Mitosomes in Entamoeba histolytica contain a sulfateactivation pathwayFumika 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 reviewJune 25, 2009)

Hydrogenosomes and mitosomes are mitochondrion-related or-ganelles in anaerobic/microaerophilic eukaryotes with highly re-duced and divergent functions. The full diversity of their contentand function, however, has not been fully determined. To under-stand the central role of mitosomes in Entamoeba histolytica, aparasitic protozoon that causes amoebic dysentery and liver ab-scesses, we examined the proteomic profile of purified mitosomes.Using 2 discontinuous Percoll gradient centrifugation and MSanalysis, we identified 95 putative mitosomal proteins. Immuno-fluorescence assay showed that 3 proteins involved in sulfateactivation, ATP sulfurylase, APS kinase, and inorganic pyrophos-phatase, as well as sodium/sulfate symporter, involved in sulfateuptake, were compartmentalized to mitosomes. We have alsoprovided biochemical evidence that activated sulfate deriva-tives, adenosine-5�-phosphosulfate and 3�-phosphoadenosine-5�-phosphosulfate, were produced in mitosomes. Phylogenetic anal-ysis showed that the aforementioned proteins and chaperoneshave distinct origins, suggesting the mosaic character of mito-somes in E. histolytica consisting of proteins derived from �-proteobacterial, �-proteobacterial, and ancestral eukaryotic ori-gins. These results suggest that sulfate activation is the majorfunction of mitosomes in E. histolytica and that E. histolyticamitosomes represent a unique mitochondrion-related organellewith remarkable diversity.

anaerobic protozoa � evolution � mitochondria � organelle � proteomics

D iversification of mitochondrial structure and function hasoccurred during eukaryotic evolution, and was especially

observed in anaerobic/microaerophilic environments. Most extantanaerobic eukaryotes, which were previously considered to lackmitochondria, are now regarded to possess reduced and highlydivergent forms of mitochondrion-related organelles (1, 2). Thehydrogenosome is an organelle in which hydrogen and ATP areproduced, and is found in anaerobic protists and fungi such asTrichomonas vaginalis (3, 4), Neocallimastix patriciarum (5, 6), andNyctotherus ovalis (7). The mitosome, typically demonstrated inparasitic and free-living protists such as E. histolytica (2, 8–15),Giardia intestinalis (16, 17), diverse microsporidian species (18–20),and Cryptosporidium parvum (21), generally has reduced functionsand does not produce hydrogen or ATP. In Mastigamoeba bala-muthi, a mitochondrion-related organelle was discovered and pre-sumed to possess a unique array of biochemical properties, althoughit remains unclear whether the organelle is more similar to eitherhydrogenosomes or mitosomes (22). In contrast, the mitochondri-on-related organelle in Blastocystis contains DNA and shows char-acteristics for both hydrogenosomes and mitochondria of highereukaryotes (23). Organisms that possess hydrogenosomes andmitosomes do not cluster together in eukaryote phylogenies, indi-cating that secondary losses and changes in mitochondrial functionshave independently occurred multiple times in eukaryote evolution(1). Although hydrogenosomes and mitosomes are divergent intheir contents and functions, a number of shared characteristicshave been previously suggested, which include a double membrane,mitochondrial chaperonin 60 (Cpn60), and the iron sulfur cluster(ISC) system (1). However, recent studies indicate that E. histolytica

and M. balamuthi lack the ISC system, and instead possess thenitrogen fixation (NIF) system, which is most likely derived from anancestral nitrogen fixing �-proteobacterium by lateral gene transfer(22, 24). Only 5 proteins have been demonstrated in E. histolyticamitosomes: Cpn60 (8–10, 12), Cpn10 (13), mitochondrial Hsp70(11, 15), pyridine nucleotide transhydrogenase (PNT) (2, 8), andmitochondria carrier family (MCF, ADP/ATP transporter) (14),and the central role of mitosomes in E. histolytica remains unknown.Analysis of the genome of E. histolytica has not revealed anyadditional information regarding the function of mitosomes andthus, a proteomic analysis of mitosomes seems to be the bestapproach to understand its structure and function (1, 2).

In this study, we examined the proteomic profile of purifiedmitosomes and showed by immunofluorescence assay that a rep-ertoire of proteins were localized to mitosomes, and demonstratedby enzymological studies that some of these mitosomal proteinswere associated with sulfate activation. We further showed byphylogenetic analysis that mitosomes are a mosaic organelle con-sisting of components derived from at least 3 distinct origins. Thisstudy identifies that sulfate activation is the major function ofmitosomes in E. histolytica.

ResultsIdentification of Mitosomal Proteins. To elucidate the central role ofmitosomes in E. histolytica, we took a proteomic approach toidentify the proteins associated with mitosomes. We developed animproved purification scheme, consisting of 2 consecutive discon-tinuous Percoll gradient centrifugations that yielded an enrichedmitosomal fraction suitable for proteome analysis [supportinginformation (SI) Fig. S1]. The presence of mitosomes was moni-tored with the authentic mitosomal marker Cpn60. Mitosomes wererecovered from fractions 19 and 20 in the first centrifugation andfrom fractions I through K in the second centrifugation with a peakin fraction J (Fig. 1). A list of mitosomal proteins was made bysubtracting the proteins identified in fractions G and O from thosein fraction J, as fraction J contained traces of either lysosome or ERprotein, which were detected by markers cysteine protease 5 (CP5)and Sec61�, respectively (Fig. 1).

