Are there Rab GTPases in Archaea?

24 Pages·2016·3.55 MB·English

Are There Rab GTPases in Archaea?
Jaroslaw Surkont*,1 and Jose B. Pereira-Leal1
1InstitutoGulbenkiandeCiencia,Oeiras,Portugal
*Correspondingauthor:E-mail:[email protected].
Associateeditor:JamesMcInerney
Abstract
A complex endomembrane system is one of the hallmarks of Eukaryotes. Vesicle trafficking between compartments is
controlledbyadiverseproteinrepertoire,includingRabGTPases.ThesesmallGTP-bindingproteinscontributeidentity
andspecificitytothesystem,andbyworkingasmolecularswitches,triggermultipleeventsinvesiclebudding,transport,
and fusion. A diverse collection of Rab GTPases already existed in the ancestral Eukaryote, yet, it is unclear how such
D
elaboraterepertoireemerged.Anovelarchaealphylum,theLokiarchaeota,revealedthatseveraleukaryotic-likeprotein ow
n
systems, including small GTPases, are present in Archaea. Here, we test the hypothesis that the Rab family of small lo
a
GTPases predates the origin of Eukaryotes. Our bioinformatic pipeline detected multiple putative Rab-like proteins in d
e
d
severalarchaealspecies.Ouranalysesrevealedthepresenceandstrictconservationofsequencefeaturesthatdistinguish fro
eukaryotic Rabs from other small GTPases (Rab family motifs), mapping to the same regions in the structure as in m
eukaryotic Rabs. These mediate Rab-specific interactions with regulators of the REP/GDI (Rab Escort Protein/GDP http
dinissAorccihataieoan, Iinnhvoiblviteodr)infamisoilpy.reSneynlsimtiveetasbtroulicstmu.reO-buarseadnamlyestihsosduspfpuorrtthserarsecveenaalerdiotwheheerxeistReanbcsedoifffReErePn/tGiaDtIe-dlikientgoenaens s://ac
a
independentfamilyinArchaea,interactingwithproteinsinvolvedinmembranebiogenesis.Theseresultsfurthersupport de
m
the archaeal nature of the eukaryotic ancestor and provide a new insight into the intermediate stages and the evolu- ic
tionarypathtowardthecomplexmembrane-associatedsignalingcircuitsthatcharacterizetheRassuperfamilyofsmall .ou
p
GTPases,andspecificallyRabproteins. .c
o
m
Keywords:eukaryogenesis,Lokiarchaeum,endomembranetraffickingsystem,Ras,LECA,proteinprenylation. /m
b
e
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rtic
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Introduction -ab
grouptoallArchaea(GuyandEttema2011;Kellyetal.2011; s
Amajorquestioninevolutionarybiologyistheoriginofthe Williams et al. 2012, 2013; Williams and Embley 2014; trac
Eukaryoticcellplan,whichischaracterizedbyamultitudeof Raymannetal.2015).Thisscenariosuggeststhatthesearch t/33
intracellularorganelles,includingtheenergyproducingendo- for transitional states should be carried out within the ar- /7/1
8
symbioticorganelles,complexendomembranetraffickingsys- chaealdomain,andspecificallytheTACKsuperphylum. 3
3
tem,andanucleuscontainingalargegenomethatencodes Arecentmetagenomicsurveyofadeepoceansediment A/2
5
thousands of genes. The protein repertoires associated with sample from the Arctic Mid-Ocean Ridge revealed the r79
t2
these organelles have been found in most Eukaryotes, sug- existenceofanewarchaealphylumwithintheTACKsuper- i55
gCeosmtinmgotnhatAtnhceeystwoerre(LaElrCeAad)y(per.ge.s,enFtieilndthanedLasDtaEcukksar2y0o0t9ic; prehpyolurtmed,thtehaLtoksiaervcehraaelobtaui(lSdpinagngbeltocakl.s20c1h5a)r.aTchteeraisutitchoorsf cle by gu
e
Schlacht et al. 2014). Likein other areas of the evolutionary Eukaryotes are present in this taxon, suggesting that st o
biology, the search for intermediate, transitional forms has Lokiarchaeota and Eukaryotes share a common ancestor n 2
attractedtheattentionofmany,andeukaryotic-likecellular andthatLokiarchaeotaisamoderndescendantofthatan- 4 N
featuresorgenerepertoireshavebeenidentifiedindifferent cestor. Small GTPase gene families are highly expanded in ov
e
prokaryotes, for example, having been termed as the “dis- Lokiarchaeota compared with other Archaea, including m
b
persedeukaryome”inArchaea(KooninandYutin2014). many small GTPases from the RAS superfamily; they form er 2
InferringancienteventssuchastheoriginofEukaryotesor severaldistinctclusters,yettheirrelationshiptotheeukary- 0
1
theoriginoftheirspecificmoleculartraitsisaverychallenging oticGTPasesremainsunclear. 8
taskgiventhetimescale,datascarcity,andinsufficientmeth- TheeukaryoticRASsuperfamilycontainsfivemajorfam-
ods.Despitethis,mountingevidencesuggeststhattheances- ilies Arf, Ras, Rho, Ran, and Rab that are involved in the
tralhostcellthataccommodatedtheendosymbioticbacteria, intracellular signaling and share the common G domain
whichgaverisetomitochondria,was fromthearchaeallin- core(GTPaseactivity),responsiblefortheswitchingmecha-
eage (Lake et al. 1984; Cox et al. 2008, reviewed in L(cid:2)opez- nismbetweentheGTP-boundactiveandGDP-boundinac-
Garc(cid:2)ıa and Moreira 2015). This host cell may have in fact tivestate.TheArffamilyisinvolvedinregulationofvesicular
evolved from within Archaea (the TACK superphylum), transport,Rasinresponsetodiverseextracellularstimuli,Rho
rather than result from a much earlier branching as a sister in actin dynamics, and Ran in nucleocytoplasmic transport
(cid:2)TheAuthor2016.PublishedbyOxfordUniversityPressonbehalfoftheSocietyforMolecularBiologyandEvolution.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/4.0/),whichpermitsunrestrictedreuse,distribution,andreproductioninanymedium,providedtheoriginalworkis Open Access
properlycited.
