Metabolic connectivity as a driver of host andendosymbiont integrationSlim Karkara,1, Fabio Facchinellib,1, Dana C. Pricea, Andreas P. M. Weberb,2, and Debashish Bhattacharyaa,2

aDepartment of Ecology, Evolution, and Natural Resources, Rutgers University, New Brunswick, NJ 08901; and bInstitut für Biochemie der Pflanzen, Clusterof Excellence on Plant Sciences, Heinrich-Heine Universität, D-40225 Düsseldorf, Germany

Edited by Patrick J. Keeling, University of British Columbia, Vancouver, Canada, and accepted by the Editorial Board March 6, 2015 (received for reviewDecember 19, 2014)

The origin of oxygenic photosynthesis in the Archaeplastida com-mon ancestor was foundational for the evolution of multicel-lular life. It is very likely that the primary endosymbiosis thatexplains plastid origin relied initially on the establishment of ametabolic connection between the host cell and captured cyano-bacterium. We posit that these connections were derived primarilyfrom existing host-derived components. To test this idea, we usedphylogenomic and network analysis to infer the phylogeneticorigin and evolutionary history of 37 validated plastid innermostmembrane (permeome) metabolite transporters from the modelplant Arabidopsis thaliana. Our results show that 57% of thesetransporter genes are of eukaryotic origin and that the capturedcyanobacterium made a relatively minor (albeit important) con-tribution to the process. We also tested the hypothesis that thebacterium-derived hexose-phosphate transporter UhpC mighthave been the primordial sugar transporter in the Archaeplastidaancestor. Bioinformatic and protein localization studies demon-strate that this protein in the extremophilic red algae Galdieriasulphuraria and Cyanidioschyzon merolae are plastid targeted.Given this protein is also localized in plastids in the glaucophytealga Cyanophora paradoxa, we suggest it played a crucial role inearly plastid endosymbiosis by connecting the endosymbiont andhost carbon storage networks. In summary, our work significantlyadvances understanding of plastid integration and favors a host-centric view of endosymbiosis. Under this view, nuclear genes ofeither eukaryotic or bacterial (noncyanobacterial) origin providedkey elements of the toolkit needed for establishing metabolic con-nections in the primordial Archaeplastida lineage.

Arabidopsis thaliana | endosymbiosis | evolution | network analysis |symbiont integration

The origin and establishment of the photosynthetic organelle,the plastid, is heralded as one of the most important bi-

ological innovations on our planet (1, 2). This primary endosym-biosis occurred more than a billion years ago and resulted fromthe engulfment and enslavement of a once free-living cyanobac-terium by a phagotrophic protist (3). Primary plastid captureputatively occurred a single time in the common ancestor of theeukaryotic supergroup Archaeplastida (also known as Plantae)that comprises the green algae and land plants (Viridiplantae),red algae, and glaucophyte algae (4–6). Once established in theselineages, the plastid spread to other lineages such as diatoms,haptophytes, most dinoflagellates, and euglenids, through red orgreen algal secondary endosymbiosis, and in some dinoflagellates,through tertiary endosymbiosis of a secondary endosymbiont-containing alga (7, 8). The exceptional rarity of primary plastidendosymbiosis is supported by there being only one other knowncase of a cyanobacterium-derived photosynthetic organelle (9).This “chromatophore” is found in a single lineage of photosyn-thetic filose amoebae that includes Paulinella chromatophora andits sister taxa (10–14). This independent primary endosymbiosislikely occurred ∼60 Mya, and the plastid donor was a member ofthe α-cyanobacterium clade (15, 16).

Given the fundamental role of algae and plants as primaryproducers in aquatic and terrestrial habitats (17, 18), much at-tention has focused on elucidating the rules that underlie pri-mary plastid origin in Archaeplastida and, more recently, inPaulinella. We have previously made the argument that a key,and likely fundamental, step in endosymbiont integration (i.e.,enslavement) was linking the metabolism of the host and endo-symbiont, thereby allowing regulatory pathways to evolve thatwould maximize connectivity of the partners, and as a result, hostfitness (19–21). The major players in this process are trans-porters located in the innermost envelope membrane of plastids(the plastid envelope permeome) that are responsible for thecontrolled movement of metabolites to and from the endosym-biont (e.g., energy as photosynthetically fixed carbon; the pre-sumed raison d’être for plastid origin). Our previous work showedthat members of the nucleotide sugar transporter family [NST;within the drug/metabolite superfamily (DMS)] gave rise throughgene duplication and divergence to a variety of plastidic sugartransporters in red algae and Viridiplantae (Fig. S1) (19, 22,23). These genes encode the plastidic phosphate translocators(pPTs) that facilitate the strict counter exchange of a host-derivedinorganic orthophosphate (Pi) for an endosymbiont-derived phos-phorylated C3, C5, or C6 carbon compound (e.g., triose phosphate,xylulose-5-phosphate, glucose-6-phosphate). Along with the sharedancestry of the plastid protein import system (6, 24), this in-novation provides one of the strongest pieces of evidencethat two major members of the Archaeplastida (red algae andViridiplantae) are monophyletic. The tree also shows that membersof the “chromalveolates” (e.g., stramenopiles, apicomplexans,cryptophytes) gained their pPT homologs through red algalendosymbiosis. The retention of hexose phosphate transport asthe primary carbon export mechanism in the third arm of theArchaeplastida, the Glaucophyta (6), provides another in-triguing twist in the story of primary endosymbiosis and will bediscussed in detail below. This transporter (UhpC) originatedthrough horizontal gene transfer (HGT) from a bacterialsource. The work on pPTs inspired us to look in more detailinto the evolutionary history and functional diversification

This paper results from the Arthur M. Sackler Colloquium of the National Academy ofSciences, “Symbioses Becoming Permanent: The Origins and Evolutionary Trajectories ofOrganelles,” held October 15–17, 2014, at the Arnold and Mabel Beckman Center of theNational Academies of Sciences and Engineering in Irvine, CA. The complete program andvideo recordings of most presentations are available on the NAS website at www.nasonline.org/Symbioses.

