Mitosomes in Entamoeba Histolytica Contain a Sulfate Activation Pathway

Mitosomes in Entamoeba histolytica contain a sulfate activation pathway

Fumika Mi-ichi, Mohammad Abu Yousuf, Kumiko Nakada-Tsukui, and Tomoyoshi Nozaki1

Department of Parasitology, National Institute of Infectious Diseases, Tokyo 162-8640, Japan

Edited by Andrew Roger, Dalhousie University, Halifax, NS, Canada, and accepted by the Editorial Board October 19, 2009 (received for review June 25, 2009) Hydrogenosomes and mitosomes are mitochondrion-related or- and M. balamuthi lack the ISC system, and instead possess the ganelles in anaerobic/microaerophilic eukaryotes with highly re- nitrogen fixation (NIF) system, which is most likely derived from an duced and divergent functions. The full diversity of their content ancestral nitrogen fixing ␧-proteobacterium by lateral gene transfer and function, however, has not been fully determined. To under- (22, 24). Only 5 proteins have been demonstrated in E. histolytica stand the central role of mitosomes in Entamoeba histolytica,a mitosomes: Cpn60 (8–10, 12), Cpn10 (13), mitochondrial Hsp70 parasitic protozoon that causes amoebic dysentery and liver ab- (11, 15), pyridine nucleotide transhydrogenase (PNT) (2, 8), and scesses, we examined the proteomic profile of purified mitosomes. mitochondria carrier family (MCF, ADP/ATP transporter) (14), Using 2 discontinuous Percoll gradient centrifugation and MS and the central role of mitosomes in E. histolytica remains unknown. analysis, we identified 95 putative mitosomal proteins. Immuno- Analysis of the genome of E. histolytica has not revealed any fluorescence assay showed that 3 proteins involved in sulfate additional information regarding the function of mitosomes and activation, ATP sulfurylase, APS kinase, and inorganic pyrophos- thus, a proteomic analysis of mitosomes seems to be the best phatase, as well as sodium/sulfate symporter, involved in sulfate approach to understand its structure and function (1, 2). uptake, were compartmentalized to mitosomes. We have also In this study, we examined the proteomic profile of purified provided biochemical evidence that activated sulfate deriva- mitosomes and showed by immunofluorescence assay that a rep- tives, adenosine-5؅-phosphosulfate and 3؅-phosphoadenosine-5؅- ertoire of proteins were localized to mitosomes, and demonstrated