Mol.Biol.Evol.33(7):1833–1842 doi:10.1093/molbev/msw061 AdvanceAccesspublicationMarch31,2016 1833
MBE
SurkontandPereira-Leal . doi:10.1093/molbev/msw061
(reviewed in Wennerberg et al. 2005). Here, we focused on
RabGTPases,criticalregulatorsofvesiculartraffickingsystems
(Fukuda2008;Stenmark2009;Kellyetal.2012;Pfeffer2013),
included in the list of eukaryotic signature proteins, that is,
“proteinsthatarefoundineukaryoticcellsbuthavenosig-
nificant homology to proteins in Archaea and Bacteria”
(Hartman and Fedorov 2002). This family has experienced
extensiveuniversalandtaxon-specificduplicationsassociated
with the emergence of major organelles and organelle spe-
cializationsoftheendomembranesystem;eachRabsubfamily
providesspecificitytoaparticularcomponentofthetraffick-
ingsystemandthisfunctionisgenerallyconservedthrough- D
o
out evolution (Dacks and Field 2007; Dacks et al. 2009; w
n
Brighouseetal.2010;Diekmannet al.2011). Theyformthe lo
a
largest RAS family, with more than 60 Rab homologues in de
d
human (Pereira-Leal and Seabra 2001), and several studies fro
point to the existence of a rich Rab repertoire at the LECA m
h
(Diekmannetal.2011;Eliasetal.2012;Klo¨pperetal.2012); ttp
s
however, they have been so far restricted to the eukaryotic ://a
domain.Here,wetestthehypothesisthatRabGTPasespre- c
a
d
date Eukaryogenesis, by investigating the small GTPase rep- e
m
ertoire in Archaea, and in particular the expanded small ic
.o
GTPasefamilyintherecentlydescribedLokiarchaea. u
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.c
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Results e
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MultipleRab-likeSequencesinArchaea rticle
IntheoriginalmetagenomicstudybySpangetal.(2015)the FIG.1. PhylogeneticprofileoftheRabfamilyinrepresentativespecies -ab
s
assembly of a complete archaeal genome defined a novel ofeukaryotes(magenta),Archaea(red),andbacteria(blue).There- tra
archaeal phylum, the Lokiarchaeota. In this Lokiarchaeum mainingarchaealspeciesthatwereusedintheanalysis,withoutRab- ct/3
genome, more than 90 members of the RAS superfamily likeproteinpredictions,arenotshowninthefigure.Afull(hollow) 3/7
were predicted, yet it is unclear whether these proteins be- square indicates the presence (absence) of at least one predicted /18
eukaryotic Rab protein (black) or archaeal Rab-like protein (gray). 3
longtoanyspecific,previouslydescribedRASfamilyorcon- 3
ThetotalnumberofRabhomologuesisshownnexttothesquare. /2
stitute a novel group. Here, we systematically searched all 5
TACK refers to the superphylum that comprises the 7
9
complete archaeal genomes, including the Lokiarchaeum, Thaumarchaeota, Aigarchaeota, Crenarchaeota, and Korarchaeota 25
for members of the RAS superfamily of small GTPases and phyla.TreetopologyisconsistentwithSpangetal.(2015). 5 b
y
specificallyannotatedRab-likeproteins.WeusedtheRabifier g
u
(Diekmannetal.2011),abioinformatic pipelinethatrunsa (fig. 1) does not reveal any obvious pattern of vertical es
series of consecutive classification steps as follows: 1) deter- inheritance. t on
mining if a protein contains the small GTPase domain, 2) 2
4
whetheritbelongstotheRabfamilyoranothermemberof N
o
theRASsuperfamily,and3)whatisthemostlikelyRabsub- InconclusivePhylogeneticPositioningofArchaeal ve
m
familyassignmentoftheprotein.Wedetectedatotalof3,152 Rab-likeSequences b
e
proteinscontainingthesmallGTPasedomain,ofwhich133 Our bioinformatic analysis confirms the presence of many r 2
0
withintheLokiarachaeumgenome(theremaininganaverage small GTPases in Archaea and identifies multiple Rab-like 18
of 13.663.4 proteins per genome). Of this total, 42 were GTPasesindiversearchaealspecies,yetwithoutanysubfamily
predictedasRab-likeGTPaseswithoutanyspecificsubfamily assignment. To determine the position of archaeal Rab-like
annotation,thatis,noneoftheRab-likeproteinsissufficiently proteins within the superfamily of small GTPases and their
similar to any of the established eukaryotic subfamilies. relationshiptoeukaryoticRabs,weconductedaphylogenetic
Among the 42 Rab-like proteins 37 belong to analysis of archaeal Rab-like proteins together with the eu-
Lokiarchaeum, the remaining five (one copy per species) karyoticRabswhicharelikelypresentintheLECA(Diekmann
were identified in Thermofilum pendens, Thermofilum sp., et al. 2011; Elias et al. 2012), also including representative
