| Summary: | Mitochondrial matrix proteins synthesized in the cytosol often contain amino (N)-terminal targeting sequences (NTSs), or alternately internal targeting sequences (ITSs), which enable them to be properly translocated to the organelle. Such sequences are also required for proteins targeted to mitochondrion-related organelles (MROs) that are present in a few species of anaerobic eukaryotes. Similar to other MROs, the mitosomes of the human intestinal parasite <i>Entamoeba histolytica</i> are highly degenerate, because a majority of the components involved in various processes occurring in the canonical mitochondria are either missing or modified. As of yet, sulfate activation continues to be the only identified role of the relic mitochondria of <i>Entamoeba</i>. Mitosomes influence the parasitic nature of <i>E. histolytica</i>, as the downstream cytosolic products of sulfate activation have been reported to be essential in proliferation and encystation. Here, we investigated the position of the targeting sequence of one of the mitosomal matrix enzymes involved in the sulfate activation pathway, ATP sulfurylase (AS). We confirmed by immunofluorescence assay and subcellular fractionation that hemagluttinin (HA)-tagged <i>Eh</i>AS was targeted to mitosomes. However, its ortholog in the δ-proteobacterium <i>Desulfovibrio vulgaris</i>, expressed as <i>Dv</i>AS-HA in amoebic trophozoites, indicated cytosolic localization, suggesting a lack of recognizable mitosome targeting sequence in this protein. By expressing chimeric proteins containing swapped sequences between <i>Eh</i>AS and <i>Dv</i>AS in amoebic cells, we identified the ITSs responsible for mitosome targeting of <i>Eh</i>AS. This observation is similar to other parasitic protozoans that harbor MROs, suggesting a convergent feature among various MROs in favoring ITS for the recognition and translocation of targeted proteins.
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