phosphosulfate, were produced in mitosomes. Phylogenetic anal- by enzymological studies that some of these mitosomal proteins EVOLUTION ysis showed that the aforementioned proteins and chaperones were associated with sulfate activation. We further showed by have distinct origins, suggesting the mosaic character of mito- phylogenetic analysis that mitosomes are a mosaic organelle con- somes in E. histolytica consisting of proteins derived from ␣- sisting of components derived from at least 3 distinct origins. This proteobacterial, ␦-proteobacterial, and ancestral eukaryotic ori- study identifies that sulfate activation is the major function of gins. These results suggest that sulfate activation is the major mitosomes in E. histolytica. function of mitosomes in E. histolytica and that E. histolytica mitosomes represent a unique mitochondrion-related organelle Results with remarkable diversity. Identification of Mitosomal Proteins. To elucidate the central role of mitosomes in E. histolytica, we took a proteomic approach to anaerobic protozoa ͉ evolution ͉ mitochondria ͉ organelle ͉ proteomics identify the proteins associated with mitosomes. We developed an improved purification scheme, consisting of 2 consecutive discon- iversification of mitochondrial structure and function has tinuous Percoll gradient centrifugations that yielded an enriched Doccurred during eukaryotic evolution, and was especially mitosomal fraction suitable for proteome analysis [supporting observed in anaerobic/microaerophilic environments. Most extant information (SI) Fig. S1]. The presence of mitosomes was moni- anaerobic eukaryotes, which were previously considered to lack tored with the authentic mitosomal marker Cpn60. Mitosomes were mitochondria, are now regarded to possess reduced and highly recovered from fractions 19 and 20 in the first centrifugation and divergent forms of mitochondrion-related organelles (1, 2). The from fractions I through K in the second centrifugation with a peak hydrogenosome is an organelle in which hydrogen and ATP are in fraction J (Fig. 1). A list of mitosomal proteins was made by produced, and is found in anaerobic protists and fungi such as subtracting the proteins identified in fractions G and O from those Trichomonas vaginalis (3, 4), Neocallimastix patriciarum (5, 6), and in fraction J, as fraction J contained traces of either lysosome or ER protein, which were detected by markers cysteine protease 5 (CP5) Nyctotherus ovalis (7). The mitosome, typically demonstrated in ␣ parasitic and free-living protists such as E. histolytica (2, 8–15), and Sec61 , respectively (Fig. 1). Giardia intestinalis (16, 17), diverse microsporidian species (18–20), Survey of the Mitosomal Proteome. Three independent mitosomal and Cryptosporidium parvum (21), generally has reduced functions purifications and MS analysis reproducibly identified 95 putative and does not produce hydrogen or ATP. In Mastigamoeba bala- mitosomal proteins (Table S1). Although 64 of the proteins iden- muthi, a mitochondrion-related organelle was discovered and pre- tified were annotated in the E. histolytica genome database as sumed to possess a unique array of biochemical properties, although ‘‘hypothetical protein,’’ 3 enzymes involved in sulfate activation— it remains unclear whether the organelle is more similar to either ATP sulfurylase (AS) (25), APS kinase (APSK), and inorganic hydrogenosomes or mitosomes (22). In contrast, the mitochondri- pyrophosphatase (IPP)—were identified as dominant constituents, on-related organelle in Blastocystis contains DNA and shows char- based on the high coverage obtained for each protein (as described acteristics for both hydrogenosomes and mitochondria of higher eukaryotes (23). Organisms that possess hydrogenosomes and

mitosomes do not cluster together in eukaryote phylogenies, indi- Author contributions: F.M. and T.N. designed research; F.M., M.A.Y., and K.N.-T. performed cating that secondary losses and changes in mitochondrial functions research; F.M. contributed new reagents/analytic tools; F.M. and T.N. analyzed data; and have independently occurred multiple times in eukaryote evolution F.M. and T.N. wrote the paper. (1). Although hydrogenosomes and mitosomes are divergent in The authors declare no conﬂict of interest. their contents and functions, a number of shared characteristics This article is a PNAS Direct Submission. A.R. is a guest editor invited by the Editorial Board. have been previously suggested, which include a double membrane, 1To whom correspondence should be addressed. E-mail: [email protected]. mitochondrial chaperonin 60 (Cpn60), and the iron sulfur cluster This article contains supporting information online at www.pnas.org/cgi/content/full/ (ISC) system (1). However, recent studies indicate that E. histolytica 0907106106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0907106106 PNAS Early Edition ͉ 1of6 Downloaded by guest on October 2, 2021 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 sub- jected to immunoblot analyses using antibodies against well established organelle markers: Cpn60 (mitosome), cysteine synthase 1 (CS1; cytoplasm), Sec61␣ (endoplasmic reticulum), and CP5 (lysosome).