Caldiarchaeum subterraneum, Thermoplasmatales archaeon, sequences of other RAS families. We used both Bayesian
andAciduliprofundumsp.Thesespeciesaredistributedacross and Maximum Likelihood approaches for the phylogenetic
Archaea, they belong to one of two major superphyla, inference(seeMaterialsandMethodsfordetails).
Euryarchaeota and TACK. This raises a question about the Aspreviouslyobserved(Dongetal.2007;Rojasetal.2012),
originoftheseRab-likeproteins,astheirphylogeneticprofile treesofsmallGTPaseshaveveryweakstatisticalsupportfor
1834
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FIG.2. PhylogenyofsmallGTPasesfromEukaryaandArchaeausing(a)Bayesianand(b)maximum-likelihoodinference.Representativeeukaryotic ttps
membersofallRASfamilies(Rab,Ran,Rho,Ras,andArf)andputativearchaealRab-likeareincluded.Black(gray)circleindicatesaBayesian ://a
c
posteriorprobabilityvalueabove0.9(0.6)andabootstrapsupportvalueabove90(60)forabranchsplit.Branchlengthsareproportionaltothe a
d
expectednumberofsubstitutionspersite,asindicatedbythescalebar. em
ic
.o
basal branches (Rho vs. Rab vs. Ras, etc.), and Rabs may alignment of the small GTPase domain (Pfam:PF00071) to u
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appearinmultipleindependentbasalbranches(fig.2,supple guidethealignmentprocessandimproveanoverallquality .c
o
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mentary fig. S1, Supplementary Material online). Archaeal of the alignment, the seed sequences were then removed /m
Rab-like sequences are monophyletic with the eukaryotic fromthefinalalignment.Thealignmentsweresubsequently b
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proteins,indicating that they aremoresimilartosequences used to construct profile hidden Markov models (pHMMs) /a
fromEukaryotesthantoothersmallGTPasesfromArchaea andgenerateplurality-ruleconsensussequencesthatdescribe rticle
(supplementaryfig.S2,SupplementaryMaterialonline).They eachfamily. -a
b
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are however not monophyletic with any one specific small Wefirstcalculatedtheoverall,pairwisesimilaritybetween tra
c
GTPasefamily,beingpartofabasalpolytomy(fig.2). thefamilies(supplementarytableS2,SupplementaryMaterial t/3
TogainamoredetailedviewontheRab-likefamilystruc- online) and observed a remarkable similarity of 78% (60% 3/7
ture,weconstructedaphylogenetictreeusingonlyarchaeal identity, local alignment) between eukaryotic Rab and ar- /1
8
3
Rab-like sequences (supplementary fig. S3, Supplementary chaealRab-likeGTPases(71%and55%,respectively,forglobal 3
/2
Materialonline).Althoughthedeepbranchingpatterncould alignment, supplementary tableS3, Supplementary Material 5
7
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not be reliably resolved, we observed that most of the se- online), much higher than between the archaeal Rab-like 2
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quencesclusterwithinseveralhighlysupportedgroups.Short family and any other member of the RAS superfamily. 5 b
y
terminal branches suggest recent duplication of several We subsequently focused on a more specific comparison g
u
Lokiarchaean proteins. Proteins from Thermoplasmatales, between Rab-like and Rab proteins; we compared amino e
s
Aciduliprofundum, and Caldiarchaeum form long branches acid variation along the sequence across Rab paralogues t o
n
indicating very divergent sequences, which do not cluster in Lokiarchaeum and representative species from different 2
4
together with Lokiarchaeum. In contrast, proteins from majoreukaryoticgroups(Homosapiens,Trypanosomabrucei, N
o
both Thermofilum species form a distinct cluster with two andGuillardiatheta).Weobservedsimilarpatternsofvaria- ve
m
otherLokiarchaeansequences. tion for all analyzed species (supplementary fig. S4, b
e
Overall, this analysis suggests that phylogenetic methods Supplementary Material online): regions of both low and r 2
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aloneareinsufficienttodeterminetherelationshipbetween high sequence conservation belong to the corresponding 1
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archaealRab-likeGTPasesandtheeukaryoticmembersofthe positions in the Rab sequences from different species, sug-
RAS superfamily. This, however, raises the question of why gesting that archaeal Rab-like sequences are evolutionarily
thesesequenceswereclassifiedasRab-like. constrainedinthesameregionsastheeukaryoticRabs.