Survey of the Mitosomal Proteome. Three independent mitosomalpurifications and MS analysis reproducibly identified 95 putativemitosomal proteins (Table S1). Although 64 of the proteins iden-tified were annotated in the E. histolytica genome database as‘‘hypothetical protein,’’ 3 enzymes involved in sulfate activation—ATP sulfurylase (AS) (25), APS kinase (APSK), and inorganicpyrophosphatase (IPP)—were identified as dominant constituents,based on the high coverage obtained for each protein (as described

Author contributions: F.M. and T.N. designed research; F.M., M.A.Y., and K.N.-T. performedresearch; F.M. contributed new reagents/analytic tools; F.M. and T.N. analyzed data; andF.M. and T.N. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. A.R. is a guest editor invited by the Editorial Board.

1To whom correspondence should be addressed. E-mail: nozaki@nih.go.jp.

This article contains supporting information online at www.pnas.org/cgi/content/full/0907106106/DCSupplemental.

www.pnas.org�cgi�doi�10.1073�pnas.0907106106 PNAS � December 22, 2009 � vol. 106 � no. 51 � 21731–21736

EVO

LUTI

ON

Dow

nloa

ded

by g

uest

on

July

2, 2

020

later). Three transporters and 7 metabolic enzymes were alsoidentified. In addition, 7 proteins involved in membrane trafficking,including 4 Rab family GTPases, were detected. Furthermore, aweak homologue of Tom40 (expectation value of 0.13), a compo-nent of the transport complex of the outer membrane of themitochondria, was identified. Three chaperones, Cpn60, Cpn10,mitochondrial Hsp70, and MCF, which were previously reported asEntamoeba mitosomal proteins (12–15), were also confirmed. Con-versely, PNT and 2 proteins involved in the NIF system—NifS andNifU—were not detected. The proteome described here is likely tobe partial, as only proteins enriched in fraction J were consideredas mitosomal proteins, although Cpn60 was detected throughoutfractions I through S in the second gradient (Fig. 1). The hetero-geneity displayed by mitosomes in the immunofluorescence assay(described later) also indicates that other subfractions of mitosomeshave likely been excluded from the list.

Verification of the Mitosomal Localization of the Identified Proteins. Toconfirm the cellular localization of these proteins, 21 randomlychosen putative mitosomal proteins and 4 known mitosomal pro-teins were examined by immunofluorescence assay in E. histolyticacell lines expressing HA-tagged proteins. Among the 25 proteinsexamined, 22 proteins—including MCF, mitochondrial Hsp70, andCpn10—colocalized with the mitosomal marker Cpn60 (Fig. 2, Fig.S2, and Table S1). These results validated our proteomic approachfor the identification of mitosomal proteins.

The distribution of each protein in mitosomes was not uniform.We often observed variations in the signal intensity between Cpn60and other mitosomal proteins including mitochondrial Hsp70 andMCF (Fig. 2 C and G and Fig. S2 C, G, K, and O). Theseobservations indicated that the composition of mitosomes is nothomogeneous. The heterogeneity in the distribution of individualmitosomal proteins was not a result of the overexpression of theHA-tagged proteins, because endogenous and HA-tagged Cpn60were well co-localized (Fig. 2 I–L).

Compartmentalization of the Sulfate Activation Pathway in Mitosomes.AS, APSK, and IPP were identified as dominant constituents ofmitosomes by proteomic analysis. Immunofluorescence imaging ofE. histolytica cell lines expressing HA-tagged AS, APSK, and IPPrevealed small punctate signals throughout the cytoplasm, whichco-localized well with Cpn60 (Fig. 2 A–H and Fig. S2 A–D).Furthermore, XP�655928, one of the 5 putative sodium/sulfate

symporters identified in the E. histolytica genome (XP�649603,XP�654527, XP�654503, XP�655928, and XP�657578), colocalizedwith Cpn60, suggesting its involvement in the uptake and transportof sulfate into mitosomes (Fig. S2 E–H).

To further demonstrate the presence of a functional sulfateactivation pathway compartmentalized to mitosomes, AS andAPSK activities were measured in the Percoll gradient centrifuga-tion fractions using Na2[35S]O4. Both [35S]-labeled adenosine-5�-phosphosulfate (APS) and 3�-phosphoadenosine-5�-phosphosul-fate (PAPS) were detected (Fig. 3). PAPS was predominantlydetected in fractions 19 and 20 of the first centrifugation andfractions I through K of the second centrifugation (Fig. 3). Thedistribution of the PAPS-forming activity was similar to Cpn60 (Fig.1). In contrast, APS was detected in nearly all fractions (i.e., D-S)of the second centrifugation. This suggests that AS is not exclusivelylocalized to mitosomes, despite its clear mitosomal localizationobserved by immunofluorescence analysis. Alternatively, APS syn-thesis may be partially catalyzed by an unidentified protein.