later). Three transporters and 7 metabolic enzymes were also symporters identified in the E. histolytica genome (XP࿝649603, identified. In addition, 7 proteins involved in membrane trafficking, XP࿝654527, XP࿝654503, XP࿝655928, and XP࿝657578), colocalized including 4 Rab family GTPases, were detected. Furthermore, a with Cpn60, suggesting its involvement in the uptake and transport weak homologue of Tom40 (expectation value of 0.13), a compo- of sulfate into mitosomes (Fig. S2 E–H). nent of the transport complex of the outer membrane of the To further demonstrate the presence of a functional sulfate mitochondria, was identified. Three chaperones, Cpn60, Cpn10, activation pathway compartmentalized to mitosomes, AS and mitochondrial Hsp70, and MCF, which were previously reported as APSK activities were measured in the Percoll gradient centrifugation fractions using Na [35S]O . Both [35S]-labeled adenosine-5Ј- Entamoeba mitosomal proteins (12–15), were also confirmed. Con- 2 4 phosphosulfate (APS) and 3Ј-phosphoadenosine-5Ј-phosphosul- versely, PNT and 2 proteins involved in the NIF system—NifS and fate (PAPS) were detected (Fig. 3). PAPS was predominantly NifU—were not detected. The proteome described here is likely to detected in fractions 19 and 20 of the first centrifugation and be partial, as only proteins enriched in fraction J were considered fractions I through K of the second centrifugation (Fig. 3). The as mitosomal proteins, although Cpn60 was detected throughout distribution of the PAPS-forming activity was similar to Cpn60 (Fig. fractions I through S in the second gradient (Fig. 1). The hetero- 1). In contrast, APS was detected in nearly all fractions (i.e., D-S) geneity displayed by mitosomes in the immunofluorescence assay of the second centrifugation. This suggests that AS is not exclusively (described later) also indicates that other subfractions of mitosomes localized to mitosomes, despite its clear mitosomal localization have likely been excluded from the list. observed by immunofluorescence analysis. Alternatively, APS syn- thesis may be partially catalyzed by an unidentified protein. Verification of the Mitosomal Localization of the Identified Proteins. To confirm the cellular localization of these proteins, 21 randomly Identification of Metabolites of Activated Sulfate. To examine the fate chosen putative mitosomal proteins and 4 known mitosomal pro- of activated sulfate in E. histolytica, we incubated trophozoites in teins were examined by immunofluorescence assay in E. histolytica cell lines expressing HA-tagged proteins. Among the 25 proteins examined, 22 proteins—including MCF, mitochondrial Hsp70, and Cpn10—colocalized with the mitosomal marker Cpn60 (Fig. 2, Fig. A B C D S2, and Table S1). These results validated our proteomic approach for the identification of mitosomal proteins. The distribution of each protein in mitosomes was not uniform. We often observed variations in the signal intensity between Cpn60 and other mitosomal proteins including mitochondrial Hsp70 and E FGH MCF (Fig. 2 C and G and Fig. S2 C, G, K, and O). These observations indicated that the composition of mitosomes is not homogeneous. The heterogeneity in the distribution of individual mitosomal proteins was not a result of the overexpression of the HA-tagged proteins, because endogenous and HA-tagged Cpn60 I J K L were well co-localized (Fig. 2 I–L).

Compartmentalization of the Sulfate Activation Pathway in Mitosomes. AS, APSK, and IPP were identified as dominant constituents of mitosomes by proteomic analysis. Immunofluorescence imaging of Fig. 2. Immunolocalization of representative mitosomal proteins. Colocal- E. histolytica cell lines expressing HA-tagged AS, APSK, and IPP ization of individual mitosomal proteins with the HA epitope (anti-HA anti- revealed small punctate signals throughout the cytoplasm, which body, red) and the authentic mitosomal protein marker Cpn60 (native Cpn60 co-localized well with Cpn60 (Fig. 2 A–H and Fig. S2 A–D). antiserum, green) is shown (A–H). Colocalization of endogenous Cpn60 and Furthermore, XP࿝655928, one of the 5 putative sodium/sulfate exogenous HA-tagged Cpn60 is also shown (I–L). (Scale bars: 10 ␮m.)