Author contributions: A.P.M.W. and D.B. designed research; S.K., F.F., D.C.P., and D.B.performed research; S.K. and F.F. contributed new reagents/analytic tools; S.K., D.C.P.,and D.B. analyzed data; and F.F., A.P.M.W., and D.B. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. P.J.K. is a guest editor invited by the EditorialBoard.1S.K. and F.F. contributed equally to this work.2To whom correspondence may be addressed. Email: andreas.weber@uni-duesseldorf.deor debash.bhattacharya@gmail.com.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1421375112/-/DCSupplemental.

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of other plastid-targeted transporters and here we present ananalysis of these proteins in Archaeplastida. Our approachwas to use phylogenomic and protein similarity networkanalysis of the validated plastidic transporters from Arabi-dopsis thaliana to deduce their evolutionary histories and or-igins (25). We also studied the phylogeny and cellularlocalization of UhpC proteins in red algae to gain insights intowhat may have been the ancestral pathway of sugar transportin Archaeplastida. These data, combined with recent evidenceof apparent translocon-independent protein import to thephotosynthetic organelle in Paulinella (26), provide a novelperspective on endosymbiont integration. Based on these data,we suggest that metabolic connectivity, whereby recruitment ofexisting host-derived transporters to the plastid innermost mem-brane, was likely an early and fundamental step in unlocking themetabolic potential of the captured cyanobacterium.

Results and DiscussionPhylogenomic Analysis of Arabidopsis Plastidic Transporters. Phylo-genomic analysis of the 34 Arabidopsis thaliana plastid envelopetransporters listed in ref. 25 and 3 others that were more recentlydescribed in this species [nitrite transporters At5g62720 (AtNITR2;1)and At3g47980 (AtNITR2;2), and the glycolate/glycerate trans-porter At1g32080 (PLGG1)] (27, 28) were used as queries againsta comprehensive local genome database (for details, see Methodsand Table S1). The resulting alignments and trees, using eitherthe full alignment length or a gap-trimmed version (available fordownload at cyanophora.rutgers.edu/transporters/) were inspectedto determine the phylogenetic origins of the plant transporterfamilies. Most of these Viridiplantae proteins were found to beeither nested with strong bootstrap support (generally >90%;Table S1) within a variety of eukaryotic lineages or to be asso-ciated with prokaryotes. A total of 57% of Arabidopsis plastidictransporters were of host (ancient eukaryotic; nine families)origin, only 8% (three families) were of cyanobacterial (putativeendosymbiont) origin, 24% were derived from noncyanobacterialprokaryotes (presumably many via HGT) with four families pu-tatively derived from Chlamydiae [i.e., PHT2.1, (NTT1, NTT2),(DiT1, DiT2.1, DiT2.2), HMA1], and a small number were eitherplant-specific or of uncertain affiliation (Fig. 1). The divergentorigin of some transporter families is exemplified by the phy-logeny of plastidic phosphate transporters (PHTs) shown inFig. S2.These results (and previous work) support the scenario that

the current, and presumably the primordial, contribution toplastid metabolite transport was dominated by the retargeting of

existing host-derived proteins to the plastid envelope permeomerather than by the wholesale repurposing of endosymbiont genes(22, 25, 29). The genes encoding these ancient transporterspresumably underwent duplication(s) with one or more copiestaking on plastid-specific functions (Figs. S1 and S2). This host-centric perspective has also been taken to suggest that the eu-karyote rather than the endosymbiont was the major contributorto protein sorting components with the endosymbiont outermembrane being the initial target for integration (30, 31). Acontrasting view (32) relies on genetic tinkering with endosym-biont genes to derive basic components of mitochondrial trans-locons (31). This lively discussion is far from settled, but it isclear that distinguishing between these hypotheses with regard todifferent endosymbiont traits depends not only on identifying theputative genetic toolkit for endosymbiont integration (with sol-ute transport and protein import being obvious candidates) butequally importantly, on elucidating their phylogenetic history.Whereas explaining the origins of plastid protein translocon com-ponents still remain a challenge (33), here we are able to providestrong evidence for a host-dominated process with regard to plastidmetabolite transport. This hypothesis is buttressed by the fact thatpermanent endosymbionts (e.g., the Paulinella chromatophore ge-nome) are characterized by massive genome decay through outrightloss or endosymbiotic gene transfer (EGT) (12–14). Therefore,innovations relating to the compartment are more likely to origi-nate in the eukaryote gene/function-rich nuclear host genome orvia HGT to the host from foreign sources.