Wenexttestedthehypothesisthatsequenceconservation
Rab-likeProteinsContainTypicalEukaryoticRab betweenarchaealandeukaryoticsequencesisassociatedwith
Motifs theRabFmotifs—sequencemotifsuniquetotheRabfamily
We next analyzed sequence properties of archaeal Rab-like that are important for the interaction with Rab effectors
GTPasesatthefamilyleveltofurtherassesstheirsimilarityto (Pereira-Leal and Seabra 2000). The results of this analysis
other members of the RAS superfamily. We constructed a aresummarizedinfigure3.Allpositionsthatcorrespondto
sequencemodelforeachfamily(Rab,archaealRab-like,Ran, theRabF1andRabF2motifsineukaryoticRabsareconserved
Rho, Ras, Arf). We first built multiple sequence alignments in the archaeal Rab-like sequence. For comparison, in other
using representative sequences for each family and a seed families at most two amino acids are conserved at the
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FIG.3.SequencecomparisonofsmallGTPasefamilies.(a)SequencelogocomparisonofRabFmotifsbetweenRabandRab-likefamilies.(b) 33
AlignmentoftheconsensussequencesgeneratedwithpHMMsoftheeukaryoticRASfamiliesandthearchaealRab-likefamily.RabFmotifsinthe /2
5
Rabfamilyandidenticalresiduesatthecorrespondingpositionsinotherfamiliesarehighlightedinblue.Orangehighlightdenotestheguanine 79
2
nucleotide-bindingpositions.RedindicatespositivelychargedC-terminalaminoacids.YellowindicatestheC-terminalcysteines,whichareoften 5
5
posttranslationallymodified.Uppercaseindicatesresidueswithprobabilitygreaterthan0.5intheHMMprofile.Secondarystructureelementsare b
y
denotedbybars(a-helices)andarrows(b-sheets). g
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correspondingpositions.Intheremainingthreemotifsmost Rab-likeProteinsAreStructurallySimilar t on
2
of the residues are identically conserved between Rab and toEukaryoticRabs 4
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Rab-like sequences, some are similar, for example, positively Givenahighleveloftheprimarysequencesimilaritybetween o
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chargedarginineandlysineinRabF4,aliphaticisoleucineand thearchaealRab-likeproteins andtheireukaryotic counter- em
b
leucineinRabF5,andaromatictyrosineandphenylalaninein parts, we modeled a putative 3D structure of a Rab-like e
RabF5(tyrosineisalsothesecondmostcommonaminoacid GTPase and compared the location of Rab-specific features r 20
1
atthispositioninthearchaealsequences).Fromthesequence at the structural level. We chose a Lokiarchaeum sequence 8
perspective,archaealRab-likeproteinshaveallthehallmarks that contains all five RabF motifs (GenBank:KKK40223), as
ofRabs,includingthemotifsinvolvedinbindingRabregula- predicted by the Rabifier. To ensure a high quality of the
torsandeffectors. model,weselectedfourtemplatesfromdifferentRabsubfa-
ThemajordifferencebetweeneukaryoticRabandarchaeal miliesthatbothhaveahighlevelofsequenceidentitytothe
Rab-likesequencesistheabsenceofC-terminalcysteineres- archaealhomologueandagoodcrystallographicresolutionof
idues,theprenylationsitesoftheeukaryoticRabs,inallofthe the 3D structure: Rab8 (H. sapiens, PDB:4LHW), Rab26
analyzedarchaealsequences.Rab-likesequencestendtohave (H. sapiens, PDB:2G6B), Rab30 (H. sapiens, PDB:2EW1), and
a shorter C-terminal sequence, missing most of what is Ypt1 (Saccharomyces cerevisiae, PDB:1YZN). All template
termed the (flexible) hypervariable region in eukaryotic structures were in the active state, that is, bound to a GTP
Rabs, known to be involved in associations with the molecule. We used Modeller (Sali and Blundell 1993), a ho-
membrane. mologymodelingplatformtopredictaputativestructureof
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FIG.4. StructurecomparisonbetweenyeastYpt1(left,PDB:1YZN)andamodelofanarchaealRab-likeprotein(right).(a)LocationofRabFmotifs em
andguaninenucleotide-bindingmotifsattheproteinsurface,(b)surfacedistributionofhydrophobic(Ala,Gly,Val,Ile,Leu,Phe,Met),and(c) be
chargedresidues(positivelychargedArg,His,LysandnegativelyAsp,Glu). r 2
0
1
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thearchaealprotein(usingallfourtemplatessimultaneously) (fig.4b)andcharged(fig.4c)aminoacidsattheproteinsur-
and subsequently assessed its quality and stability. We ob- face and observed a similar distribution of the residues in
tainedasimilarstructureusingPhyre2(Kelleyetal.2015),an bothstructures.