Identification of Metabolites of Activated Sulfate. To examine the fateof activated sulfate in E. histolytica, we incubated trophozoites in

Fig. 1. Purification of mitosomes. Fractions derivedfrom the first (fractions 1–20) and second (fractionsA-S) discontinuous Percoll gradient centrifugationwere electrophoresed on 5%–20% SDS/PAGE and sub-jected to immunoblot analyses using antibodiesagainst well established organelle markers: Cpn60 (mi-tosome), cysteine synthase 1 (CS1; cytoplasm), Sec61�

(endoplasmic reticulum), and CP5 (lysosome).

A B C D

E F G H

I J K L

Fig. 2. Immunolocalization of representative mitosomal proteins. Colocal-ization of individual mitosomal proteins with the HA epitope (anti-HA anti-body, red) and the authentic mitosomal protein marker Cpn60 (native Cpn60antiserum, green) is shown (A–H). Colocalization of endogenous Cpn60 andexogenous HA-tagged Cpn60 is also shown (I–L). (Scale bars: 10 �m.)

21732 � www.pnas.org�cgi�doi�10.1073�pnas.0907106106 Mi-ichi et al.

Dow

nloa

ded

by g

uest

on

July

2, 2

020

BI-S-33 medium containing Na2[35S]O4 for up to 2 h, or labeled for1 h and chased for up to 24 h. The cell lysate was separated intomethanol-soluble and insoluble fractions. Labeled [35S] was de-tected predominantly in the methanol-soluble fraction. At least 5polar lipids, separated on a silica HPTLC plate in 25:65:10 (vol/vol/vol) methanol/chloroform/acetic acid (Fig. 4), accounted formost of the [35S] detected (81.6% � 6.2% at 1 h pulse). These lipidswere distinct from commonly observed phospholipid species (phos-phatidylethanolamine, phosphatidylserine, phosphatidylinositol,and phosphatidylcholine). These data indicate that activated sulfateis mainly used to synthesize sulfur-containing polar lipids.

Phylogenetic Analysis of the Sulfate Activation Pathway. To investigatethe origin of sulfate activation in E. histolytica, phylogenetic analysesof the proteins involved in the pathway and established mitosomalchaperones were conducted. The origins of AS, APSK, IPP, sodi-um/sulfate symporter, Cpn60, and mitochondrial Hsp70 were not

identical. E. histolytica AS showed strong affinity to AS from�-proteobacteria (Fig. 5), whereas E. histolytica IPP clustered withIPP from other eukaryotes (Fig. S4). E. histolytica APSK was closelyrelated to �-proteobacteria, �-proteobacteria, and Dictyosteliumdiscoideum (with low bootstrap values), and E. histolytica sodium/sulfate symporters clustered with a limited group of eukaryotes(diatoms and green algae) and bacteria, although their exact originswere not clearly resolved (Fig. S3 and Fig. S5). In contrast, E.histolytica Cpn60 and mitochondrial Hsp70 showed monophyly withthe �-proteobacteria (8, 11, 22). The data indicate the phylogeneti-cally mosaic nature of mitosomes in E. histolytica.

DiscussionAlthough the diversity in structure and function of mitochondrion-related organelles have been demonstrated in a wide range oforganisms, E. histolytica remains as one of the anaerobic/microaerophilic eukaryotes, in which the function of the mitochon-drion-related organelle remains unknown. In this study, we haveprovided biochemical and microscopic evidence demonstrating thatsulfate activation, which generally occurs in the cytoplasm andplastids in eukaryotes, is the major function of mitosomes in E.histolytica.

Genes encoding for all of the major components necessary forsulfate activation and transport of substrates and products acrossthe mitosomal membrane are identifiable in the genome (Fig. 6).Sodium/sulfate symporter, which is necessary for the uptake ofsulfate into mitosomes, was identified. ATP that is necessary for theactivation of sulfate by both AS and APSK, and the quality controlof heat-sensitive AS by chaperones, is incorporated via MCF tomitosomes with a concomitant cytosolic export of ADP or AMP.E. histolytica MCF transports both ADP and AMP to the cytosol forthe incorporation of ATP to mitosomes (14). In other organisms,PAPS transporter (PAPST), which transports PAPS and AMP inopposite directions, is required for shuttling PAPS between the

A B C D E F G H I J K L M N O P Q S

Fractions

APS

PAPS

0

5000

10000

15000

20000

25000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Fractions

Arb

itrar

y U

nits

APS

PAPS

APS

PAPS

First Percoll Fractions1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Second Percoll FractionsA B C D E F G H I J K L M N O P Q S

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 reactionmixture 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 wasinitiated by the addition of the freeze-thawed fraction (3 �L), carried out for 2 h at 25 °C, and terminated by the addition of 88 �L methanol. Ten-microlitersamples were analyzed by PEI-cellulose TLC to determine 35S-labeled APS or PAPS as previously described (27). The amount of each product was quantified bydensitometric analysis using an image analyzer (Fuji) and the results are expressed in arbitrary units.

SO42-, APS, PAPS

ContinuousLabeling (min) Pulse (1h) + Chase (h)

PC

PS/PI

PE5 30 60 120 0 1 2 4 8 24

Fig. 4. Incorporation of [35S]-labeled sulfate into polar lipids. 35S was incor-porated into at least 5 polar lipids (arrowheads), which were distinct fromcommonly observed phospholipid species. PE, phosphatidylethanolamine; PS,phosphatidylserine; PI, phosphatidylinositol; PC, phosphatidylcholine.