Network Analysis of Arabidopsis Plastidic Transporters. To gain aphylogeny-independent perspective on plastid transporter evo-lution, we generated protein similarity networks with an all vs. allBLASTP analysis of the nonredundant list of database hits to the34 Arabidopsis transporters described in ref. 25 (see Methods fordetails). Using a 70% query coverage cutoff resulted in thesedata forming 16 major (i.e., containing many members) connectedcomponents (gene families) that together include all of thetransporter families, as well as 5 minor components that includehighly diverged family members (e.g., CLT2, PIC1; Fig. 2).Placement of the major components (boxed) into transporter su-perfamilies using the Transporter Classification Database (TCDB;www.tcdb.org/) shows that most plastidic transporters are an-ciently diverged and derive from distinct superfamilies, with sixcomponents (boxed in gray field) comprised solely of eukaryotic orplant-specific sequences (Fig. 2). Nonetheless, several interestingcomponents were identified, including the expected pPT family(e.g., PPT, TPT, XPT) that is derived from a single gene du-plication of an existing NST in the ancestor of red algae andViridiplantae (Fig. S1). Another family of interest containedthe copper transporting P-type ATPases HMA1 and PAA1(34–36). This component included the eukaryote derivedACA1/PEA1 (a calcium ATPase; Table S1) family that sharessome links with the HMA1/PAA1 cluster (Figs. 2 and 3). Totest this latter connection, we imposed a minimum pairwiseprotein identity threshold of 40%. This restriction was appliedtogether with the standard e-value cutoff ≤10−5 and a minimumof 70% query hit coverage to produce a new network. Underthis stringent condition (Fig. S3), the ACA1/PEA1 family be-came an independent component, whereas links remained be-tween many of the copper transporters. Inspection of theprotein alignment suggested that the links to ACA1/PEA1 werelikely explained by the presence of two shared domains thatresulted in the network interaction (Fig. 3A).Labeling this network in two different ways (Fig. 3 A and B)

shows that both of the copper transporters in Arabidopsis and inother eukaryotes are derived from prokaryotic ancestors. Phy-logenetic analysis of the combined alignment resolves gene ori-gins with PAA1 having a cyanobacterial (endosymbiont) provenance,whereas HMA1 has a chlamydial origin (Fig. 3C). Intriguingly,

Host (eukaryote) Cyanobacteria Other prokaryotes Plant specific Unclear

57% 24%

8%

8%

Prokaryotic [8 families]: Permease (Pic1) Copper (PAA1) Folate (FT) Phosphate (PHT2.1) Dicarboxylate (Dit1, 2.1, 2.2) Copper (HMA1) Nucleotide (NTT1, 2) Nitrite (AtNITR2;1, 2;2)

Host (eukaryote) derived [9 families]: Sugar/phosphate (TPT, PPTs, GPTs, XPT) Phosphate (PHT4.2-4.5) MCF (SAMT/C, BT1, NDT1) Calcium ATPase (ACA1/PEA1) Stress response (CLT1-3) Glycolate/glycerate (PLGG1)Glucose (pGlcT)Magnesium (MGT10) Cationic proton (CHX23)

Plant-specific [2 families]: Nitrate (Nitr1) Folate (FLT1)

Unclear [2 families]: Bile acid (BAT5) Maltose (Mex1)

Total number of genes:

Fig. 1. Results of phylogenomic analysis of A. thaliana plastid-targetedtransporters. The specific contributions and their numbers made by the eu-karyote host (brown text), cyanobacteria (blue text), prokaryotes other thancyanobacteria (gray text), and other sources are shown.

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both of these prokaryotes are implicated in plastid origin withChlamydiae, providing some key genes required for starch syn-thesis and metabolic integration of the endosymbiont (37). Thebiological meaning of this network result is, however, unclear.Copper is an essential micronutrient that is a component ofphotosystems (cofactor of plastocyanin) and Cu/Zn superoxidedismutase that is involved in the dismutation of superoxide tohydrogen peroxide (38). However, due to its high toxicity (e.g., itcan catalyze the production of free radicals), in plant cells,copper is associated with Cu chaperones (39, 40). Recent worksuggests that the HMA1 and PAA1 transporters operate as dis-tinct pathways for copper import into plastids (36), thereby pu-tatively explaining the maintenance of two diverged copies inArchaeplastida.Another intriguing connection uncovered by the network

analysis was between three members of the mitochondrialcarrier superfamily (MCF) that includes the SAMT/C [coun-ter exchange of S-adenosylmethionine (SAMT; cytosol) withS-adenosylhomocysteine (SAHC; plastid)], NDT1 [counter exchangeof NAD+ (cytosol) with AMP or ADP (plastid)], and BT1 (inArabidopsis, unidirectional flow of plastid AMP, ADP, ATP to

the cytosol; Figs. S4 and S5]. These three adenosine-basedtransporters are of eukaryotic origin and have evolved differingtransport activities during Archaeplastida evolution. Phyloge-netic analysis of the combined alignment (Fig. S4C) shows thatall of these plastid (re)targeted transporter families likely tracetheir origin to a single gene that was present in the commonancestor of eukaryotes. This hypothesis is supported by the ab-sence of prokaryotic homologs of this gene family in the tree(i.e., using our search parameters) and the observation that thetransporter subtrees contain a wide array of eukaryotic lineages(e.g., fungi, metazoans, red algae), suggesting ancient prove-nance in the ancestor of these taxa.In summary, the phylogenomic and network analyses point to

a fundamental role for the host cell in the evolution of metabolicconnectivity of the endosymbiont. This development is not onlyin terms of the number of proteins that have been recruited tothe envelope permeome but also in the crucial roles they play,from fixed carbon transport to the delivery of methyl donors(SAMT/C) to the plastid to facilitate prenyllipid biogenesis or toregulate the synthesis of aspartate-derived amino acids (41).