automaticserverforproteinstructurepredictionandanalysis WeassessedtheputativeGTPaseactivityandthenucleo-
(notshown).Figure4showsstructuresofboththemodeland tide-dependentconformationalchangeofthearchaealRab-
theyeasttemplate.Rabmotifsarehighlightedinblue(RabF likeproteinbyanalyzingitsthermodynamicstabilityatboth
motifs) and orange (guanine nucleotide-binding residues). the GDP and GTP-bound state and predicting interactions
Both structures are very similar (0.41 A˚ root-mean-square betweentheproteinandthephosphategroupsofthenucle-
deviationoftheCaatomiccoordinates),motifsarelocalized otide.InadditiontothemodeloftheGTP-boundstate,we
atthesamestructuralelementsandsimilarlyexposedtothe modeledthestructureoftheGDP-boundform,againusing
environment.Wealsocomparedthelocationofhydrophobic severaltemplatesbelongingtodifferentRabsubfamilies:Rab1
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(Cryptosporidium parvum, PDB:2RHD), Rab2 (H. sapiens, divergedfromtheeukaryoticcounterpartsbeyondthedetec-
PDB:2A5J),Rab8(H.sapiens,PDB:4LHV),andRab43(H.sapi- tionlevelofstandardautomatedmethods.Inonecase,how-
ens, PDB:2HUP). The analysis of the structural predictions ever, that ofREP/GDI, even though the statistics ofthe hits
shows that the archaeal Rab-like protein is thermodynami- werepoor,weobservedrepeatedpositivehits,whichwethen
callystableinbothconformations(bothpredictedstructures investigatedfurther.
areshowninsupplementaryfig.S5,SupplementaryMaterial We manually inspected putative GDI/REP domains in
online).Theinteractionbetweenthephosphategroupsand Archaea.TheprimarysequenceofGDIandREPdomaincon-
the protein is stabilized by several residues present in the tainingproteinsisgenerallyweaklyconservedinEukaryotes,
protein active site. The presence of Gln68 and its relative both within each family and between GDI-REP paralogues
position to the GTP molecule enables the interaction be- (e.g., 30% human and fruit fly REP, 21% human GDI1 and
tween a water molecule and the phosphate, necessary for REP1,localalignmentidentity).Hence,giventheevolutionary
theGTPhydrolysis(Dumasetal.1999).Theanalysisofstruc- distancebetweenEukaryotesandArchaeaweexpectthatany D
o
turalmodelsofthearchaealRab-likeGTPaseindicatesthatit putative archaeal homologs would be within the “twilight w
n
canexistintwostableconformationsanditisabletocycle zone”ofsequencesimilarity,whichprecludesanyautomatic lo
a
betweenan“on”and“off”statelikeothersmallGTPasesand, sequence-basedanalysis.Weusedafoldrecognitionmethod de
d
inparticular,eukaryoticRabs. (Jones1999)withthebestscoring(HMMER)archealGDI/REP fro
protein to detect candidate proteins with determined 3D m
ARabEscortProtein/GDPDissociation structures. The best predictions belong to eukaryotic GDIs http
InhibitorAncestorinArchaea and archaeal proteins without experimentally determined s://a
OuranalysissofarsuggeststhatRab-likesequencespredate function (top three hits correspond to proteins from Bos c
a
d
Eukaryogenesis.Surprisingly,wefoundmotifsinarchaealRab- taurusPDB:1D5T,PyrococcusfuriosusPDB:3NRN,andS.cer- e
m
like sequences that are known to mediate interactions be- evisiae PDB:2BCG). These structures are also very similar to ic
.o
tween eukaryotic Rabs and their regulators and effectors. FAD-containingmonooxygenasesandoxidases(Schalketal. u
p
Eukaryotic Rabs are prenylated on the C-terminus, a 1996),includingarchaealgeranylgeranylreductases.Whilethe .co
m
posttranslational modification catalyzed by theenzymeRab sequence identity between putative archaeal GDI/REP and /m
geranylgeranyltransferase,whichrequiresachaperonetermed eukaryotic GDI is very low, at the structural level both do- b
e
2R0E0P6()R;aabpEascraolrotgPureotoefinR)E(PP,erteeirrma-LeedalGeDtIal.(G20D0P1;dLiesusoncgiaettioanl. mareainsism(i3laNrR, iNncalunddin1gUKthVe,aRaybea-bstinGdDinIginplcaotmforpmlex(fwigi.th5cY);PoTu1)r /article
Inhibitor) recycles Rabs in and out of membranes (Wu structural comparison revealed several residues that may -ab
s
etal.1996;fig.5a).BindingofRabstoREPandGDIismediated form interactions with Rab switch regions (not shown). tra
c