Mi-ichi et al. PNAS � December 22, 2009 � vol. 106 � no. 51 � 21733

EVO

LUTI

ON

Dow

nloa

ded

by g

uest

on

July

2, 2

020

organelle and the cytosol (28). Although the E. histolytica genomecontains a gene encoding for putative PAPST (XP�654175; expec-tation value, 4.2�14) (29, 30), we failed to demonstrate the mito-somal localization of the protein (PAPST; Fig. S6). In addition, aputative phosphate transporter (XP�654379; expectation value,1.23�61) (31) also failed to localize to mitosomes (PHT; Fig. S6).Thus, the identity of the presumptive PAPS and phosphate trans-porters in E. histolytica mitosomes remains unknown. Generally,PAPS is used in 2 different pathways. In one route, the sulfatemoiety of PAPS is transferred to various acceptors to yield muco-polysaccharides, sulfolipids, and sulfoproteins by sulfotransferases.Alternatively, sulfate is reduced and assimilated into cysteine (32,33). The E. histolytica genome contains 11 potential genes encodingfor sulfotransferases, but lacks the enzymes for sulfate reduction(34, 35). Therefore, together with the results of [35S]O4 metabolic

labeling (Fig. 4), it is conceivable to assume that PAPS predomi-nantly serves as the sulfo-donor of sulfotransferases to synthesizesulfur-containing lipids. Two of the most highly expressed sulfo-transferases (SULT1, XP�654200, expectation value, 1�12; SULT2,XP�654101, expectation value, 1�13) were distributed to the cyto-plasm (SULT1 and SULT2; Fig. S6), suggesting that sulfo-transferreactions occur in the cytosol, and thus reinforcing the premise thatPAPS needs to be exported from mitosomes. Further character-ization of these sulfur-containing lipid species in E. histolytica isnecessary to understand the physiological significance of the com-partmentalized sulfate activation in mitosomes of this organism.

As sulfate activation in eukaryotes generally occurs in thecytoplasm and plastids (36, 37), our finding has raised an importantquestion on whether compartmentalization of sulfate activation inmitosomes is unique to E. histolytica. The acquisition and compart-

0.1

Monosiga brevicollis_KS

Cyanidioschyzon merolae_SOryza sativa_SAllium sepa_S

Glycine max_SBrassica napus_SArabadopsis thaliana_S

Anopholes gambia_KSDrosophila melanogaster_KSCiona instestinalis_KS

Mus musculus_KS-2Homo sapiens_KS-2Mus musculus_KS-1Homo sapiens_KS-1

Phytophthora infestans_KSPThalassiosira pseudonana_KSP

Phaeodactylum tricornutum_KSPRiftia pachyptila symbiont_S

Thiobacillus denitrificans_SPsychrobacter arcticum_S

Entamoeba hisolytica_SEntamoeba invadens_S

Desulfovibrio vulgaris_SDesulfotalea psychrophila_S

Chlorobium chlorochromatii_SChlorobium tepidum_S

Chloroflexus aurantiacus_SDeinococcus geothermalis_S

Deinococcus radiodurans_SBacillus subtilis_S

Staphylococcus epidermidis_SRubrobacter xylanophilus_S

Gloeobacter violaceus_SSynechococcus sp.ja-3-3a_S

Anabaena variabilis_STrichodesmium erythraeum_S

Synechococcus sp.wh8102_SCrocosphaera watsonii_S

Campylobacter jejuni_SThermus thermophilus_S

Toxoplasma gondii_SKAquifex aeolicus_SK

Chloroflexus aurantiacus_SKCoxiella burnetii_SK

Desulfotalea psychrophila_SKDictyostelium discoideum_SKRhodobacter sphaeroides_SK

Silicibacter pomeroyi_SKOceanicola granulosus_SK

Jannaschia sp. CCS1_SK

Ashbya gossypii_SKluyveromyces lactis_SSaccharomyces cervesiae_SCandida glabrata_S

Mucor circinelloides_SKUstilago maydis_SK

Gibberella zeae_SKYarrowia lipolytica_SKNeurospora crassa_SKPenicillium chrysogenum_SK

Aspergillus oryzae_SK

Prochlorococcus marinus_SSynechocystis sp.6803_S

Chlamydomonas reinhardtii_SPhaeodactylum tricornutum_SThalassiosira pseudonana_SEuglena gracilis_S

99

99

9950

53

94

100

52

98

85

73

100

92

99

99

89

87

84

92

62

56

71

100

56

90

100

30

46

100

99

ML DM MP

96 95 68

ML DM MP

99 100 100

ML DM MP

67 95 73

10088

85

60

82

88

72

100

89

Proteobacteria

δ-Proteobacteria

Bacteria

Cyanobacteria

Bacteria

Bacteria

Eukaryote

Eukaryote

☆☆

SK fusion group

Fungi

Fungi

α-Proteobacteria

Eukaryote

Eukaryote

Proteobacteria

Bacteria

KSP fusion group

KS fusion group

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 intoconsideration. The �-value of the �-shaped parameter used in the analysis of AS was 0.50723. Bootstrap proportion (BP) values are attached to the internalbranches. 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 estimatednumber 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 to178, 182 to 196, 200 to 226, 231 to 262, 264 to 325m and 333 to 399 of the E. histolytica sequence.

21734 � www.pnas.org�cgi�doi�10.1073�pnas.0907106106 Mi-ichi et al.

