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

Herbert J Santos,Kenichiro Imai,Tomoyoshi Nozaki,Saki Arakawa,Yoshitaka Murakami,Kentaro Tomii,Takashi Makiuchi,Yoko Chiba

doi:10.3390/microorganisms8081229

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.

Highlights

Two billion years of coevolution have established a complete co-dependence between the host archaeon and its endosymbiont of α-proteobacterial origin, presently known as the mitochondrion [1]
Carboxy-terminal HA-tagged wildtype EhAS-HA was expressed in amoebic trophozoites
The results of the immunofluorescence assay (IFA) indicate that EhAS-HA is localized to mitosomes, as reported previously [17]

Summary

Introduction

Two billion years of coevolution have established a complete co-dependence between the host archaeon and its endosymbiont of α-proteobacterial origin, presently known as the mitochondrion [1] Cells depend on their mitochondria for a multitude of functions including aerobic respiration and metabolisms of carbohydrates, lipids, and amino acids [2]. Translocation mechanisms for mitochondrial proteins synthesized in the cytosol are essential Such a process begins with the recognition of mitochondrial transport signal sequence contained in the protein [1]. NTSs usually have high frequencies of arginine [4], lysine, and hydroxylated amino acids; low frequencies of aspartate and glutamate; and low hydrophobicity scores [5] They are able to form positively charged amphipathic α-helices which are important for recognition by components of the translocase of the outer mitochondrial membrane (TOM) [6] such as Tom and Tom22 [7]. These proteins are targeted through the recognition of a single or multiple internal targeting signal(s) (ITS) [15] by its receptor, Tom70 [1]

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