Import of Entamoeba histolytica Mitosomal ATP Sulfurylase Relies on Internal Targeting Sequences

Abstract

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 Entamoeba histolytica 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 Entamoeba. Mitosomes influence the parasitic nature of E. histolytica, 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 EhAS was targeted to mitosomes. However, its ortholog in the δ-proteobacterium Desulfovibrio vulgaris, expressed as DvAS-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 EhAS and DvAS in amoebic cells, we identified the ITSs responsible for mitosome targeting of EhAS. 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.

The mitosome of the anaerobic parasitic protist Entamoeba histolytica: A peculiar and minimalist mitochondrion‐related organelle

“…histolytica mitosomal matrix proteins identified in the proteome (Mi‐ichi et al ., 2009). One example is AS whose sequence is 60% identical to that of the sulfate‐reducing δ‐proteobacterium Desulfovibrio vulgaris (Santos et al ., 2020). By swapping low homology sequences of AS in E .…”

“…One example is AS whose sequence is 60% identical to that of the sulfate-reducing δ-proteobacterium Desulfovibrio vulgaris (Santos et al, 2020). By swapping low homology sequences of AS in E. histolytica and D. vulgaris, respectively, two ITSs were found to enable traffic of AS into the mitosomal matrix (Santos et al, 2020). It is of note that the presence of two ITS segments in EhAS is a feature that is different from the AS homolog of M. balamuthi, wherein the protein targeting the hydrogenosome matrix occurs likely through its predicted presequence (Santos et al, 2020).…”

“…Due to a lack of detectable homologs, we are still unable to identify even a single member of the TIM complex. Given the fact that soluble proteins are targeted to the matrix (Mi‐ichi et al ., 2009; Santos et al ., 2020), we posit that a highly divergent protein import machinery likely exists as exemplified by the discovery of Tom60 (Makiuchi et al ., 2013). Proteins destined to be in the matrix of mitosomes are recognized through the presence of either or both an amino‐terminus located presequence and/or a single or multiple internal targeting sequence (ITS).…”

“…histolytica and D . vulgaris , respectively, two ITSs were found to enable traffic of AS into the mitosomal matrix (Santos et al ., 2020). It is of note that the presence of two ITS segments in Eh AS is a feature that is different from the AS homolog of M .…”

“…It is of note that the presence of two ITS segments in Eh AS is a feature that is different from the AS homolog of M . balamuthi , wherein the protein targeting the hydrogenosome matrix occurs likely through its predicted presequence (Santos et al ., 2020).…”