2of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0907106106 Mi-ichi et al. Downloaded by guest on October 2, 2021 First Percoll Fractions Second Percoll Fractions

1 2 3 4 5 6 7 8 9 10 111213 14 15 16 17 18 19 20 A B C D E F G H I J K L M N O P Q S

APS

PAPS

25000 APS APS 20000 PAPS PAPS

15000

10000 Arbitrary Units Arbitrary

5000

0 1234567891011 121314151617181920 ABCDEFGHIJKLMNOPQS Fr actions Fr actions EVOLUTION 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 ␮L 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 ␮L), carried out for2hat25°C,andterminated by the addition of 88 ␮L 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.

35 BI-S-33 medium containing Na2[ S]O4 for up to 2 h, or labeled for identical. E. histolytica AS showed strong affinity to AS from 1 h and chased for up to 24 h. The cell lysate was separated into ␦-proteobacteria (Fig. 5), whereas E. histolytica IPP clustered with methanol-soluble and insoluble fractions. Labeled [35S] was de- IPP from other eukaryotes (Fig. S4). E. histolytica APSK was closely tected predominantly in the methanol-soluble fraction. At least 5 related to ␦-proteobacteria, ␣-proteobacteria, and Dictyostelium polar lipids, separated on a silica HPTLC plate in 25:65:10 (vol/ discoideum (with low bootstrap values), and E. histolytica sodium/ vol/vol) methanol/chloroform/acetic acid (Fig. 4), accounted for sulfate symporters clustered with a limited group of eukaryotes most of the [35S] detected (81.6% Ϯ 6.2% at 1 h pulse). These lipids (diatoms and green algae) and bacteria, although their exact origins were distinct from commonly observed phospholipid species (phos- were not clearly resolved (Fig. S3 and Fig. S5). In contrast, E. phatidylethanolamine, phosphatidylserine, phosphatidylinositol, histolytica Cpn60 and mitochondrial Hsp70 showed monophyly with and phosphatidylcholine). These data indicate that activated sulfate the ␣-proteobacteria (8, 11, 22). The data indicate the phylogeneti- is mainly used to synthesize sulfur-containing polar lipids. cally mosaic nature of mitosomes in E. histolytica.

Phylogenetic Analysis of the Sulfate Activation Pathway. To investigate Discussion the origin of sulfate activation in E. histolytica, phylogenetic analyses Although the diversity in structure and function of mitochondrion- of the proteins involved in the pathway and established mitosomal related organelles have been demonstrated in a wide range of chaperones were conducted. The origins of AS, APSK, IPP, sodi- organisms, E. histolytica remains as one of the anaerobic/ um/sulfate symporter, Cpn60, and mitochondrial Hsp70 were not microaerophilic eukaryotes, in which the function of the mitochondrion-related organelle remains unknown. In this study, we have provided biochemical and microscopic evidence demonstrating that Continuous Labeling (min) Pulse (1h) + Chase (h) sulfate activation, which generally occurs in the cytoplasm and plastids in eukaryotes, is the major function of mitosomes in E. 5 30 60 120 0 1 2 4 8 24 histolytica. PE Genes encoding for all of the major components necessary for sulfate activation and transport of substrates and products across PS/PI the mitosomal membrane are identifiable in the genome (Fig. 6). Sodium/sulfate symporter, which is necessary for the uptake of PC sulfate into mitosomes, was identified. ATP that is necessary for the activation of sulfate by both AS and APSK, and the quality control SO 2-, APS, PAPS of heat-sensitive AS by chaperones, is incorporated via MCF to 4 mitosomes with a concomitant cytosolic export of ADP or AMP. Fig. 4. Incorporation of [35S]-labeled sulfate into polar lipids. 35S was incor- E. histolytica MCF transports both ADP and AMP to the cytosol for porated into at least 5 polar lipids (arrowheads), which were distinct from the incorporation of ATP to mitosomes (14). In other organisms, commonly observed phospholipid species. PE, phosphatidylethanolamine; PS, PAPS transporter (PAPST), which transports PAPS and AMP in phosphatidylserine; PI, phosphatidylinositol; PC, phosphatidylcholine. opposite directions, is required for shuttling PAPS between the