POT/PTR

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FOLT1 MGT10

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2.A.12 ATP:ADP Antiporter (AAA) Family2.A.28 Bile Acid:Na Symporter (BASS) Family2.A.37 Monovalent Cation:Proton Antiporter-2 (CPA2) Family2.A.47 Divalent Anion:Na+ Symporter (DASS) Family2.A.7 Drug/Metabolite Transporter (DMT) Superfamily1.A.35 CorA Metal Ion Transporter (MIT) Family2.A.29 Mitochondrial Carrier (MC) Family2.A.84 Chloroplast Maltose Exporter (MEX) Family2.A.1 Major Facilitator Superfamily (MFS)2.A.20 Inorganic Phosphate Transporter (PiT) Family3.A.3 P-type ATPase (P-ATPase) Superfamily2.A.17 Proton-dependent Oligopeptide Transporter (POT/PTR) Family

Fig. 2. Network analysis of 34 Arabidopsis plastid-targeted transporters. Each connected component is identified with respect to transport function,superfamily classification, and the taxonomic composition of network nodes. Highly diverged family members that form independent componentsunder the cutoff used for pairwise comparison (i.e., 70% coverage) are also shown. Components comprised solely of eukaryotic sequences are shown inthe gray boxes.

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Proteobacteria-Sphingomonas echinoides ATCC 14820 gi393720128Planctomycetes-Pirellula staleyi DSM 6068 gi283778995

Bacteroidetes-Anaerophaga thermohalophila DSM 12881 gi346225162Bacteroidetes-Echinicola vietnamensis DSM 17526 gi431799419Bacteroidetes-Spirosoma linguale DSM 74 gi284005704

10096

100Proteobacteria-Gallionella capsiferriformans ES-2 gi302878165

100

Deinococci-Deinococcus radiodurans R1 gi15807741Proteobacteria-Nitrobacter winogradskyi Nb-255 gi75677312

Deinococci-Deinococcus peraridilitoris DSM 19664 gi429221623Deinococci-Deinococcus geothermalis DSM 11300 gi94972049Deinococci-Deinococcus gobiensis I-0 gi38685447810053Deinococci-Deinococcus maricopensis DSM 21211 gi320333774

Deinococci-Meiothermus silvanus DSM 9946 gi297566049Proteobacteria-Shewanella sp. W3-18-1 gi120597953Proteobacteria-Alteromonas macleodii AltDE1 gi410860838

9510079

Chloroflexi-Anaerolineae-Anaerolinea thermophila UNI-1 gi320160161Chloroflexi-Chloroflexus sp. Y-400-fl gi222524221Chloroflexi-Chloroflexus aurantiacus J-10-fl gi163846446

Chloroflexi-Chloroflexus aggregans DSM 9485 gi219849569100100

100

80Proteobacteria-Pseudomonas sp. GM16 gi399013333

Planctomycetes-Rhodopirellula sallentina SM41 gi470883805Planctomycetes-Rhodopirellula sp. SWK7 gi470886103

Proteobacteria-Syntrophobacter fumaroxidans MPOB gi116751190100100 Chloroflexi-Ktedonobacteria-Ktedonobacter racemifer DSM 44963 gi298241596Bacteroidetes-Prevotella amnii CRIS 21A-A gi307564735Bacteroidetes-Prevotella histicola F0411 gi357043465

Synergistetes-Anaerobaculum hydrogeniformans gi289522690Fusobacteria-Fusobacterium sp. 7 1 gi237745313

10095100

88Euryarchaeota-Haloarcula japonica DSM 6131 gi448688914Euryarchaeota-Halogranum salarium B-1 gi399579023

85100

Firmicutes-Geobacillus sp. Y412MC52 gi319765801Firmicutes-Amphibacillus xylanus NBRC 15112 gi408355524

Proteobacteria-Betaproteobacteria-Kingella denitrificans ATCC 33394 gi325267405Firmicutes-Geobacillus sp. WCH70 gi239826094unclassifiedBacteria-Thermobaculum-Thermobaculum terrenum ATCC BAA-798 gi269926252

100

99100

Firmicutes-Caldalkalibacillus thermarum TA2 A1 gi335038349Firmicutes-Thermincola potens JR gi296131980

99

52 Firmicutes-Anoxybacillus sp. DT3-1 gi470088449

7494

51

Proteobacteria-Legionella pneumophila subsp. pneumophila str Philadelphia 1 gi52841244Chlamydiae-Chlamydia muridarum MopnTet14 gi301336271

Chlamydiae-Chlamydia psittaci NJ1 gi406593848Chlamydiae-Chlamydophila psittaci 01DC11 gi384451969Chlamydiae-Chlamydia psittaci M56 gi407459716Chlamydiae-Chlamydophila abortus LLG gi424825512

99100

Chlamydiae-Simkania negevensis Z gi338732433Chlamydiae-Parachlamydia acanthamoebae str. Hall's coccus gi282891102

Chlamydiae-Waddlia chondrophila WSU 86-1044 gi297621848100

100

99Rhodophyta-Chondrus crispus T00006183001Rhodophyta-Galdieria sulphuraria A stig 29-Gs35650 1

Chlorophyta Picocystis salinarum-MMETSP0807 0183833482Viridiplantae-Oryza sativa Japonica Group gi115469636Viridiplantae-Brachypodium distachyon gi357117442

Viridiplantae-Solanum lycopersicum gi460372126Viridiplantae-Vitis vinifera gi225438839

Viridiplantae-Glycine max gi356565533Viridiplantae-Cucumis sativus gi449468396

Viridiplantae-Fragaria vesca subsp vesca gi470103122Viridiplantae-Ricinus communis gi255567899Viridiplantae-Populus trichocarpa gi224094264

Viridiplantae-Arabidopsis thaliana gi15235511Viridiplantae-Arabidopsis lyrata jgi49089310061

62

9391

100

66100

10098

76

98100

100

Cyanobacteria-Moorea producens 3L gi332710730Cyanobacteria-Cyanothece sp. CCY0110 gi126658952Cyanobacteria-Cyanothece sp. ATCC 51472 gi354552434

