Unlike mitosomes, however, hydrogenosomes are metabolically active organelles that produce ATP by substrate level phosphorylation. The limited knowledge of mitosomal proteomes has been gained mainly from analyses of genome sequences and localization studies of a few model mitosomal proteins. The only published proteomics study that focused on Ginsenoside-Ro mitosomes was that recently reported for the amoeba E. histolytica, identifying a unique Gomisin-D sulfate activation pathway. To increase our 
understanding of the function and origin of these enigmatic organelles, we established a large-scale proteomic approach to analyze the mitosomes of Giardia intestinalis. This organism was selected because Giardia intestinalis is a common human intestinal pathogen, its genome sequence has been published, and it is considered to be among the most basal eukaryotes. Moreover, previous analysis of the G. intestinalis genome provided little new information pertaining to the putative mitosomal proteome, so there are substantial gaps in our knowledge of the structure and function of this essential organelle. Here, we quantitatively analyzed the presence of isobarically-tagged proteins in mitosome enriched fractions. This technique allowed us to discriminate the mitosomal proteins from those of contaminating cellular structures. Combined with an exhaustive bioinformatics analysis, this strategy identified 139 putative mitosomal proteins; 20 of which were experimentally confirmed to be localized in mitosomes. Our results revealed that the proteome of the G. intestinalis mitosome is selectively reduced and houses a single metabolic pathway for FeS cluster assembly, a novel diflavin protein with NADPH reductase activity, a minimal protein import machinery and proteins that may be important for the interaction of mitosomes with other cellular compartments. Typically, it functions together with a TIM complex that forms the translocation pore for protein passage across the membrane. In representative organisms from all lineages of eukaryotes, the TIM complex is built from one or two proteins of the Tim17/22/23 family. Surprisingly, we find no evidence for a member of this protein in our proteomics data, and sensitive hidden Markov model searches detected no related sequences in the Giardia genome. In eukaryotes, the Sec61 channel catalyzes protein transport across the endoplasmic reticulum, while a highlyrelated protein called SecY is the translocation channel in the inner membrane of bacteria, including the alpha-proteobacteria from which mitochondria are derived. Interestingly, Reclinomonas americana encodes a bacterial-type SecY protein translocation channel in its mitochondrial genome, and our proteomics analysis detected what appeared to be contamination of the mitosomal membranes with GiSecY/Sec61. We expressed a tagged version of this protein in Giardia but it localized to the endoplasmic reticulum, as expected for a cognate Sec61, rather than to the mitosomes. The nature of the mitosomal inner membrane protein translocation channel remains unknown, and yet must exist given that at least 17 of the proteins detected in the mitosomal proteome are likely to reside in the matrix. Another surprising result, one that can only be explained by a secondary gene loss, is the absence of the outer membrane protein Sam50 in Giardia. Sam50 is a component of the SAM complex, which is required for the assembly of both Tom40 and VDAC. The apparent absence of Sam50 from the Giardia genome and from our proteomics data is unique among eukaryotes. A putative Sam50 homologue has been predicted in the genomes of all eukaryotes, including trypanosomatids and mitosome- and hydrogenosome-containing protists. Numerous phylogenetic and functional analyses indicate that Sam50 was derived from the Omp85/BamA protein present in the outer membrane of the ancestral, alpha-proteobacterial endosymbiont and it must, therefore, have been present in the earliest mitochondria. It is not clear how Giardia Tom40 is assembled within the outer membrane without the assistance of the SAM complex.