Mi-ichi et al. PNAS Early Edition ͉ 3of6 Downloaded by guest on October 2, 2021 Cyanidioschyzon merolae_S Oryza sativa_S 92 Allium sepa_S 62 Glycine max_S 56 Brassica napus_S 71 Arabadopsis thaliana_S 56 Monosiga brevicollis_KS 84 Anopholes gambia_KS KS fusion group Drosophila melanogaster_KS

100 Ciona instestinalis_KS Eukaryote 99 Mus musculus_KS-2 87 Homo sapiens_KS-2 89 99 Mus musculus_KS-1 Homo sapiens_KS-1 Phytophthora infestans_KSP 92 KSP fusion group 100 Thalassiosira pseudonana_KSP Phaeodactylum tricornutum_KSP ML DM MP Riftia pachyptila symbiont_S 96 95 68 Thiobacillus denitrificans_S Proteobacteria 90 Psychrobacter arcticum_S 100 Entamoeba hisolytica_S ML DM MP ☆ 99 100 100 46 Entamoeba invadens_S ☆ 99 Desulfovibrio vulgaris_S δ-Proteobacteria 30 Desulfotalea psychrophila_S 100 Chlorobium chlorochromatii_S Chlorobium tepidum_S

88 Chloroflexus aurantiacus_S 100 Deinococcus geothermalis_S Bacteria Deinococcus radiodurans_S Bacillus subtilis_S ML DM MP 67 95 73 Staphylococcus epidermidis_S Rubrobacter xylanophilus_S

85 Gloeobacter violaceus_S Synechococcus sp.ja-3-3a_S Cyanobacteria 60 Anabaena variabilis_S 88 Trichodesmium erythraeum_S 82 Synechococcus sp.wh8102_S 72 Crocosphaera watsonii_S Campylobacter jejuni_S Thermus thermophilus_S Bacteria Toxoplasma gondii_SK Eukaryote 94 Aquifex aeolicus_SK SK fusion group Chloroflexus aurantiacus_SK Bacteria Coxiella burnetii_SK Proteobacteria Desulfotalea psychrophila_SK Dictyostelium discoideum_SK Eukaryote

100 Rhodobacter sphaeroides_SK Silicibacter pomeroyi_SK α-Proteobacteria Oceanicola granulosus_SK 52 Jannaschia sp. CCS1_SK Mucor circinelloides_SK Ustilago maydis_SK 89 53 Gibberella zeae_SK

73 Yarrowia lipolytica_SK Neurospora crassa_SK Fungi 50 99 Penicillium chrysogenum_SK Aspergillus oryzae_SK

98 Ashbya gossypii_S Kluyveromyces lactis_S Saccharomyces cervesiae_S Fungi 85 Candida glabrata_S 99 Prochlorococcus marinus_S Bacteria 100 Synechocystis sp.6803_S Chlamydomonas reinhardtii_S 99 Phaeodactylum tricornutum_S Thalassiosira pseudonana_S Eukaryote Euglena gracilis_S 0.1 substitutions/site

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 ␣-value of the ⌫-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, 151to 178, 182 to 196, 200 to 226, 231 to 262, 264 to 325m and 333 to 399 of the E. histolytica sequence.