Dow

nloa

ded

by g

uest

on

July

2, 2

020

mentalization of the enzymes involved in sulfate activation mayhave occurred exclusively in Entamoeba among the anaerobic/microaerophilic organisms possessing mitochondrion-related or-ganelles. T. vaginalis, G. intestinalis, and C. parvum apparently lackgenes encoding for the enzymes. Encephalitozoon cuniculi, one ofthe microsporidia, has the gene for IPP (Fig. S4), but lacks genesencoding for AS and APSK. Although the genome and ESTdatabases are not complete for M. balamuthi and N. patriciarum,none of AS, APSK, and IPP genes was found so far. It is worthexamining if genes involved in sulfate activation are present in M.balamuthi because the 2 species share the NIF system for FeScluster biosynthesis (22). There is only one eukaryote, other than E.histolytica, that possesses sulfate activation in the mitochondria. InEuglena gracilis, the enzymatic activities involved in sulfate activa-tion and reduction have been detected in the mitochondria (36, 38,39). The origin of E. gracilis AS, however, is presumed to be thesecondary green algal plastid and not the �-proteobacteria (36),whereas E. gracilis IPP is thought to be derived from bacteria andnot an ancestral eukaryote (Fig. S4). These results suggest that theorigins of E. gracilis mitochondria are distinct from those of E.histolytica mitosomes. Based on the fact that the 4 E. histolyticaproteins involved in sulfate transport and activation have originsdistinct from �-proteobacterium, we propose a hypothesis wherebyAS, APSK, and sodium/sulfate symporter were acquired by theancestor of Entamoeba from �-proteobacterium (and other bacte-ria) by lateral gene transfer (40). We examined whether AS andAPSK were found in an operon in �-proteobacteria, which wouldsupport the hypothesis whereby both AS and APSK of �-proteobac-terial origin were transferred to the ancestor of Entamoeba. How-ever, this is not a case in at least 2 representative species (Desul-fotalea psychrophila and Desulfovibrio vulgaris).

Various diverse or sometimes shared characteristics are reportedin other eukaryotes possessing either hydrogenosomes or mito-somes. These include hydrogenase and pyruvate:ferredoxin oxi-doreductase for ATP generation in hydrogenosomes; NADH de-hydrogenase part of mitochondrial complex I in T. vaginalis, N.ovalis, and Blastocystis; TCA cycle enzymes in M. balamuthi, Blas-tocystis, N. patriciarum, and N. ovalis; glycine cleavage complex in M.balamuthi, T. vaginalis, and Blastocystis; and alternative oxidase inBlastocystis and C. parvum (1). Cpn60 and the ISC system werethought to be shared characteristics of hydrogenosomes and mito-

somes in anaerobic/microaerophilic eukaryotes (1). However, E.histolytica and M. balamuthi possess only the NIF system instead ofthe ISC system (22, 24). In the mitosomal proteome, neither NifSnor NifU was identified in the mitosome-enriched fraction. Wepreviously reported that NifS and NifU, both of which lack amitochondrion-targeting signal, were fractionated on an anionexchange chromatography from the soluble fraction obtained by0.45-�m filtration and 45,000 � g ultracentrifugation of amoebiclysate, suggesting the predominantly cytoplasmic localization of theNIF system in E. histolytica (24). Similarly, both NifS and NifU inM. balamuthi lack a mitochondria targeting signal (22). These datasuggest 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. histolyticamitosomes also partially contain the NIF system.

The targeting mechanisms to the E. histolytica mitosomes remainunsolved and need further investigation. Although Entamoeba PNTwas presumed to be imported into mitosomes based on the resem-blance of its amino-terminal portion rich in hydroxylated and basicamino acids to the canonical mitochondrial targeting peptide (2),our proteomic and immunofluorescence studies clearly showed thatPNT is not imported to mitosomes, but is distributed to vesicles andvacuoles (Fig. S7). Commonly available prediction programs suchas Mitoprot (41) and PSORT II (42) were unable to correctlypredict the mitosomal localization of proteins whose localizationwas verified by immunofluorescence assay. Similarly, the remainingputative mitosomal proteins have no predictable mitochondrialtargeting signal. As described earlier, our mitosomal proteomecontained a weak homologue of Tom40 (expectation value, 0.13).Thus, there is a need to verify the identity of this potential Tom40and the existence of the Tom complex in E. histolytica. In otheranaerobic eukaryotes, protein import machinery to the mitochon-drion-related organelles appears to be conserved (43, 44), and themitosomal targeting signal from G. lamblia and the hydrogenosometargeting signal from T. vaginalis were interchangeable (45). On thecontrary, cryptic mitochondrial targeting signals have also beendiscovered in G. lamblia, T. vaginalis, and microsporidian species,and unique aspects of mitochondrial processing peptidases havebeen reported in G. lamblia (1, 46). Together, the machinery andmechanisms of protein import into mitosomes require furtherinvestigation.

In summary, we have shown that E. histolytica mitosomes are amitochondrion-related organelle that is highly divergent and rep-resents a mosaic organelle consisting of proteins derived from�-proteobacteria, �-proteobacteria, and eukaryotes. It is primarilyinvolved in sulfate activation. This study should shed light on thediversification of mitochondrion-related organelles in eukaryoticevolution.

