The mitosomes of Giardia intestinalis are thought to be mitochondria highly-reduced in response to the oxygen-poor niche. We performed a quantitative proteomic assessment of Giardia mitosomes to increase understanding of the function and evolutionary origin of these enigmatic organelles. Mitosome-enriched fractions were obtained from cell homogenate using Optiprep gradient centrifugation. To distinguish mitosomal proteins from contamination, we used a quantitative shot-gun strategy based on isobaric tagging of peptides with iTRAQ and tandem mass spectrometry. Altogether, 638 proteins were identified in mitosome-enriched fractions. Of these, 139 proteins had iTRAQ ratio similar to that of the six known mitosomal markers. Proteins were selected for expression in Giardia to verify their cellular localizations and the mitosomal localization of 20 proteins was confirmed. These proteins include nine components of the FeS cluster assembly machinery, a novel diflavo-protein with NADPH reductase activity, a novel VAMP-associated protein, and a key component of the outer membrane protein translocase. None of the novel mitosomal proteins was predicted by previous genome analyses. The small proteome of the Giardia mitosome reflects the reduction in mitochondrial metabolism, which is limited to the FeS cluster assembly pathway, and a simplicity in the protein import pathway required for organelle biogenesis.

Mitochondria are archetypal organelles of endosymbiotic origin in eukaryotic cells. Some unicellular eukaryotes (protists) were considered to be primarily amitochondrial organisms that diverged from the eukaryotic lineage before the acquisition of the premitochondrial endosymbiont, but their amitochondrial status was recently challenged by the discovery of mitochondria-like double membrane-bound organelles called mitosomes. Here, we report that proteins targeted into mitosomes of Giardia intestinalis have targeting signals necessary and sufficient to be recognized by the mitosomal protein import machinery. Expression of these mitosomal proteins in Trichomonas vaginalis results in targeting to hydrogenosomes, a hydrogen-producing form of mitochondria. We identify, in Giardia and Trichomonas, proteins related to the component of the translocase in the inner membrane from mitochondria and the processing peptidase. A shared mode of protein targeting supports the hypothesis that mitosomes, hydrogenosomes, and mitochondria represent different forms of the same fundamental organelle having evolved under distinct selection pressures.biogenesis ͉ FeS cluster assembly ͉ Pam18 ͉ matrix-located processing peptidase ͉ ferredoxin

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.