byresiduesintheRabFmotifs(Raketal.2003,2004;Goody OurresultsstronglysupporttheexistenceofaREP/GDI-like t/3
et al.2005).Thesameregions areinvolvedinbindingother molecule in the TACK group, whose function implies an 3/7
generalRabregulators—Rabactivityisregulatedbyguanine- isoprenyl-bindingability. /18
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nucleotide-exchange factors (GEF) that turn Rabs “on” by Wefurtherusedthesamestrategytoinvestigatewhether 3
/2
promoting the GDP to GTP exchange, and by GTPase- the isoprenylation machinery, specifically the two subunits 5
7
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activating proteins (GAP) that increase GTP hydrolysis rate (aandb)oftheeukaryoticisoprenyltransferases,ispresent 2
5
and turn Rabs off. Both sets of proteins interact with Rabs inArchaea.Bothapproacheswereinconclusivetodetermine 5 b
y
withresiduesincludedintheRabFmotifs(thosewithinthe theexistenceoftheasubunit,asthetetratricopeptiderepeat g
u
switchregions).TheidentificationofRabFmotifsinArchaea thatcharacterizesthisdomainiswidespreadandfunctionally e
s
raises the hypothesis that such proteins and interactions promiscuous, precluding any conclusion about function. t o
n
couldalsopredateEukaryogenesis. However, we detected archaeal proteins whose predicted 2
4
We used two approaches to test if homologues of these foldmatchesseveralisoprenoidmetabolismenzymesinclud- N
o
eukaryotic proteins can be detected in Archaea, indicating ingthegeranylgeranyltransferasesubunitb.Wefoundmul- ve
m
thatsomeofthecomplexRabregulatorycyclescouldpredate tipleinstancesofgenescontainingthesedomains,observing b
e
Eukaryogenesis. First, we used sequences of several human somespecieswheretheyco-occur(fig.5d). r 2
0
regulators (GEFs, GAPs, FNT, PGGT1B, REP, RABGGT), per- 18
formedBLAST(Altschuletal.1990)similaritysearchesagainst
Discussion
archaeal genomes and found only hits with insignificant se-
quence similarity (not shown). As BLAST is known to lack Inthiswork,weinvestigatedthehypothesisthatthesepa-
sensitivitytodetectremotehomologies,wethenusedamore ration of the eukaryotic signature Rab sequences predates
sensitive approach based on pHMM. We retrieved pHMMs theemergenceofEukaryotes.Thishypothesisfollowsfrom
(Pfam)ofthedomainsthatarefoundinRab-bindingproteins the recent discovery of a new archaeal group, the
(Mss4, Sec2, VPS9, DENN, RabGAP-TBC, GDI/REP, prenyl- Lokiarchaeota, that was claimed to be a sister group of
transferase,PPTA),whichwethenusedasqueriesforasim- Eukaryotes. Our Rabifier pipeline identified 42 candidate
ilarity search using the HMMER package. In most cases, we Rab-like sequences that have multiple features related to
foundonlyscatteredhitsonthetreewithmarginalsequence eukaryotic Rabs, they exist in several Archaea of both the
similarity(fig.5b),suggestingthateithercanonicalRabregu- TACK group and Euryarchaeota but are particularly abun-
latory proteins are absent from Archaea or their sequences dant in Lokiarchaeum. Although phylogenetic methods
1838
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AreThereRabGTPasesinArchaea? . doi:10.1093/molbev/msw061
(a) (b)
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FIG.5.IdentificationofRabregulatoryproteins.(a)SchematicrepresentationoftheRabactivationpathway.(b)Homologydetectionbythe o
v
similaritysearchofstructuraldomainscharacteristictotheRabregulatoryproteins.Numbersrepresente-valuesofthebestscoringproteinsina em
speciesforeachdomain.BoldfontindicatesspeciespredictedtocontainRaborRab-likeGTPases.GEF,guaninenucleotideexchangefactor;GAP, be
GTPase-activating protein; GDI, GDP dissociation inhibitor; GGT, geranylgeranyl transferase; GDF,GDI displacement factor; REP, Rabescort r 2
0
protein;PPTA,Proteinprenyltransferasealphasubunit;PT,Prenyltransferase.(c)StructuralalignmentofGDI/REP-likeproteinsfromPyrococcus 1
8
furiosusandaGDI-YPT1complexfromSaccharomycescerevisiae.(d)Totalnumberofproteinscontainingtheprenylationcomplexdomains
encodedinagenome.Eacharchaealfamilyisrepresentedbyaspecieswiththebiggestnumberofproteinscontainingselecteddomains.Tree
topologyisconsistentwithNCBITaxonomy.
