The Ancient and Widespread Nature of the ER–Mitochondria
Encounter Structure
Jeremy G. Wideman,*,1,2 Ryan M.R. Gawryluk,3 Michael W. Gray,3 and Joel B. Dacks1
1
Department of Cell Biology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada
Department of Science, Augustana Faculty, University of Alberta, Camrose, Alberta, Canada
3
Department of Biochemistry and Molecular Biology, Centre for Comparative Genomics and Evolutionary Bioinformatics,
Dalhousie University, Halifax, Nova Scotia, Canada
*Corresponding author: E-mail: [email protected].
Associate editor: Charles Delwiche
2
Abstract
Mitochondria are the result of a billion years of integrative evolution, converting a once free-living bacterium to an
organelle deeply linked to diverse cellular processes. One way in which mitochondria are integrated with nonendosymbiotically derived organelles is via endoplasmic reticulum (ER)–mitochondria contact sites. The ER membrane is physically tethered to the mitochondrial outer membrane by the ER–mitochondria encounter structure (ERMES). However, to
date, ERMES has only ever been found in the fungal lineage. Here, we bioinformatically demonstrate that ERMES is
present in lineages outside Fungi and validate this inference by mass spectrometric identification of ERMES components
in Acanthamoeba castellanii mitochondria. We further demonstrate that ERMES is retained in hydrogenosome-bearing
but not mitosome-bearing organisms, yielding insight into the process of reductive mitochondrial evolution. Finally, we
find that the taxonomic distribution of ERMES is most consistent with rooting the eukaryotic tree between Amorphea
(Animals + Fungi + Amoebozoa) + Excavata and all other eukaryotes (Diaphoratickes).
Key words: ERMES complex, organelle evolution, hydrogenosomes and mitosomes, ER-mitochondria contacts.
Letter
Mitochondria (or homologous organelles) are ubiquitous
eukaryotic features and therefore can be inferred to have
been present in the last eukaryotic common ancestor
(LECA). One aspect of mitochondrial evolution that has not
been previously studied is the process by which mitochondria
were integrated with nonendosymbiotically derived cellular
systems like the membrane-trafficking system. In fungal and
animal cells, mitochondria are linked to the membranetrafficking system by the physical contact of mitochondria
and the endoplasmic reticulum (ER) via ER–mitochondria
contact sites (Helle et al. 2013). Although in animals the molecular nature of such contact sites remains an open question
(Hayashi et al. 2009; Raturi and Simmen 2013), in fungi a protein assemblage termed the “ER-mitochondria encounter
structure” (ERMES) tethers the ER to mitochondria
(Kornmann et al. 2009). ERMES is composed of four proteins:
the mitochondrial outer membrane (MOM) b-barrel Mdm10
and three SMP (“synaptotagmin-like, mitochondrial, lipidbinding protein”) domain-containing proteins (Kopec et al.
2010), namely, the ER transmembrane protein, Mmm1;
the MOM transmembrane protein, Mmm2 (Mdm34); and
the cytosolic bridge protein, Mdm12 (fig. 1). In addition to the
four structural proteins, it has been suggested that the ubiquitous (Vlahou et al. 2011) dynamin-related MOM-anchored
GTPase, Gem1 (Miro in animals), may regulate the activity of
ERMES (Kornmann et al. 2011; Stroud et al. 2011), but this
inference has recently been disputed (Nguyen et al. 2012). In
addition to membrane tethering, ERMES has been implicated
in numerous cellular processes including maintenance of
mitochondrial morphology, mitochondrial protein import,
phospholipid transport between the ER and mitochondria,
mitochondrial attachment to the actin cytoskeleton, and mitochondrial division and inheritance of mitochondrial DNA
(Boldogh et al. 2003; Meisinger et al. 2004, 2007; Kornmann
et al. 2009; Wideman et al. 2010; Lackey et al. 2011; Voss et al.
2012; Murley et al. 2013). It is generally accepted that ERMES
does not have a clear homolog in multicellular animals (metazoans) and has been proposed as a fungal-specific novelty
(Helle et al. 2013). However, presence or absence of ERMES
components in eukaryotes outside animals and fungi has not
been investigated.