Cyanobacteria-Crocosphaera watsonii WH 8501 gi6792267898Cyanobacteria-Crinalium epipsammum PCC 9333 gi428305746

100

Cyanobacteria-Nodularia spumigena CCY9414 gi119509118Cyanobacteria-'Nostoc azollae' 0708 gi298490874Cyanobacteria-Nostoc sp. PCC 7120 gi17231274Cyanobacteria-Anabaena variabilis ATCC 29413 gi75907770100Cyanobacteria-Lyngbya sp. PCC 8106 gi119486994

Cyanobacteria-Arthrospira platensis NIES-39 gi479127846Cyanobacteria-Trichodesmium erythraeum IMS101 gi113475254

60100

68

53

Chlorophyta Ostreococcus mediterraneus-MMETSP0938 0179713786Viridiplantae-Physcomitrella patens subsp. patens gi168035237

Viridiplantae-Brachypodium distachyon gi357148204Viridiplantae-Sorghum bicolor gi242082423

Viridiplantae-Glycine max jgiGlyma08g07710Viridiplantae-Fragaria vesca subsp vesca gi470108222

Viridiplantae-Arabidopsis thaliana gi42573157Viridiplantae-Arabidopsis lyrata jgi857226Viridiplantae-Cucumis sativus gi449438779100

Viridiplantae-Populus trichocarpa gi224073351Viridiplantae-Ricinus communis gi255581361

Viridiplantae-Vitis vinifera gi225448275Viridiplantae-Solanum lycopersicum gi460369361

100

51

10054

Rhizaria-Paulinella chromatophora gi194476775

100

99

90

Euryarchaeota-Pyrococcus sp. ST04 gi389852347Euryarchaeota-Thermococcus litoralis DSM 5473 gi375082994

Planctomycetes-planctomycete KSU-1 gi386811127100

Firmicutes-Thermacetogenium phaeum DSM 12270 gi410668389Chloroflexi-Dehalogenimonas lykanthroporepellens BL-DC-9 gi300088297

Euryarchaeota-Methanohalobium evestigatum Z-7303 gi298674971Euryarchaeota-Methanococcoides burtonii DSM 6242 gi91772641

Euryarchaeota-Methanohalophilus mahii DSM 5219 gi294495676

77100

87100

98

53

100

0.2 substitutions/site

otherprokaryotes

otherprokaryotes

y100Euryarchaeota

HM

A1 clade

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ACA1/PEA1: calcium ATPase(At1g27770)

HMA1: heavy metal ATPase(At4g37270)

PAA1: copper P-type ATPase(At4g33520)

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Other prokaryotes

ChlamydiaeCyanobacteria

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E1-E2_ATPase (pfam00122) HAD_like, haloacid dehalogenase-like (cd04127)

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Arabidopsis query“Chromalveolates”Excavates

Prokaryotes

RhodophytesUnikontsViridiplants

Fig. 3. Network and phylogenetic analysis of the plastid targeted HMA1-PAA1 copper transporters. (A) The network that includes the ACA1/PEA1 calciumATPase of apparent eukaryotic origin that shares a weak connection to HMA1/PAA1 transporters based on domain sharing (Fig. S3). The nodes in this networkare labeled according to taxonomic origin within prokaryotes and eukaryotes. (B) The same network was relabeled with nodes indicating distribution indifferent eukaryotic phyla. This image highlights the independent prokaryotic origins of the HMA/PAA1 family in eukaryotes. (C) RaxML tree (WAG + Γ modelof evolution) of the HMA1/PAA1 data with redundant sequences removed. The results of 100 bootstrap replicates are shown at the branches. Cyanobacteria are inblue text, Viridiplantae are in green text, red algae are in red text, Chlamydiae are in magenta text, and all other taxa are in black text. The query transportersequences from Arabidopsis are shown in boldface black text within each Viridiplantae clade.

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Evolution of the UhpC family. A surprising finding of the analysisof the genome of the glaucophyte Cyanophora paradoxa is thatin contrast to red algae and Viridiplantae, it does not encodeNST-derived plastid-targeted sugar-phosphate transporters (Fig.S1) (6). Six nonplastidial, likely endomembrane-targeted, NSTproteins are present in the Cyanophora genome, suggestingthat the glaucophytes had split from the Archaeplastida beforethe gene duplications and acquisition of plastid-targeting signalsthat propelled pPT evolution in red algae and Viridiplantae. Acloser inspection of Cyanophora gene models, however, turnedup two novel candidates for plastid sugar-phosphate transport inthis species. These genes encode homologs of bacterial mem-brane bound UhpC-type hexose-phosphate sensors, which inbacteria are part of an operon that is responsible for the uptakeof glucose-6-phosphate (G-6-P) (42). In contrast to their bacte-rial ancestors, both Cyanophora UhpC homologs feature anN-terminal extension that displays characteristic features ofglaucophyte plastid-targeting sequences (6). Analysis of the plastidinner envelope proteome of Cyanophora confirmed the presenceof the two UhpCs in the envelope fraction (43). This finding wasfurther corroborated by transient expression of fluorescent-tagged CyanophoraUhpCs in tobacco cells, where they localized tothe periphery of the plastids (43). Whereas NST-derived plastidsugar-phosphate transporters are restricted to red algae andViridiplantae, it now becomes clear that UhpC is widespread inalgal members of the Archaeplastida (Fig. 4A; but lost in plants).In Cyanophora, UhpC is putatively responsible for the counterexchange of orthophosphate for G-6-P, thereby likely constitutingthe ancestral path of carbon export from plastids in the Archae-plastida (43).