alonewereinsufficienttodeterminethepositionofRab-like regulatescellpolarityandmotilitybyaccumulatingatacell
proteins within the RAS superfamily, our results indicate pole in its active GTP-bound state (Zhang et al. 2010). The
that these GTPases may be Rab precursors. Surprisingly, closest group to eukaryotic Rab/Rho/Ras/Ran are the Rup
wealsofoundevidencefor aGDI/REP-likeproteinexisting proteins (Ras superfamily GTPase of unknown function in
in Archaea, raising the possibility that this interaction prokaryotes; Wuichet and Søgaard-Andersen 2015).
predatesEukaryogenesis. Phylogenetic analysis is not able to resolve the relationship
Small GTPases are well known to exist in prokaryotes, between eukaryotic small GTPases and prokaryotic ones, so
where they mediate diverse functions, for example, MglA noclaimcanbemadewhetherthesesequencesareRup-like
1839
MBE
SurkontandPereira-Leal . doi:10.1093/molbev/msw061
or a new independent branch (supplementary fig. S6, thealphaandbetasubunitsofeukaryoticprenyltransferases
SupplementaryMaterialonline). arecommon,althoughthereisnoevidencethattheyareable
We concentrated on characterizing sequence and toformaheterodimerwiththeprenyltransferaseactivity.The
structural features that could shed light on the relation- betasubunithomologuesareinvolvedintheisoprenoidme-
shipbetweenthesesequencesandeukaryoticRabs.Atthe tabolismandtheirstructureispredictedtobesimilartoeu-
familylevel,theyaremoresimilartotheRabfamilythan karyotic prenyltransferases, which further supports the
toothereukaryoticsmallGTPases(Arf/Ras/Rho/Ran).We notion that some components of the prenylation complex
foundextensiveRabFmotifsconservation,motifsthatin arepresentinArchaea.
Eukaryotesarediagnosticofthisfamily,andthatmediate Small GTPases are molecular switches that can cycle be-
importantproteininteractionscharacteristicofRabs.On tweentwomembrane-associatedstates,aswellascycleinan
thestructuralmodelsofarchaealRab-likeproteins,these out of membrane. Our results suggest that these Archaea
motifs map to the same positions as their eukaryotic represent a snapshot of the evolution of this circuit, that D
o
counterparts, suggesting that they could mediate similar resolves part oftheevolutionary pathintomembrane-asso- w
n
interactions,whichlendsfurthersupporttotheirRab-like ciatedproteintraffickingregulators.TheRabproteinfamilyis lo
a
classification. Our results thus point to Archaea having alreadyindividualized,eventhoughwelackanyknowninter- de
d
Rab-likesequences,whichalthoughnotbeingfull-fledged nal membranes in the TACK Archaea. These proteins are fro
Rabs, as we will discuss below, are already differentiated apparently activeGTPases ableto cyclebetween two struc- m
h
intermediates to this small GTPasefamily. turalstates,butitisuncleariftheydoitinthecytosolorifan ttp
s
The presence of Rab motifs that are known to mediate “in”and“out”ofmembraneswitchwasalreadyestablished.In ://a
interactionswithotherEukaryote-specificRabregulatorswas thisscenario,aninteractionwiththeproteinthatwillbecome c
a
d
puzzlingandledustotestthehypothesisthatoneormoreof thechaperonethatcatalysesthissecondpartoftheRabcycle e
m
theseinteractionscouldhavepredatedeukaryogenesis.Using isalreadyestablished,butintheabsenceoflipidmodification. ic
.o
sensitive methods we found convincing REP/GDI-like pro- It is plausible that localization to membranes may exist via u
p
teinsinmultipleArchaeathatareinvolvedinthebiosynthesis protein-proteininteractions.Finally,thebuildingblocksfora .c
o
m
of membrane lipids (geranylgeranyl reductase, EC 1.3.1.101). protein prenylation machinery are also found in multiple /m
An archaeal form of this enzyme had its crystal structure Archaea,suggestingthateventheemergenceofthiscompo- b
e
scoolmvepdleaxn.IdtiaslitghnusswveerllywpritohbathbelectrhyasttatlhestcruocntsuerrevaotfioGnDoIf:Rthabe nenOtuorfctohnecRluasbiocnysclaerempaoysasilbsolepbreecdaautseeewukeawryeorgeeanbelseis.togo /article
RabF motifs in archaeal Rab-like sequences points to an es- beyondphylogeneticmethods,whichareclearlyinsufficiently -ab
s
tablishedinteractionwiththisenzyme.Thefunctionalmean- sensitive to resolve events at this order of temporal diver- tra
c
ingofthisinteractionisunclear,butthefactthatthisenzyme gence,usinginsteadourmotif/domain-basedtooltoidentify t/3
isinvolvedinthesynthesisoftheisoprenoidsthatareusedin Rabs,theRabifier.Itisimportantnowtolookintoothersmall 3/7