Results
We therefore searched for evidence of ERMES proteins in
diverse eukaryotes outside the fungal lineage, using iterative
Basic Local Alignment Search Tool (Blast) and HMMER
homology-searching methods followed by verification by
phylogenetic analysis (see Materials and Methods). We first
examined available genome sequences from the clades
Apusozoa and Amoebozoa, as proximal outgroups to the
fungi (see supplementary fig. S1, Supplementary Material
online, for a tree depicting all of the taxonomic nomenclature
used in this study). In the apusozoan Thecamonas trahens,
we found evidence for the presence of a single ERMES
protein, Mmm1 (fig. 2, column 7, and supplementary fig.
S2, Supplementary Material online). Considering the incomplete nature of the T. trahens genome sequence, this observation is encouraging but preliminary. In Amoebozoa,
ß The Author 2013. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please
e-mail: [email protected]
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Mol. Biol. Evol. 30(9):2044–2049 doi:10.1093/molbev/mst120 Advance Access publication June 28, 2013
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Ancient and Widespread Nature of the ERMES . doi:10.1093/molbev/mst120
ERMembrane
Mmm1
Mdm12
ERMES
Mmm2
Gem1?
MOM
Mdm10
FIG. 1. The ERMES complex. ERMES is composed of Mmm1, Mdm12,
Mmm2, and Mdm10. Gem1 is a potential substoichiometric member of
ERMES.
Metazoa
Mm Rn
Gg
Hs
Oa
Xt
Dr Md
Ce
Dm
Nv
Bf
Fungi
Choanozoa
Mb
Ta
Co
Sr Sa
however, we detected a full complement of ERMES proteins
in Dictyostelium discoideum, Polysphondylium pallidum, and
Acanthamoeba castellanii (fig. 2, column 8, and supplementary fig. S3A and B, Supplementary Material online). In A.
castellanii, direct analysis of purified mitochondria by
tandem mass spectrometry validated the presence of
Mdm10, Mmm1, and Mmm2 (supplementary table S1,
Supplementary Material online). Notably, Mmm1 is formally
an ER protein in Saccharomyces cerevisiae, and in A. castellanii,
it has a strongly predicted ER signal peptide.
Having established the presence of ERMES in nonfungal
lineages, we revisited the hypothesis of ERMES absence in the
Holozoa (metazoans and their unicellular relatives). In metazoans, all proteins containing potential SMP or Porin_3
domains that we retrieved by Blast or HMMER searching
were considered as candidate ERMES homologs, but these
hits were confirmed by phylogenetic analysis as Nvj2, Tcb,
PDZ-8, or Tom40 homologs (supplementary fig. S4A–D,
Supplementary Material online). This result confirms previous
reports that no ERMES homologs exist in metazoans (fig. 2,
columns 1–3). We also failed to identify ERMES components
in the unicellular choanoflagellates Monosiga brevicollis and
Apusozoa Amoebozoa
Sp
Ro
Um
Bd
Sc Amac
Spu
Nce
Cn Ca
Pir Ec
Nc
Ppal
Ttr
Ac
Dd
Ehis
Mmm1
Excavata
Tb
Tv
Lm
Bs
Ng
Gi
Plants
Cr At Ppat
Vc
Mpus
Ot Cm
SAR
Tg
Cp
Pf
Py
Tpar
Bb
CCTH
Bn Tt
Ptet
Es
Tps
Pt Ps
Ehux
Gt
?
Mmm2
?
Mdm12
?