Materials and MethodsMitosome Purification. Approximately 0.5–1 � 108 trophozoites of E. histolyticastrain 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, proteaseinhibitor mixture (Roche), 300 �M E-64, 10 mM Mops-KOH (pH 7.4)], and homog-enized with a Dounce homogenizer. Unbroken cells, nuclei, and large vacuoleswere removed by centrifugation at 5,000 � g at 4 °C for 10 min, and thesupernatant was gently layered onto 3 mL of homogenization buffer containing30% (vol/vol) Percoll. After centrifugation at 120,000 � g at 4 °C for 1 h, 200 �Lfractions were collected from top to bottom (fractions 1–20). Fractions 19 and 20(density �1.054) were collected, mixed, and applied onto 2 mL of homogeniza-tion buffer containing 70% (vol/vol) Percoll. The gradient was subsequentlyoverlaid with 1 mL of the homogenization buffer containing 15% (vol/vol)Percoll.Aftercentrifugationat120,000�gat4 °Cfor1h,200�Loffractionswerecollected from top to bottom (fractions A-S; Fig. S1).

Immunoblot Analysis. Fractions prepared as previously mentioned were mixedwith 4� SDS/PAGE sample buffer. Samples were boiled at 95 °C for 5 min fol-lowedbycentrifugationat13,000�gat4 °Cfor20mintoremovethePercoll.Thefractions were analyzed by SDS/PAGE, silver stained, and immunoblot analysis as

Fig. 6. A compartmentalized sulfate activation pathway in E. histolyticamitosomes. The mitosome proteins whose localization was confirmed byimmunofluorescence assay are shown in red, whereas the putative transport-ers are shown in gray. PHT, phosphate transporter; SULT, sulfotransferase.Two of the most highly expressed sulfotransferases were demonstrated to belocalized to the cytoplasm (Fig. S6).

Mi-ichi et al. PNAS � December 22, 2009 � vol. 106 � no. 51 � 21735

EVO

LUTI

ON

Dow

nloa

ded

by g

uest

on

July

2, 2

020

previously described (49). The dilution of the primary antibodies was 1:1,000 foranti-Cpn60 and anti-cysteine synthase 1 antiserum, and 1:100 for anti-CP5 andanti-Sec61� antiserum (50, 51).

Production of E. histolytica Lines Expressing Epitope-Tagged Mitosomal Proteins.Plasmids for the production of amoeba lines expressing engineered mitosomalproteins containing 3 tandem HA-epitopes were constructed essentially as pre-viously described (52). Lipofection of trophozoites and selection of transformantswere also performed as previously described (53).

Indirect Immunofluorescence Assay. Indirect immunofluorescence assay was per-formed as previously described (49) with some modifications. Briefly, the amoe-bae were washed and fixed with acetone/methanol (1:1) for 10 min. Afterwashing with PBS solution, cells were permeabilized with 0.3% Triton X-100 for15 min and reacted with primary antibody diluted at 1:500 (anti-Cpn60 anti-serum) and 1:1,000 (anti-HA monoclonal antibody) in PBS solution. The sampleswere then reacted with Alexa Fluor 488- or 568-conjugated anti-rabbit or anti-mouse secondary antibody (1:1,000) for 1 h. The samples were examined aspreviously described (49).

Metabolic Labeling. Approximately 106 trophozoites were labeled with [35S]-labeled sulfate (25 mCi/m mole) in 0.5 mL of the Diamond BI-S-33 medium eithercontinuously for 5 to 120 min or labeled for 1 h and chased for 1 to 24 h (54). Cellswere collected and lipids were extracted with 0.5 mL of methanol and separatedon a silica high-performance thin-layer chromatography plate in 25:65:10 (vol/vol/vol) methanol/chloroform/acetic acid. Dried thin-layer chromatographyplates were analyzed by autoradiography.

ACKNOWLEDGMENTS. We thank Dr. Takeshi Makiuchi for discussions of phylo-genetic analysis. We thank Yoko Yamada, Kyoko Masuda, Kayoko Hashimoto,and Rumiko Kosugi for technical assistance. We thank Jorge Tovar, Royal Hollo-wayUniversityofLondon,foranti-Cpn60antibodyforaninitialanalysisofCpn60.We also thank Rosana Sanchez-Lopez, UNAM, Cuernavaca, Mexico, for anti-Sec61� antiserum. This work was supported by Creative Scientific Research Grant18GS0314 from the Japanese Ministry of Education, Science, Culture, Sports, andTechnology (to T.N.); Grant-in-Aid for Scientific Research (18GS0314, 18050006,18073001) from the Ministry of Education, Culture, Sports, Science and Technol-ogy of Japan; a grant for research on emerging and re-emerging infectiousdiseases from the Ministry of Health, Labour and Welfare of Japan; and a grantfor research to promote the development of anti-AIDS pharmaceuticals from theJapan Health Sciences Foundation (to T.N.).

1. van der Giezen M (2009) Hydrogenosomes and mitosomes: conservation and evolutionof functions. J Eukaryot Microbiol 56:221–231.

2. Aguilera P, Barry T, Tovar J (2008) Entamoeba histolytica mitosomes: organelles insearch of a function. Exp Parasitol 118:10–16.