Here we extended this analysis by generating in silico targetingpredictions for the other Archaeplastida UhpCs to determinewhether any of these might also be plastid destined. Our hy-pothesis was that if red and/or green algal UhpCs are also plastidtargeted, then the common ancestor of Archaeplastida was likelyto have relied on this bacterium-derived protein as the primor-dial transporter of a C6 carbon compound that can be fed di-rectly into cytosolic glycogen biosynthesis after being convertedinto UDP-glucose (i.e., the ancestral location of starch synthesisin Archaeplastida before relocation to the Viridiplantae chlo-roplast). This HGT event could have occurred before (or per-haps coincident) with the retargeting of members of the NSTfamily and before origin of the pPT genes (43). Use of theprogram ChloroP 1.1 (44) suggested that many complete UhpCsequences in red and green algae are likely to be plastid targeted(Fig. 4A). We tested this hypothesis for UhpCs in the red algaeGaldieria sulphuraria (45) and Cyanidioschyzon merolae (46). Tothis end, we expressed fluorescent-tagged fusion proteins fromthese algae in Nicotiana benthamiana and observed their locali-zation by fluorescence microscopy. The full-length proteins ofG. sulphuraria (gene Gasu_03960) and of C. merolae (geneCYME_CMQ264C), as well as the first 112 and 200 amino acidsof each protein corresponding to their putative transit peptides,were cloned in front of the N terminus of the yellow fluorescentprotein (YFP). Observation of the protoplast isolated fromleaves infiltrated with the constructs carrying the predictedtransit peptides fused to the YFP shows that the fluorescentsignal colocalizes with the chlorophyll autofluorescence (Fig. 4B,Plates 1 and 3). This result indicates that the red algal predictedtransit peptides are indeed sufficient to target the YFP to the

Aggregatibacter actinomycetemcomitans D11S 1 261866887Legionella drancourtii LLAP12 gi374261936Legionella longbeachae D-4968 gi270157197Fluoribacter dumoffii Tex-KL gi388455407

Legionella pneumophila str. Corby gi148359980Legionella pneumophila 2300/99 Alcoy gi296108199GammaLegionella pneumophila str. Lens gi54295389

Chlamydia psittaci NJ1 gi406593001Chlamydia psittaci 01DC12 gi410858063Chlamydophila psittaci 01DC11 gi384451181Chlamydia psittaci CP3 gi406591890Chlamydophila psittaci Cal10 gi329942393Chlamydophila abortus S26/3 gi62184720Chlamydophila caviae GPIC gi29839842Chlamydophila felis Fe/C-56 gi89898737

Chlamydophila pneumoniae AR39 gi16752375Waddlia chondrophila WSU 86-1044 gi297621742

Parachlamydia acanthamoebae UV-7 gi338176229Candidatus Protochlamydia amoebophila UWE25 gi46446021

Dixoniella grisea contig 7817 3 (incomplete)Porphyridium purpureum contig2054.9 (-) 0.44

Chondrus crispus T00007675001 (-) 0.49Calliarthron tuberculosum ORF 2866 133 (incomplete)

Hildenbrandia pulchella contig 40013 4 (+) 0.57Boldia erythrosiphon contig 6072 2 (incomplete)

Cyanidioschyzon merolae CMQ264C (+) 0.52Galdieria sulphuraria stig 24-Gs31310.1 (+) 0.51

Gloeochaete wittrockiana-MMETSP1089 0184335576 (+) 0.55Cyanophora paradoxa contig54038 (+) 0.55

Cyanophora paradoxa contig37408 (incomplete)Ostreococcus lucimarinus CCE9901 gi145343717 (+) 0.56Ostreococcus tauri gi308800194 (+) 0.52)

Ostreococcus RCC809 jgi90209 (-) 0.46Micromonas pusilla CCMP1545 gi303273388 (+) 0.51Micromonas sp. RCC299 gi255070251 (+) 0.55

Asterochloris sp. jgi24630 (-) 0.44Chlorella vulgaris jgi81415 (+) 0.55)Coccomyxa subellipsoida jgi52341 (+) 0.55

Chlorella NC64A jgi141550 (-) 0.46Volvox carteri f. nagariensis gi302840937 (+) 0.57)Chlamydomonas reinhardtii gi159482274 (+) 0.53Chlamydomonas reinhardtii 159482272 (+) 0.52

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Fig. 4. Analysis of UhpC proteins in Archaeplastida. (A) RaxML tree (WAG + Γ model of evolution) with the results of 100 bootstrap replicates shown at thebranches. Glaucophyta are in blue text, Viridiplantae are in green text, red algae are in red text, Chlamydiae are in magenta text, and all other prokaryotesare in black text. The four proteins for which localization data exist to indicate plastid inner envelope membrane targeting are shown in large boldface blacktext. The results of bioinformatic targeting predictions using ChloroP (cTP score; marked with [+] when predicted to be plastid targeted or [−] when not) areshown for each Achaeplastida UhpC, when complete proteins are available. (B) Expression of YFP-fusion constructs in Nicotiana benthamiana protoplasts.Confocal microscope pictures on isolated Nicotiana protoplasts expressing the YFP-fusion constructs driven by the ubiquitin 10 promoter. (Plate 1) Expressionpattern of the predicted transit peptide of the UhpC homolog Gasu_03960 from Galdieria fused to YFP. Shown are YFP fluorescence (YFP) in green, chlo-rophyll autofluorescence (chlorophyll) in red and an overlay of the two pictures (merge). (Plate 2) Expression pattern of the full-length Gasu_03960 fused toYFP. (Plate 3) Expression pattern of the predicted transit peptide of the UhpC homolog CYME_CMQ264C from Cyanidioschyzon fused to YFP. (Plate 4) Ex-pression pattern of the full-length CYME_CMQ264C fused to YFP.