Mdm10
Nvj2
*
Tom40
**
*
*
Porin
Abbreviations:
Metazoa:
Rn, Rattus norvegicus; Hs, Homo sapiens; Oa,
Ornithorhynchus anatinus; Md, Monodelphis domesticus;
Dr, Danio rerio; Xt, Xenopus tropicalis; Gg, Gallus gallus;
Mm, Mus musculus; Ce, Caenorhabditis elegans; Dm,
Drosophila melanogaster; Bf, Branchiostoma floridae;
Nv, Nematostella vectensis; Ta, Trichoplax adhaerens
Choanozoa:
Mb, Monosiga brevicollis; Co, Capsaspora owczarzaki;
Sa, Sphaeroforma arctica; Sr, Salpingoeca rosetta
Apusozoa:
Ttr, Thecamonas trahens
Amoebozoa:
Ppal, Polysphondylium pallidum; Dd, Dictyostelium
Fungi:
discoideum; Ehis, Entamoeba histolytica; Ac,
Sp, Schizosaccharomyces pombe; Sc, Saccharomyces
cerevisiae; Ca, Candida albicans; Nc, Neurospora crassa; Acanthamoeba castellanii
Cn, Cryptococcus neoformans; Um, Ustilago maydis; Ro,
Rhizopus oryzae; Bd, Batrachochytrium dendrobatidis;
Excavata:
Nce, Nosema ceranae; Ec, Encephalitozoon cuniculi; Pir, Lm, Leishmania major; Ng, Naegleria gruberi; Gi,
Piromyces sp.; Spu, Spizellomyces punctatus; Amac,
Giardia intesinalis; Bs, Bodo saltans; Tv, Trichomonas
Allomyces macrogynus
vaginalis; Tb, Trypanosoma brucei
**
* **
**
*
Archaeplastida:
Cr, Chlamydomonas reinhardtii; At, Arabidopsis thaliana; Ppat,
Physcomitrella patens; Mpus, Micromonas pusilla; Cm,
Cyanidioschyzon merolae; Ot, Ostreococcus tauri; Vc,
Volvox carteri
SAR:
Tg, Toxoplasma gondii; Tpar, Theileria parva; Bb, Babesia
bovis; Py, Plasmodium yoelli; Pf, Plasmodium falciparum; Cp,
Cryptosporidium parvum; Tt, Tetrahymena thermophila; Ptet,
Paramecium tetraurelia; Tps, Thalassiosira pseudonana; Ps,
Phytophthora sojae; Pt, Phaeodactylum tricornutum; Es,
Ectocarpus siliculosus; Bn, Bigelowiella natans
CCTH:
Ehux, Emiliana huxleyi; Gt, Guillardia theta
FIG. 2. Coulson plot of ERMES components and related proteins. Presence is indicated by colored pie sectors. Black arrows indicate Acanthamoeba
castellanii proteins identified by mass spectrometric analysis. White asterisks denote proteins reported previously (Dagley et al. 2009; Pusnik et al. 2009,
2011; Waller et al. 2009). White question marks indicate proteins that were validated as ERMES components by phylogenetic analyses but where the
precise subunit classification could not be specified.
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Wideman et al. . doi:10.1093/molbev/mst120
Salpingoeca rosetta. However, we report the presence of a
complete ERMES in Capsaspora owczarzaki (a filasterian)
and a near-complete ERMES in Sphaeroforma arctica (an
ichthyosporean), two organisms that represent basally diverging single-celled relatives of metazoans (fig. 2, column 4,
and supplementary fig. S3A and B, Supplementary Material
online). Thus, we hypothesize a loss/replacement of the
ERMES complex at the transition leading to choanoflagellates
and metazoans. This inference, however, remains to be tested
by elucidation of the precise nature of the metazoan ER–
mitochondria contact machinery.
With ERMES components potentially present in the
ancestor of unikonts (animals, fungi, and amoebozoans), we
searched genomes from the remaining eukaryotic supergroups for these same components. Starting with the supergroup Excavata, we uncovered evidence of a complete ERMES
in the heterolobosean Naegleria gruberi (fig. 2 and supplementary fig. S3A and B, Supplementary Material online). We also
found evidence for various ERMES components in the kinetoplastids Bodo saltans, Leishmania major, and Trypanosoma
brucei and a near-complete ERMES in the metamonad
Trichomonas vaginalis (fig. 2, column 9, and supplementary
fig. S5A and B, Supplementary Material online). In corroboration of our bioinformatic analyses, two of the identified T.
vaginalis ERMES proteins (Mdm12 and Mmm2) have been
independently identified as hydrogenosomal proteins in separate studies of the T. vaginalis hydrogenosomal proteome
(Mdm12 in Rada et al. [2011] and Mmm2 in Schneider et al.
[2011]). Additionally, the protein we identified as T. brucei
Mmm2 was detected in a proteomic study of the T. brucei
mitochondrial outer membrane (Niemann et al. 2013).