3. Lindmark DG, Muller M (1973) Hydrogenosome, a cytoplasmic organelle of the anaer-obic flagellate Tritrichomonas foetus, and its role in pyruvate metabolism. J Biol Chem248:7724–7728.

4. Hrdy I, et al. (2004) Trichomonas hydrogenosomes contain the NADH dehydrogenasemodule of mitochondrial complex I. Nature 432:618–622.

5. Yarlett N, Orpin CG, Munn EA, Yarlett NC, Greenwood CA (1986) Hydrogenosomes inthe rumen fungus Neocallimastix patriciarum. Biochem J 236:729–739.

6. van der Giezen M, et al. (2003) Fungal hydrogenosomes contain mitochondrial heat-shock proteins. Mol Biol Evol 20:1051–1061.

7. Hackstein JH, Tjaden J, Huynen M Mitochondria, hydrogenosomes and mitosomes:products of evolutionary tinkering! Curr Genet 50:225–245, 2006.

8. Clark CG, Roger AJ (1995) Direct evidence for secondary loss of mitochondria inEntamoeba histolytica. Proc Natl Acad Sci USA 92:6518–6521.

9. Mai, Z, et al. (1999) Hsp60 is targeted to a cryptic mitochondrion-derived organelle(‘‘crypton’’) in the microaerophilic protozoan parasite Entamoeba histolytica. Mol CellBiol 19:2198–2205.

10. Tovar J, Fischer A, Clark CG (1999) The mitosome, a novel organelle related tomitochondria in the amitochondrial parasite Entamoeba histolytica. Mol Microbiol32:1013–1021.

11. Bakatselou C, Kidgell C, Clark CC (2000) A mitochondrial-type hsp70 gene of Entam-oeba histolytica. Mol Biochem Parasitol 110:177–182.

12. Leon-Avila G, Tovar J (2004) Mitosomes of Entamoeba histolytica are abundantmitochondrion-related remnant organelles that lack a detectable organellar genome.Microbiology 150:1245–1250.

13. van der Giezen M, Leon-Avila G, Tovar J (2005) Characterization of chaperonin 10(Cpn10) from the intestinal human pathogen Entamoeba histolytica. Microbiology151:3107–3115.

14. Chan KW, et al. (2005) A novel ADP/ATP transporter in the mitosome of the microaero-philic human parasite Entamoeba histolytica. Curr Biol 15:737–742.

15. Tovar J, Cox SS, van der Giezen M (2007) A mitosome purification protocol based onpercoll density gradients and its use in validating the mitosomal nature of Entamoebahistolytica mitochondrial Hsp70. Methods Mol Biol 390:167–177.

16. Tovar J, et al. (2003) Mitochondrial remnant organelles of Giardia function in iron-sulphur protein maturation. Nature 426:172–176.

17. Dolezal P, et al. (2005) Giardia mitosomes and trichomonad hydrogenosomes share acommon mode of protein targeting. Proc Natl Acad Sci USA 102:10924–10929.

18. Burri L, Williams BA, Bursac D, Lithgow T, Keeling PJ (2006) Microsporidian mitosomesretain elements of the general mitochondrial targeting system. Proc Natl Acad Sci USA103:15916–15920.

19. Goldberg AV, et al. (2008) Localization and functionality of microsporidian iron-sulphur cluster assembly proteins. Nature 452:624–628.

20. Tsaousis AD, et al. (2008) A novel route for ATP acquisition by the remnant mitochon-dria of. Encephalitozoon cuniculi. Nature 453:553–556.

21. Henriquez FL, Richards TA, Roberts F, McLeod R, Roberts CW (2005) The unusualmitochondrial compartment of Cryptosporidium parvum. Trends Parasitol 21:68–74.

22. Gill EE, et al. (2007) Novel mitochondrion-related organelles in the anaerobic amoebaMastigamoeba balamuthi. Mol Microbiol 66:1306–1320.

23. Stechmann A, et al. (2008) Organelles in Blastocystis that blur the distinction betweenmitochondria and hydrogenosomes. Curr Biol 18:580–585.

24. Ali V, Shigeta Y, Tokumoto U, Takahashi Y, Nozaki T (2004) An intestinal parasitic protist,Entamoeba histolytica, possesses a non-redundant nitrogen fixation-like system for iron-sulfur cluster assembly under anaerobic conditions J Biol Chem 279:16863–16874.

25. Nozaki T, et al. (1998) Cloning and bacterial expression of adenosine-5�-triphosphatesulfurylase from the enteric protozoan parasite Entamoeba histolytica. Biochim Bio-phys Acta 1429:284–291.

26. Leyh TS, Taylor JC, Markham GD (1988) The sulfate activation locus of Escherichia coliK12: cloning, genetic, and enzymatic characterization. J Biol Chem 263:2409–2416.

27. Yanagisawa K, et al. (1998) cDNA cloning, expression, and characterization of thehuman bifunctional ATP sulfurylase/adenosine 5�-phosphosulfate kinase enzyme. Bio-sci Biotechnol Biochem 62:1037–1040.

28. Capasso JM, Hirschberg CB (1984) Mechanisms of glycosylation and sulfation in theGolgi apparatus: evidence for nucleotide sugar/nucleoside monophosphate and nu-cleotide sulfate/nucleoside monophosphate antiports in the Golgi apparatus mem-brane. Proc Natl Acad Sci USA 81:7051–7055.

