas vaginalis

Conservation of Transit Peptide-Independent Protein Import into the Mitochondrial and Hydrogenosomal Matrix

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

Giardia intestinalis incorporates heme into cytosolic cytochrome b₅

An Advanced System of the Mitochondrial Processing Peptidase and Core Protein Family in Trypanosoma brucei and Multiple Origins of the Core I Subunit in Eukaryotes

Iron-induced changes in the proteome of Trichomonas vaginalis hydrogenosomes

NIF-type iron-sulfur cluster assembly system is duplicated and distributed in the mitochondria and cytosol of Mastigamoeba balamuthi

The mitochondrion-like organelle of Trimastix pyriformis contains the complete glycine cleavage system

Divergence of Erv1-associated mitochondrial import and export pathways in trypanosomes and anaerobic protists

Transcriptomic identification of iron-regulated and iron-independent gene copies within the heavily duplicated Trichomonas vaginalis genome

Live imaging of mitosomes and hydrogenosomes by HaloTag technology