Although Mdm10 was not identified in excavate lineages
beyond Naegleria, two porin-like b-barrel proteins with unknown function exist in the kinetoplastid lineage (Flinner et al.
2012) including B. saltans. Similarly, several divergent Tom40
paralogs exist in T. vaginalis (Rada et al. 2011). Thus, it is
conceivable that these proteins could have functionally
replaced Mdm10; however, further analyses would be required to determine the role of these proteins (in ERMES or
otherwise) and their relation to other MOM b-barrel proteins.
Although the close proximity of the ER and hydrogenosomes in T. vaginalis has been previously reported (Benchimol
et al. 1996), this cellular feature is considerably understudied.
To better understand the distribution of ERMES machinery
with relation to the various types of mitochondria-related
organelles (MROs), we searched the genomes of organisms
within unikont and excavate lineages that contain either
hydrogenosomes or the further-reduced form, mitosomes.
We were unable to identify ERMES components in any of
the organisms possessing mitosomes, including Giardia lamblia (Excavata), Entamoeba histolytica (Amoebozoa), and the
microsporidians Nosema ceranae and Encephalitozoon cuniculi (Fungi). However, consistent with the data from Tri. vaginalis, we did identify all four ERMES proteins in the
hydrogenosome-containing fungus Piromyces sp. (fig. 2).
This distribution establishes an additional demarcation in
the transitional continuum from aerobic mitochondria and
2046
hydrogenosome-like organelles to the genome-less, nonenergy-producing mitosomes.
Finally, we searched genome databases of organisms classified in the Diaphoretickes (Adl et al. 2012) mega-assembly
(which includes the Archaeplastida, SAR, and CCTH groups
[see supplementary fig. S1, Supplementary Material online])
for encoded ERMES components. Even with exhaustive
searching, no ERMES homologs could be found in any of
these groups (fig. 2, columns 10–13). Any candidate ERMES
proteins containing potential SMP or Porin_3 domains found
using HMMER or BlastP were subjected to phylogenetic
analyses and were determined to be Nvj2, Tcb (plant synaptotagmins), or porin homologs (supplementary fig. S3A–D,
Supplementary Material online). The absence of ERMES in all
Diaphoretickes genomes suggests that an ERMES complex
does not operate in these organisms and, most simply, that
an ERMES was never present in their common ancestor.
Discussion
Although ERMES has been proposed to be a fungal-specific
protein complex, our results indicate that it is an ancient
complex present in several eukaryotic supergroups. As cell
biological and genetic tools are improved in organisms such
as A. castellanii, D. discoideum, and N. gruberi, comparative
studies with the goal of understanding ER–mitochondria
contacts will be invaluable in understanding the nature of
ER–mitochondria integration as a general eukaryotic phenomenon. Additionally, because ERMES is not found in
Archaeplastida or Metazoa, this complex represents a good
candidate drug target in plant or animal diseases caused by
Fungi, Ichthyosporea, trypanosomes, Acanthamoeba,
Trichomonas, and potentially several other pathogens.
The absence of ERMES in Metazoa and Diaphoretickes
leaves open two possibilities regarding alternative
ER–mitochondria contact machinery. First, the machinery
might be distinct, with independent origins of analogous
ERMES-like complexes. In this case, if such components
could be identified in Diaphoretickes members, particularly
in the parasitic apicomplexans, such a finding would again
offer a potential avenue for therapeutic intervention.
Alternatively, there might exist as-yet-undiscovered
common cellular machinery for ER–mitochondria contact
sites. Of the various proposed machineries for such a role in
Metazoa (e.g., VDAC/Grp75/IP3R, Mfn1/2, Bap31/Fis1, and
PTPIP51/VapB [Helle et al. 2013]), many are broadly distributed in eukaryotes and could be candidates for machinery
present in the LECA. This avenue will be exciting to pursue in
more detail once a more robust front runner for the ER–
mitochondria contact machinery in animals is established.
For now, ERMES remains the best-characterized and supported machinery for maintaining ER–mitochondria contact.
