Mitosomes in Entamoeba histolytica contain a sulfate activation pathway

Abstract

Hydrogenosomes and mitosomes are mitochondrion-related organelles in anaerobic/microaerophilic eukaryotes with highly reduced and divergent functions. The full diversity of their content and function, however, has not been fully determined. To understand the central role of mitosomes in Entamoeba histolytica, a parasitic protozoon that causes amoebic dysentery and liver abscesses, we examined the proteomic profile of purified mitosomes. Using 2 discontinuous Percoll gradient centrifugation and MS analysis, we identified 95 putative mitosomal proteins. Immunofluorescence assay showed that 3 proteins involved in sulfate activation, ATP sulfurylase, APS kinase, and inorganic pyrophosphatase, as well as sodium/sulfate symporter, involved in sulfate uptake, were compartmentalized to mitosomes. We have also provided biochemical evidence that activated sulfate derivatives, adenosine-5'-phosphosulfate and 3'-phosphoadenosine-5'-phosphosulfate, were produced in mitosomes. Phylogenetic analysis showed that the aforementioned proteins and chaperones have distinct origins, suggesting the mosaic character of mitosomes in E. histolytica consisting of proteins derived from alpha-proteobacterial, delta-proteobacterial, and ancestral eukaryotic origins. These results suggest that sulfate activation is the major function of mitosomes in E. histolytica and that E. histolytica mitosomes represent a unique mitochondrion-related organelle with remarkable diversity.

Fig. 1.

Purification of mitosomes. Fractions derived from the first (fractions 1–20) and second (fractions A-S) discontinuous Percoll gradient centrifugation were electrophoresed on 5%–20% SDS/PAGE and subjected to immunoblot analyses using antibodies against well established organelle markers: Cpn60 (mitosome), cysteine synthase 1 (CS1; cytoplasm), Sec61α (endoplasmic reticulum), and CP5 (lysosome).

Fig. 2.

Immunolocalization of representative mitosomal proteins. Colocalization of individual mitosomal proteins with the HA epitope (anti-HA antibody, red) and the authentic mitosomal protein marker Cpn60 (native Cpn60 antiserum, green) is shown (A–H). Colocalization of endogenous Cpn60 and exogenous HA-tagged Cpn60 is also shown (I–L). (Scale bars: 10 μm.)

Fig. 3.

Demonstration of AS and APSK activities. Mitosome-enriched fractions were tested for the ability to synthesize ³⁵S-labeled APS or PAPS (26). The reaction mixture for each sample totaled 12 μL and contained 39 mM MgCl₂, 64 mM ATP, 1.3 mM Na₂SO₄ (25 mCi/m mole), and 20 mM Tris/HCl (pH 8.0). The reaction was initiated by the addition of the freeze-thawed fraction (3 μL), carried out for 2 h at 25 °C, and terminated by the addition of 88 μL methanol. Ten-microliter samples were analyzed by PEI-cellulose TLC to determine ³⁵S-labeled APS or PAPS as previously described (27). The amount of each product was quantified by densitometric analysis using an image analyzer (Fuji) and the results are expressed in arbitrary units.

Fig. 4.

Incorporation of [³⁵S]-labeled sulfate into polar lipids. ³⁵S was incorporated into at least 5 polar lipids (arrowheads), which were distinct from commonly observed phospholipid species. PE, phosphatidylethanolamine; PS, phosphatidylserine; PI, phosphatidylinositol; PC, phosphatidylcholine.

Fig. 5.

Phylogenetic analysis of AS. The best maximum likelihood (ML) tree of AS inferred by the JTT model taking across-site rate heterogeneity into consideration. The α-value of the Γ-shaped parameter used in the analysis of AS was 0.50723. Bootstrap proportion (BP) values are attached to the internal branches. Branches with less than 50% BP support are unmarked. BP values are calculated by ML, distance matrix (DM), and maximum parsimony (MP) methods. One hundred and 1,000 resamplings were performed for ML and DM and MP analyses, respectively. The length of each branch is proportional to the estimated number of substitution. With 67 taxa, 302 aligned amino acid sites were used for analysis, corresponding to residues 55 to 103, 117 to 131, 135 to 147, 151 to 178, 182 to 196, 200 to 226, 231 to 262, 264 to 325m and 333 to 399 of the E. histolytica sequence.

Fig. 6.

A compartmentalized sulfate activation pathway in E. histolytica mitosomes. The mitosome proteins whose localization was confirmed by immunofluorescence assay are shown in red, whereas the putative transporters are shown in gray. PHT, phosphate transporter; SULT, sulfotransferase. Two of the most highly expressed sulfotransferases were demonstrated to be localized to the cytoplasm (Fig. S6).