organelle and the cytosol (28). Although the E. histolytica genome labeling (Fig. 4), it is conceivable to assume that PAPS predomi- contains a gene encoding for putative PAPST (XP࿝654175; expec- nantly serves as the sulfo-donor of sulfotransferases to synthesize tation value, 4.2Ϫ14) (29, 30), we failed to demonstrate the mito- sulfur-containing lipids. Two of the most highly expressed sulfo- somal localization of the protein (PAPST; Fig. S6). In addition, a transferases (SULT1, XP࿝654200, expectation value, 1Ϫ12; SULT2, putative phosphate transporter (XP࿝654379; expectation value, XP࿝654101, expectation value, 1Ϫ13) were distributed to the cyto- 1.23Ϫ61) (31) also failed to localize to mitosomes (PHT; Fig. S6). plasm (SULT1 and SULT2; Fig. S6), suggesting that sulfo-transfer Thus, the identity of the presumptive PAPS and phosphate trans- reactions occur in the cytosol, and thus reinforcing the premise that porters in E. histolytica mitosomes remains unknown. Generally, PAPS needs to be exported from mitosomes. Further character- PAPS is used in 2 different pathways. In one route, the sulfate ization of these sulfur-containing lipid species in E. histolytica is moiety of PAPS is transferred to various acceptors to yield muco- necessary to understand the physiological significance of the com- polysaccharides, sulfolipids, and sulfoproteins by sulfotransferases. partmentalized sulfate activation in mitosomes of this organism. Alternatively, sulfate is reduced and assimilated into cysteine (32, As sulfate activation in eukaryotes generally occurs in the 33). The E. histolytica genome contains 11 potential genes encoding cytoplasm and plastids (36, 37), our finding has raised an important for sulfotransferases, but lacks the enzymes for sulfate reduction question on whether compartmentalization of sulfate activation in 35 (34, 35). Therefore, together with the results of [ S]O4 metabolic mitosomes is unique to E. histolytica. The acquisition and compart-

4of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0907106106 Mi-ichi et al. Downloaded by guest on October 2, 2021 somes in anaerobic/microaerophilic eukaryotes (1). However, E. histolytica and M. balamuthi possess only the NIF system instead of the ISC system (22, 24). In the mitosomal proteome, neither NifS nor NifU was identified in the mitosome-enriched fraction. We previously reported that NifS and NifU, both of which lack a mitochondrion-targeting signal, were fractionated on an anion exchange chromatography from the soluble fraction obtained by 0.45-␮m filtration and 45,000 ϫ g ultracentrifugation of amoebic lysate, suggesting the predominantly cytoplasmic localization of the NIF system in E. histolytica (24). Similarly, both NifS and NifU in M. balamuthi lack a mitochondria targeting signal (22). These data suggest that FeS cluster biosynthesis in E. histolytica and M. balamuthi are mainly, if not exclusively, localized to the cytosol (22, 24). However, it remains unknown whether the E. histolytica mitosomes also partially contain the NIF system. The targeting mechanisms to the E. histolytica mitosomes remain unsolved and need further investigation. Although Entamoeba PNT was presumed to be imported into mitosomes based on the resem- blance of its amino-terminal portion rich in hydroxylated and basic Fig. 6. A compartmentalized sulfate activation pathway in E. histolytica amino acids to the canonical mitochondrial targeting peptide (2), mitosomes. The mitosome proteins whose localization was conﬁrmed by our proteomic and immunofluorescence studies clearly showed that immunoﬂuorescence assay are shown in red, whereas the putative transport- PNT is not imported to mitosomes, but is distributed to vesicles and ers are shown in gray. PHT, phosphate transporter; SULT, sulfotransferase. vacuoles (Fig. S7). Commonly available prediction programs such Two of the most highly expressed sulfotransferases were demonstrated to be as Mitoprot (41) and PSORT II (42) were unable to correctly localized to the cytoplasm (Fig. S6). predict the mitosomal localization of proteins whose localization was verified by immunofluorescence assay. Similarly, the remaining