Our results suggest that ERMES is retained in hydrogenosome-possessing lineages despite the significant functional
divergence of these MROs. This fact strongly implies the
existence of ER–hydrogenosome contacts and suggests that
they may perform many similar functions to that of ER–
mitochondria contacts including phospholipid transfer, maintenance of organellar morphology, protein import, calcium
Ancient and Widespread Nature of the ERMES . doi:10.1093/molbev/mst120
signaling, and ATP transfer. Because of their relative simplicity
and modified biology compared with mitochondria,
functional analyses of hydrogenosomes in ERMES-bearing
lineages can potentially shed light on the conserved functions
of this complex. Furthermore, ERMES represents a further
differentiating point between hydrogenosomes and the
nonenergy-generating mitosomes and provides a novel
avenue for enquiry into the process of organelle reduction
in the evolution of MROs. The genomic and cell biological
study of organisms with MROs intermediate between hydrogenosomes and mitosomes (e.g., Mastigamoeba balamuthi
[Gill et al. 2007], Trimastix [Hampl et al. 2008], and
Carpediemonas-like organisms [Takashita et al. 2012]) will
be integral in understanding organellar reduction as well as
ERMES function.
Finally, our results provide an important insight into the
contentious issue of the rooting of the eukaryotic tree (see
supplementary discussion, Supplementary Material online, for
a complete analysis of recent rooting hypotheses). The simplest explanation for the observed distribution of ERMES in
eukaryotes is that it arose as an innovation in the ancestor of a
monophyletic group containing Excavata, Amoebozoa, and
Opisthokonta (Holozoa + Fungi). Alternatively, although
slightly less parsimonious, ERMES could have been present
in the LECA and then lost in the common ancestor of the
Diaphoretickes megaclade. Either scenario places the root of
the eukaryotic tree between the Diaphoretickes and all other
eukaryotes (fig. 3).
The placement of the eukaryotic root between
Diaphoretickes and all other eukaryotes is consistent with
data that have been used to refute the well-known Bikont–
Unikont hypothesis (Roger and Simpson 2009) (see supplementary discussion, Supplementary Material online).
MBE
Furthermore, our proposed position of the root was not
strongly rejected in recent analyses using large-scale data
sets with concatenated data (Derelle and Lang 2012), analyses
of rare genomic events (Rogozin et al. 2009), or gene tree
parsimony analysis (Katz et al. 2012). One important caution
stems from the current phylogenetic distribution of available
protist genome sequences, in that we are unable to test for
the presence of ERMES in several lineages of particular interest, notably the enigmatic (putatively excavate) flagellate
Malawimonas (Derelle and Lang 2012), the newly described
Collodictyon (Zhao et al. 2012) and, perhaps most importantly, the core jakobids, the excavate lineage whose strikingly
ancestral mitochondrial genome uniquely harbors genes
encoding subunits of a bacterial-type RNA polymerase
(Burger et al. 2013). This limitation leaves open the possibility
of a rooting within excavates (i.e., Excavata might actually be
paraphyletic). Overall, we contend that our rooting placement represents the hypothesis most consistent with the
entirety of the data currently available and is thus a leading
contender for the critically important, hotly debated, and
thus-far-elusive position of the evolutionary root of
eukaryotes.
In general terms, the evolutionary origin of a eukaryotic
organelle may be either endosymbiotic or autogenous. Our
study of ERMES demonstrates broad taxonomic relevance for
an enigmatic set of cellular components, opens a new line of
enquiry into the cell biology and the evolutionary process
giving rise to MROs, and points to a new theory for the
placement of the eukaryotic root. Most significantly, rather
than telling a narrowly focused story about endosymbiotic or
autogenous organelle evolution, our study provides important insight into the highly integrated evolutionary history of
eukaryotic organelles.
Materials and Methods
Diaphoretickes
Archaeplastida
Chloroplastida
Rhodoplastida
Excavata
Kinetoplastida
Alveolata
Dikarya
Trichomonas H
Metazoa
Microsporidia
M
Choanoflagellata
Giardia M
Rhizaria
Filasterea/
Ichthyosporea
Cryptophyta
Haptophyta
H Neocallimastigomycota
Heterolobosea
Stramenopiles
SAR
Opisthokonta
Apusozoa
Slime molds
CCTH
ERMES
presumed point of origin
complete loss
partial loss
complete ERMES maintained
M mitosome
H hydrogenosome
M Entamoeba
Acanthamoebae
Amoebozoa
LECA
FIG. 3. Proposed most parsimonious evolutionary history of ERMES.