29. Kamiyama S, et al. (2003) Molecular cloning and identification of 3�-phosphoad-enosine 5�-phosphosulfate transporter. J Biol Chem 278:25958–25963.

30. Kamiyama S, et al. (2006) Molecular cloning and characterization of a novel 3�-phosphoadenosine 5�-phosphosulfate transporter, PAPST2. J Biol Chem 281:10945–10953.

31. Guo B, Irigoyen S, Fowler TB, Versaw WK (2008) Differential expression and phyloge-netic analysis suggest specialization of plastid-localized members of the PHT4 phos-phate transporter family for photosynthetic and heterotrophic tissues. Plant SignalBehav 3:784–790.

32. Strott CA (2002) Sulfonation and molecular action. Endocr Rev 23:703–732.33. Negishi M, et al. (2001) Structure and function of sulfotransferases. Arch Biochem

Biophys 390:149–157.34. Clark CG, et al. (2007) Structure and content of the Entamoeba histolytica genome. Adv

Parasitol 65:51–190.35. Ali V, Nozaki T (2007) Current therapeutics, their problems, and sulfur-containing-

amino-acid metabolism as a novel target against infections by ‘‘amitochondriate’’protozoan parasites. Clin Microbiol Rev 20:164–187.

36. Patron NJ, Durnford DG, Kopriva S (2008) Sulfate assimilation in eukaryotes: fusions,relocations and lateral transfers. BMC Evol Biol 8:1–14.

37. Bradley ME, Rest JS, Li WH, Schwartz NB (2009) Sulfate activation enzymes: phylogenyand association with pyrophosphatase. J Mol Evol 68:1–13.

38. Brunold C, Schiff JA (1976) Studies of sulfate utilization of algae: 15. Enzymes ofassimilatory sulfate reduction in Euglena and their cellular localization. Plant Physiol57:430–436.

39. Saidha T, Na SQ, Li JY, Schiff JA (1988) A sulphate metabolizing centre in Euglenamitochondria. Biochem J 253:533–539.

40. Loftus B, et al. (2005) The genome of the protist parasite. Entamoeba histolytica Nature433:865–868.

41. Claros MG, Vincens P (1996) Computational method to predict mitochondrially im-ported proteins and their targeting sequences. Eur J Biochem 241:779–786.

42. Nakai K, Horton P (1999) PSORT: a program for detecting sorting signals in proteins andpredicting their subcellular localization. Trends Biochem Sci 24:34–36.

43. Dolezal, et al. (2006) Evolution of the molecular machines for protein import intomitochondria. Science 313:314–318.

44. Dagley MJ, et al. (2009) The protein import channel in the outer mitosomal membraneof Giardia intestinalis. Mol Biol Evol 26:1941–1947.

45. Dolezal P, et al. (2005) Giardia mitosomes and trichomonad hydrogenosomes share acommon mode of protein targeting. Proc Natl Acad Sci USA 102:10924–10929.

46. Smíd O, et al. (2008) Reductive evolution of the mitochondrial processing peptidasesof the unicellular parasites Trichomonas vaginalis and Giardia intestinalis. PLoS Pathog4: e1000243.

47. Diamond LS, Mattern CF, Bartgis IL (1972) Viruses of Entamoeba histolytica. I. Identi-fication of transmissible virus-like agents. J Virol 9:326–341.

48. Diamond LS, Harlow DR, Cunnick CC (1978) A new medium for the axenic cultivationof Entamoeba histolytica and other. Entamoeba Trans R Soc Trop Med Hyg 72:431–432.

49. Nakada-Tsukui K, Saito-Nakano Y, Ali V, Nozaki T (2005) A retromerlike complex is a novelRab7 effector that is involved in the transport of the virulence factor cysteine protease inthe enteric protozoan parasite Entamoeba histolytica. Mol Biol Cell 16:5294–5303.

50. Nozaki T, et al. (1998) Molecular cloning and characterization of the genes encodingtwo isoforms of cysteine synthase in the enteric protozoan parasite Entamoebahistolytica. Mol Biochem Parasitol 97:33–44.

51. Mitra BN, Saito-Nakano Y, Nakada-Tsukui K, Sato D, Nozaki T (2007) Rab11B smallGTPase regulates secretion of cysteine proteases in the enteric protozoan parasiteEntamoeba histolytica. Cell Microbiol 9:2112–2125.

52. Saito-Nakano Y, Yasuda T, Nakada-Tsukui K, Leippe M, Nozaki T (2004) Rab5-associated vacuoles play a unique role in phagocytosis of the enteric protozoanparasite Entamoeba histolytica. J Biol Chem 279:49497–49507.

53. Nozaki T, et al. (1999) Characterization of the gene encoding serine acetyltransferase,a regulated enzyme of cysteine biosynthesis from the protist parasites Entamoebahistolytica and Entamoeba dispar. Regulation and possible function of the cysteinebiosynthetic pathway in Entamoeba. J Biol Chem 274:32445–32452.

54. Bakker-Grunwald T, Geilhorn B (1992) Sulfate metabolism in Entamoeba histolytica.Mol Biochem Parasitol 53:71–78.

21736 � www.pnas.org�cgi�doi�10.1073�pnas.0907106106 Mi-ichi et al.

Dow

nloa

ded

by g

uest

on

July

2, 2

020