mentalization of the enzymes involved in sulfate activation may putative mitosomal proteins have no predictable mitochondrial EVOLUTION have occurred exclusively in Entamoeba among the anaerobic/ targeting signal. As described earlier, our mitosomal proteome microaerophilic organisms possessing mitochondrion-related or- contained a weak homologue of Tom40 (expectation value, 0.13). ganelles. T. vaginalis, G. intestinalis, and C. parvum apparently lack Thus, there is a need to verify the identity of this potential Tom40 genes encoding for the enzymes. Encephalitozoon cuniculi, one of and the existence of the Tom complex in E. histolytica.Inother the microsporidia, has the gene for IPP (Fig. S4), but lacks genes anaerobic eukaryotes, protein import machinery to the mitochon- encoding for AS and APSK. Although the genome and EST drion-related organelles appears to be conserved (43, 44), and the databases are not complete for M. balamuthi and N. patriciarum, mitosomal targeting signal from G. lamblia and the hydrogenosome none of AS, APSK, and IPP genes was found so far. It is worth targeting signal from T. vaginalis were interchangeable (45). On the examining if genes involved in sulfate activation are present in M. contrary, cryptic mitochondrial targeting signals have also been balamuthi because the 2 species share the NIF system for FeS discovered in G. lamblia, T. vaginalis, and microsporidian species, cluster biosynthesis (22). There is only one eukaryote, other than E. and unique aspects of mitochondrial processing peptidases have histolytica, that possesses sulfate activation in the mitochondria. In been reported in G. lamblia (1, 46). Together, the machinery and Euglena gracilis, the enzymatic activities involved in sulfate activa- mechanisms of protein import into mitosomes require further tion and reduction have been detected in the mitochondria (36, 38, investigation. 39). The origin of E. gracilis AS, however, is presumed to be the In summary, we have shown that E. histolytica mitosomes are a secondary green algal plastid and not the ␦-proteobacteria (36), mitochondrion-related organelle that is highly divergent and rep- whereas E. gracilis IPP is thought to be derived from bacteria and resents a mosaic organelle consisting of proteins derived from ␣ ␦ not an ancestral eukaryote (Fig. S4). These results suggest that the -proteobacteria, -proteobacteria, and eukaryotes. It is primarily origins of E. gracilis mitochondria are distinct from those of E. involved in sulfate activation. This study should shed light on the histolytica mitosomes. Based on the fact that the 4 E. histolytica diversification of mitochondrion-related organelles in eukaryotic proteins involved in sulfate transport and activation have origins evolution. distinct from ␣-proteobacterium, we propose a hypothesis whereby Materials and Methods AS, APSK, and sodium/sulfate symporter were acquired by the ϫ 8 ancestor of Entamoeba from ␦-proteobacterium (and other bacte- Mitosome Purification. Approximately 0.5–1 10 trophozoites of E. histolytica ria) by lateral gene transfer (40). We examined whether AS and strain HM-1:IMSS cl6 (47), cultivated axenically in Diamond BI-S-33 medium (48), ␦ were resuspended in 1 mL of homogenate buffer [250 mM sucrose, protease APSK were found in an operon in -proteobacteria, which would ␮ ␦ inhibitor mixture (Roche), 300 M E-64, 10 mM Mops-KOH (pH 7.4)], and homog- support the hypothesis whereby both AS and APSK of -proteobac- enized with a Dounce homogenizer. Unbroken cells, nuclei, and large vacuoles terial origin were transferred to the ancestor of Entamoeba. How- were removed by centrifugation at 5,000 ϫ g at 4 °C for 10 min, and the ever, this is not a case in at least 2 representative species (Desul- supernatant was gently layered onto 3 mL of homogenization buffer containing fotalea psychrophila and Desulfovibrio vulgaris). 30% (vol/vol) Percoll. After centrifugation at 120,000 ϫ g at 4 °C for 1 h, 200 ␮L Various diverse or sometimes shared characteristics are reported fractions were collected from top to bottom (fractions 1–20). Fractions 19 and 20 in other eukaryotes possessing either hydrogenosomes or mito- (density Ͼ1.054) were collected, mixed, and applied onto 2 mL of homogeniza- somes. These include hydrogenase and pyruvate:ferredoxin oxi- tion buffer containing 70% (vol/vol) Percoll. The gradient was subsequently doreductase for ATP generation in hydrogenosomes; NADH de- overlaid with 1 mL of the homogenization buffer containing 15% (vol/vol) Percoll. After centrifugation at 120,000 ϫgat 4 °C for 1 h, 200 ␮L of fractions were hydrogenase part of mitochondrial complex I in T. vaginalis, N. collected from top to bottom (fractions A-S; Fig. S1). ovalis, and Blastocystis; TCA cycle enzymes in M. balamuthi, Blas- tocystis, N. patriciarum, and N. ovalis; glycine cleavage complex in M. Immunoblot Analysis. Fractions prepared as previously mentioned were mixed balamuthi, T. vaginalis, and Blastocystis; and alternative oxidase in with 4ϫ SDS/PAGE sample buffer. Samples were boiled at 95 °C for 5 min fol- Blastocystis and C. parvum (1). Cpn60 and the ISC system were lowed by centrifugation at 13,000 ϫgat 4 °C for 20 min to remove the Percoll. The thought to be shared characteristics of hydrogenosomes and mito- fractions were analyzed by SDS/PAGE, silver stained, and immunoblot analysis as