ERMES is depicted as being gained in the common ancestor to
Excavata, Opisthokonta, and Amoebozoa but lost in the common ancestor of Choanoflagellata and Metazoa. ERMES is also inferred to have
been lost in organisms that have mitosomes (red “M”) but maintained
in organisms with hydrogenosomes (green “H”). Partial loss of ERMES is
inferred as indicated. Further breakdown and explanation of taxa can be
found in supplementary figure S1, Supplementary Material online.
ERMES homologs were found using a combination of Blast
(Altschul et al. 1997) and HMMER v2.3.2 (http://hmmer.jane
lia.org/, last accessed July 14, 2013) algorithms. Protein sequences were obtained from various databases including
the National Center for Biotechnology Information, the
Joint Genome Institute, and directly from genome project
websites. Functionally validated ERMES proteins from
Neurospora crassa and Saccharomyces cerevisiae were used
to collect sequences from basal fungi. These sequences were
used as queries to find candidate ERMES proteins in other
lineages. Hidden Markov Models using fungal ERMES sequences were constructed and used to obtain additional
ERMES candidates. Proteins retrieved were subjected to reciprocal Blast analysis. Those retrieving any potential SMP- or
Porin_3-domain containing protein were classified by phylogenetics. See supplementary table S2, Supplementary Material
online, for a list of all relevant sequences obtained for this
study.
Tree building was restricted to sequences that had recognizable SMP (DUF2404) or Porin_3 domains to ensure proper
alignment of homologous sites. Sequences were aligned using
MUSCLE v3.6 (Edgar 2004) with manual adjustment using
Mesquite (http://mesquiteproject.org, last accessed July 14,
2047
Wideman et al. . doi:10.1093/molbev/mst120
2013). Model testing was performed using ProtTest v2.4
(Abascal et al. 2005) with a Gamma rate distribution and
accounting for invariant sites as appropriate. Tree building
was carried out using MrBayes 3.2.1 (Ronquist and
Huelsenbeck 2003) for Bayesian analysis. Maximum-likelihood
bootstrap values were obtained using PhyML v2.4.4 (Guindon
and Gascuel 2003) and RAxML v2.2.3 (Stamatakis 2006) with
100 pseudoreplicates. Protein classification was validated by
phylogenetic analyses using Nvj2 or Tom40 and Porin proteins as outgroups. Classifications are based on inclusion in
the relevant clade with support values greater than 0.8 posterior probability and 50% bootstrap support, with the following exception. In some instances (supplementary figs. S2,
S3A, and S5, Supplementary Material online), proteins in
question were robustly shown as ERMES orthologs, but the
specific classification was not resolved. In these cases, provisional classification is based on robust exclusion from the
other ERMES component clades.
Acanthamoeba castellanii mitochondria were purified
from axenic cultures on two successive step-sucrose gradients; whole mitochondria, or mitochondrial subfractions,
were analyzed by tandem mass spectrometry, as described
previously (Gawryluk et al. 2012). Protein identification was
carried out by the Mascot search engine (version 2.3). Ion
scores 38 (representing a P value < 0.05) were considered
significant.
Supplementary Material
Supplementary discussion, figures S1–S5, and tables S1 and S2
are available at Molecular Biology and Evolution online (http://
www.mbe.oxfordjournals.org/).
Acknowledgments
J.G.W. and R.M.R.G. conducted the experiments. J.G.W.,
R.M.R.G., M.W.G., and J.B.D. wrote the manuscript. The
authors thank the members of the Dacks lab and Frank
Nargang for discussion. This work was supported by a
National Science and Engineering Research Council of
Canada Discovery Grant and a New Faculty Award from
Alberta Innovates Technology Futures to J.B.D., by a
Canadian Institutes of Health Research Operating Grant
and funding from the Tula Foundation to M.W.G. and by a
Predoctoral Scholarship from the Killam Trusts to R.M.R.G.
J.B.D. is the Canada Research Chair in Evolutionary Cell
Biology.
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