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chloroplast. The fluorescent signal of the full-length proteinsfused to the YFP for Gasu_03960 and CYME_CMQ264C sur-rounds the plastids, a pattern typical for proteins located to theinner envelope membrane (Fig. 4B, Plates 2 and 4). This obser-vation is consistent with localization to the plastid inner envelopemembrane as previously described (43).These results can also interpreted from the perspective of the

ménage à trois hypothesis of symbiont integration (37, 47). Thishypothesis posits that plastid endosymbiosis putatively relied onthree partners: the host, the cyanobacterium, and a chlamydialsymbiont (hence, ménage à trois). Under this view, the UhpCtransporter (of bacterial origin; see below) could have beentransferred first to the cyanobacterium genome to allow the flowof fixed carbon to the inclusion vesicle (of chlamydial origin) thathoused both prokaryotes. The G-6-P that was secreted fromthe cyanobacterium, after conversion to G-1-P by phosphoglu-comutase, acted as substrate for the synthesis of either ADP- orUDP-glucose and subsequently glycogen in the vesicular space.Glycogen was also synthesized in the host cytosol with the use ofchlamydial-derived enzymes (GlgA, GlgC) secreted by their type3 secretion system (TTS). Regardless of the manner in which theancient ménage à trois interaction operated, over time the UhpC(and other endosymbiont) genes were transferred to the hostgenome and retargeted to the plastid inner membrane to allowuptake of G-6-P for glycogen synthesis. Therefore, genes pro-vided to the host by chlamydial cells, other bacteria, and thecyanobacterium forged the successful endosymbiosis, and thechlamydial symbiont (and their inclusion vesicles) was lost (47).The phylogenetic evidence for a chlamydial origin of UhpC inArchaeplastida is ambiguous because of the presence of manyProteobacteria in the tree that also forms a sister to the eukaryoteclade. Regardless of the rooting scheme that is used, the directionof HGT in this case is, however, clear. Among eukaryotes, onlyArchaeplastida contain a bacterium-derived UhpC gene; therefore,the transfer was most likely from a prokaryote to a eukaryote cell.

The Metabolic Connectivity Model. Given the strong evidence foreukaryotic or HGT-derived metabolite transporters in Archae-plastida, both of which are nuclear encoded, how might theseproteins be directly implicated in early events in endosymbiontintegration? For clues to answering this question, we can look atthe recent plastid endosymbiosis in Paulinella. Work done byNowack and Grossman (26) has shown that this amoeba appar-ently relies on the secretory system, via passage through the Golgi,to deliver essential photosystem proteins (PsaE, PsaK1, PsaK2) tothe chromatophore. These proteins have been lost from the en-dosymbiont genome and are now encoded in the nucleus, synthe-sized in the cytoplasm, and trafficked into the chromatophore. Theabsence of a detectable terminal or internal targeting signal inthese proteins suggests that the secretory system is capable of de-livering mature proteins to an endosymbiont. We take these resultsas support for the idea that the initial metabolite transporters thatserviced the Archaeplastida (as described above) plastid could alsohave been delivered by the secretory system to the inner envelopemembrane to allow exchange of metabolites with the host cell (20).Our work shows that the UhpC hexose-phosphate transporterpresent in the Archaeplastida ancestor could have been an earlyplayer in fixed carbon transport, as envisioned in the ménage àtrois hypothesis, or under a scenario that does not rely on an in-clusion vesicle as the initial platform for glycogen synthesis.The hypothesis of a host-dominated process for metabolite

transport in the nascent plastid endosymbiont as postulated herewould be greatly strengthened by evidence that crucial functionswere provided by the eukaryote. Another example of such afunction is the essential detoxification of the oxygenation prod-uct of rubisco, 2-phosphoglycolic acid (2-PG). Cyanobacteriapossess at least three distinct metabolic routes to this end,including the bacterial-type glycolate pathway and a plant-like

C2-pathway (48, 49). The capacity to detoxify 2-PG inside theplastid must have been lost early during endosymbiosis becauseall members of the Archaeplastida rely on the export of glycolicacid from plastids to the cytoplasm for conversion to glycerate inmitochondria and/or peroxisomes. This process requires the exportof glycolic acid from, and the import of glycerate into, the plastid,which is achieved by the recently discovered glycolate/glyceratetransporter PLGG1, a eukaryote-derived protein (Table S1).Some cyanobacteria are able to excrete glycolate at significantlevels, with 9% of net fixed carbon lost in air for heterocystousspecies and up to 60% lost in some strains under high oxygenconditions (50). This mechanism, however, represents a significantloss of carbon and of Calvin–Benson cycle intermediate products.Hence, recovery of part of this carbon by the eukaryotic photo-respiration pathway would offer a significant selective advantage.The advantage of PLGG1 over other (unknown) cyanobacterialglycolate exporters is that it works in a counter exchange mode,which leads to the salvage of three of four carbon units lostvia oxygenation/photorespiration.In summary, the metabolic connectivity model we present here

relies on functional diversification of organelle functions.Through targeting of host-derived transporter proteins to theorganelle membrane, two previously distinct metabolic networksbecome increasingly interwoven and interdependent. This idea issupported by previous work that shows the growth of metabolicnetworks in prokaryotes is frequently achieved by the addition oftransporter activities at the network periphery. That is, given acore set of metabolic functions, the network can be expandedby gaining access to additional substrates via the acquisition ofgenes encoding transporter proteins. These elaborations aredriven by adaptation to changing environments (e.g., extracel-lular vs. intracellular) (51). In addition, it is widely recognizedthat free-living bacteria have access to a large pan-domain genepool from which they can acquire novel metabolic and trans-porter genes. Therefore, the cyanobacterial plastid ancestorhad a chimeric genome at the time of endosymbiosis, compo-nents of which were transferred to the eukaryote host by EGT.The vast prokaryotic gene pool also contributed to Archaeplastdiaevolution via independent HGT events. The role of HGT in en-dosymbiont integration is of particular relevance because oncecells are internalized they rarely gain genes, but rather undergosignificant genome reduction (12–14). Hence, it is not surprisingthat, as we show here, growth in functional diversity of plastidsis achieved by the retargeting of host encoded proteins or theacquisition by the host of foreign genes whose products can beretargeted to the organelle.