Mi-ichi et al. PNAS Early Edition ͉ 5of6 Downloaded by guest on October 2, 2021 previously described (49). The dilution of the primary antibodies was 1:1,000 for Metabolic Labeling. Approximately 106 trophozoites were labeled with [35S]- anti-Cpn60 and anti-cysteine synthase 1 antiserum, and 1:100 for anti-CP5 and labeled sulfate (25 mCi/m mole) in 0.5 mL of the Diamond BI-S-33 medium either anti-Sec61␣ antiserum (50, 51). continuously for 5 to 120 min or labeled for 1 h and chased for 1 to 24 h (54). Cells were collected and lipids were extracted with 0.5 mL of methanol and separated Production of E. histolytica Lines Expressing Epitope-Tagged Mitosomal Proteins. on a silica high-performance thin-layer chromatography plate in 25:65:10 (vol/ Plasmids for the production of amoeba lines expressing engineered mitosomal vol/vol) methanol/chloroform/acetic acid. Dried thin-layer chromatography plates were analyzed by autoradiography. proteins containing 3 tandem HA-epitopes were constructed essentially as previously described (52). Lipofection of trophozoites and selection of transformants were also performed as previously described (53). ACKNOWLEDGMENTS. We thank Dr. Takeshi Makiuchi for discussions of phylogenetic analysis. We thank Yoko Yamada, Kyoko Masuda, Kayoko Hashimoto, and Rumiko Kosugi for technical assistance. We thank Jorge Tovar, Royal Hollo- Indirect Immunofluorescence Assay. Indirect immunofluorescence assay was per- way University of London, for anti-Cpn60 antibody for an initial analysis of Cpn60. formed as previously described (49) with some modifications. Briefly, the amoe- We also thank Rosana Sańchez-Lo´pez, UNAM, Cuernavaca, Mexico, for anti- bae were washed and fixed with acetone/methanol (1:1) for 10 min. After Sec61␣ antiserum. This work was supported by Creative Scientific Research Grant washing with PBS solution, cells were permeabilized with 0.3% Triton X-100 for 18GS0314 from the Japanese Ministry of Education, Science, Culture, Sports, and 15 min and reacted with primary antibody diluted at 1:500 (anti-Cpn60 anti- Technology (to T.N.); Grant-in-Aid for Scientific Research (18GS0314, 18050006, 18073001) from the Ministry of Education, Culture, Sports, Science and Technol- serum) and 1:1,000 (anti-HA monoclonal antibody) in PBS solution. The samples ogy of Japan; a grant for research on emerging and re-emerging infectious were then reacted with Alexa Fluor 488- or 568-conjugated anti-rabbit or anti- diseases from the Ministry of Health, Labour and Welfare of Japan; and a grant mouse secondary antibody (1:1,000) for 1 h. The samples were examined as for research to promote the development of anti-AIDS pharmaceuticals from the previously described (49). Japan Health Sciences Foundation (to T.N.).

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