the lipid modification of eukaryotic small GTPases is highly GTPase families, as our preliminary data suggest that other /1
8
3
suggestive.Inspectionofthestructureofthearchaealenzyme members of the Ras/Rho/Ran/Rab clade may have already 3
/2
suggeststhatalthoughithasabindingpocketabletoshield beenindividualizedinArchaea.Itisalsoimportanttoinves- 5
7
9
thelipidgroupsfromthecytosolasREPandGDIdo,itisina tigatewhethertheinteractionwepredictherebetweenRab- 2
5
different orientation, suggesting that it cannot chaperone likeandREP/GDI-likesequencesdoesinfactexist,andwhatis 5 b
y
lipid-modified eukaryotic Rabs that have longer C-termini the subcellular localization of these small GTPases. g
u
thanthearchaealRab-likesequences. Lokiarchaeota,areunlikelytargetorganismsfortheseexper- e
s
In Eukaryotes REP/GDI are chaperones of the lipid- iments, as they exist in a difficult to reach environment. t o
n
modified Rabs, that deliver them to the membranes, where However, organisms that areroutinely culturedinthelabo- 2
4
REPisdoingsointhecontextofthelipidmodificationreac- ratory have these sequences (see fig. 5), which makes these N
o
v
tion,asanaccessoryproteintotheRabGGTasecomplex,and experimentstractable.Furthermore,wefoundthatotheren- e
m
where GDI recycles Rabs in and out of membranes. The vironmental(marine)samples(Kawaietal.2014)alsopossess b
e
presenceofaREP/GDIhomologueinArchaearaisesthehy- Lokiarchaeota-like small GTPases and specifically abundant r 2
0
pothesis that membrane association of small GTPases via Rab-like sequences (117 proteins in the analyzed sample), 1
8
prenylation may have preceded the emergence of whichmakesthepossibilityofisolationandcultureofthese
Eukaryotes.Thereis,atleast,onereportclaimingisoprenyla- organismsmoreplausible.Ourstudygivesfurthersupportto
tion of proteins in Archaea (Konrad and Eichler 2002). the notion that Eukarya emerged from within Archaea,
However,theabsenceofanextendedC-terminalregionbe- and may be construed to support the notion that it was
yondtheGTPaseglobulardomaintogetherwiththeabsence from within organisms close to the recently identified
of the prenylateable C-terminal cysteine residues points Lokiarchaeum. We are convinced that in the near future
against this. Furthermore, we found no evidence of a poly- we will be able to resolve the origin of the in-out of
basicregionthatisknowntomediatemembraneassociation membrane cycle of small GTPases, and their association
(Williams2003),norofanyothermembraneassociationsig- withspecificeukaryoticprocesses.Itispossiblethatthiscycle
nal.OurresultsthussuggestthattheseRab-likesequencesare emerged in Archaea, even before the specific system they
unlikelytoassociatewithmembranesvialipidation.Itis,how- regulate in Eukaryotes has emerged, and that have later
ever, interesting to note that archaeal homologues of both beenco-opted.
1840
MBE
AreThereRabGTPasesinArchaea? . doi:10.1093/molbev/msw061
Materials and Methods wereevaluatedwiththeDOPEpotential(ShenandSali2006),
ProSA(Sippl1993;WiedersteinandSippl2007),andVerify3D
Sequences
(Lu¨thyetal.1992).PyMOL(ThePyMOLMolecularGraphics
All complete archaeal proteomes (231) were downloaded
System, Version 1.7.4 Schro¨dinger, LLC.) was used for struc-
fromtheUniProtdatabase(TheUniProtConsortium2015),
turevisualization.
all Lokiarchaeum proteins (5,384) were downloaded from
GenBank (Benson et al. 2014). The complete list of species
is shown in the supplementary table S1, Supplementary Supplementary Material
Material online. Eukaryotic and bacterial genomes were
SupplementaryfiguresS1–S6andtablesS1–S3areavailableat
downloadedfromEnsembl(Cunninghametal.2015).
Molecular Biology and Evolution online (http://www.mbe.
oxfordjournals.org/).
ProteinSequenceAlignments
D
MultiplesequencealignmentswerebuiltwithMAFFT7.221 o
Acknowledgments w
(Katoh and Standley 2013) using a high accuracy mode n
lo
(–maxiterate 1,000 –localpair). TrimAl v1.2 (Capella- The authors thank all members of the Computational ad
e
Gfroumtie´rarleigznemteanl.ts2.00P9a)irwwiases useseqduetnoceremaliogvnemgeanpt-sricwherreegcioonns- GfoernroemadicinsgLatbhoeramtoarnyufsocrriphte.lpTfhuilsdwiscourkssiwonass. sKurpzypsozrttoefdKbu(cid:2)ys d from
structed with water (the Smith–Waterman local alignment Fundac¸~ao para a Ci^encia e a Tecnologia (SFRH/BD/51880/ h
algorithm)andneedle(theNeedleman–Wunschglobalalign- 2012toJ.S.). ttps
mentalgorithm)fromtheEMBOSSpackage(Riceetal.2000). ://a
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