MethodsNetwork and Phylogenetic Analysis. The 34 different validated A. thalianaplastid inner membrane transporter sequences listed in Fischer (25) and thethree new transporters (27, 28) were used in our analyses. Each protein se-quence from Arabidopsis was used to query via BLASTP an in-house data-base composed of The National Center for Biotechnology InformationReference sequence database (RefSeq) v59, the 672 proteomes publiclyavailable from the Moore Marine Eukaryote Transcriptome SequencingProject (MMETSP; camera.calit2.net/mmetsp/list.php), with additional eu-karyote proteomes obtained from NCBI dbEST, (www.ncbi.nlm.nih.gov/dbEST/), and TBestDB. The BLASTP output for each transporter protein se-quence was analyzed so that a maximum of 100 individual hits was retainedwith a limit of ≤12 hits per phylum to avoid oversampling taxon-rich phyla inthe database. Each dataset was aligned using Clustal Omega (52), andphylogenetic trees were constructed with the full alignment using IQ-TREEv.0.9.6 (53) with automatic best-fit model selection and 1500 μLtra-fastbootstrap replicates (Fig. S2). We also used Gblocks (54) to trim each trans-porter alignment to conserved sites using a block size of 2 (−b4 = 2) andallowing all gaps (−b5 = a). These reduced alignments were used as input toIQ-TREE as described above to generate phylogenies and bootstrap supportvalues. For some transporters (e.g., pPTs, PTs), the individual protein align-ments were combined, and a RAxML (55) tree was inferred using the WAG + Γmodel of sequence evolution and 100 bootstrap replicates.

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For the network analysis, the complete set of transporters that wasreturned from the phylogenomic analysis was analyzed to remove all re-dundant sequences. This data set of 2,330 unique sequences was used in an allvs. all BLASTP analysis with a minimum e-value cutoff ≤10−5; i.e., a thresholdthat selected hits based on both bit score (the similarity measure) andlength. This restriction returned short hits to regions with high similaritywhile allowing hits to longer regions with lower similarity. The lowest sim-ilarity value recorded for the hits was 18.15% identity. From these results,we applied a cutoff of 70% query coverage (i.e., any hit must cover at least70% of the 2 proteins), resulting in a set of 21 connected components thatencompass 1,727 proteins. The classification of proteins in these connectedcomponents was done using a greedy clustering algorithm based on themodularity (ratio of intra- vs. intergroup links) (56) that detects denselyconnected regions of sequences. New classes of transporters were manuallydefined to regroup transporter families listed in Fischer (25) that have ho-mologs that belong the same community in the network. The taxonomiccontent of the network was analyzed and displayed using in-house R scripts(57) with the igraph package (Figs. 2 and 3).

The motivation to use both phylogenetic and network methods with thesame data reflects the fact that these approaches provide complementary in-sights into transporter evolution. Phylogenetic methods assume that sequencesare inherited via vertical descent and the goal herewas to detect these instances,aswell as subsets of the data that contradict this assumptiondue towell-resolvedcases of endosymbiotic or horizontal gene transfer. In contrast, network analysisis a straightforward clustering approach with input sequences, in which noassumption is made regarding themechanism of evolution. Sequence clusteringis based on several criteria (e.g., hit coverage, similarity score) using a robustalgorithm that detects densely connected sets of proteins. Importantly, the

clustering allows us to detect sequences that are related through intermediatesthat would not be present in the same tree due to high pairwise sequencedivergence between the most distantly related protein families. This propertypotentially allows us to merge regions of different trees into single clusters toidentify ancient connections between transporters. We have previously appliedthis principle to detect ancient signals of monophyly between redox enzymes,allowing inferences into their origin and evolution in divergent prokaryoticlineages (58–60). Here the networks were used in a more limited fashion topotentially identify transporter family relationships that may not be revealedusing phylogenetic methods.

Localization of Transporter Proteins in Transiently Transformed Tobacco Cells.The coding sequences of the UhpC homologs from Galdieria sulphuraria(Gasu_03960) and Cyanidioschyzon merolae (CYME_CMQ264C) were ampli-fied by PCR and cloned by Gibson cloning into the plant expression vectorpUBC-YFP for C-terminal YFP-fusion under the control of the ubiquitin10 promoter (61, 62). Agrobacterium tumefaciens transformation (strainGV3101), tobacco leaf infiltration, and protoplast isolation were done aspreviously described (43). Isolated protoplasts were observed 2–4 d afterinfiltration with an inverted Zeiss LSM 780 confocal laser-scanning mi-croscope. YFP and chlorophyll were excited using the 488-nm laser line ofan Argon laser, and the emission was collected at 517–578 and 590–690 nm,respectively.

ACKNOWLEDGMENTS. This research was funded by National ScienceFoundation Grants 0936884 and 1317114 (to D.B.). A.P.M.W. appreciatessupport from the Deutsche Forschungsgemeinschaft (Grants EXC 1028 andWE 2231/8-2).

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