Enzymatic systems of inorganic pyrophosphate bioenergetics in photosynthetic and heterotrophic protists: remmants or metabolic cornerstones?

REVIEW ARTICLE Jose ÂR. Pe  ez-Cas in Äei a áRosa io Go Âmez-Ga cõÂa Rosa L. Lo Âpez-Ma que ÂsáManuel Losada Au elio Se ano Enzyma ic sys ems o ino ganic py ophospha e bioene ge ics in pho osyn he ic and he e o ophic p o is s: emnan s o me abolic co ne s ones? Recei ed: 30 Ap il 2001 / Accep ed: 15 June 2001 / Published online: 1 No embe 2001 ÓSp inge -Ve lag and SEM 2001 Abs ac An inc easing body o biochemical and gene ic e idence sugges s ha ino ganic py ophospha e (PPi) plays an impo an ole in p o is bioene ge ics. In hese o ganisms, wo ypes o ino ganic py ophospha ases [EC 3.6.1.1, namely soluble PPases (sPPases) and p o- on- ansloca ing PPases (H + -PPases)] ha hyd olyse he PPi gene a ed by cell anabolism, he eby eplenish- ing he o hophospha e pool needed o phospho yla- ion eac ions, a e p esen in die en cellula compa men s. Pho osyn he ic and he e o ophic p o- is s possess sPPases loca ed in cellula o ganelles (plas ids and mi ochond ia), whe e many anabolic and biosyn he ic eac ions ake place, in addi ion o H + - PPases, which a e in eg al memb ane p o eins o he acuolysosomal memb anes and use he chemical ene gy o PPi o gene a e an elec ochemical p o on g adien use ul in cell bioene ge ics. This las ca ego y o p o on pumps was conside ed o be es ic ed o highe plan s and some p imi i e pho osyn he ic bac e ia, bu i has been ound ecen ly in many p o is s (mic oalgae and p o ozoa) and bac e ia, hus indica ing ha H + -PPases a e much mo e widesp ead han p e iously hough . No cy osolic sPPase (in bac e ia, ungi and animal cells) has been shown o occu in hese lowe euka yo es. The widesp ead occu ence o hese key enzymes o PPi me abolism among e olu iona ily di e gen p o is s s ongly suppo s he ances al cha ac e o he bioen- e ge ics based on his simple ene gy- ich compound, which may play an impo an ole in su i al unde die en bio ic and abio ic s ess condi ions. Keywo ds Ino ganic py ophospha e áSoluble ino ganic py ophopha ase áP o on- ansloca ing py ophospha ase áPho osyn he ic p o is s áPa asi ic p o is s In oduc ion Ino ganic py ophospha e (PPi) is a simple molecule composed o wo me aphospha e g oups (PO 3± ) linked by an oxygen anion (O 2± ), hus o ming he P-O-P s uc u e, also known as a ``py ophospha e b idge''. P-O-P is a chemical g oup ha s o es eadily usable ene gy o biochemical eac ions and i can be ound no only in PPi bu also in ATP, he well-known ``ene gy cu ency'' o li ing cells. PPi is p oduced in la ge amoun s by a a ie y o i al biosyn he ic eac ions, such as he syn hesis o biopolyme s (polysaccha ides, p o eins, nu- cleic acids, lipids). PPi hyd olysis is impo an o pull hese anabolic eac ions (mos o which a e e e sible) in he di ec ion o biosyn hesis. Mo eo e , an ecien PPi hyd olysis is also essen ial o eplenish he o hophos- pha e (Pi) needed o phospho yla ion [2] (Fig. 1). Al hough PPi was un il ecen ly conside ed a was e p oduc o anabolism, an inc easing body o e idence indica es ha i can play a ele an ole in cellula bio- ene ge ics. Mo eo e , i has been sugges ed ha PPi may ha e been he ances o o ATP as he ``ene gy cu ency'' du ing he ea ly s ages o biochemical e olu ion [4]. Two majo ypes o PPi-hyd olysing enzymes, known as in- o ganic py ophospha ases (PPases, EC 3.6.1.1), ha e been cha ac e ized o da e: soluble and memb ane- embedded. Soluble PPases (sPPases) a e ubiqui ous p o- eins whose ole is he emo al o he PPi p oduced by anabolic eac ions, so ha hey can ecien ly p oceed in he co ec (biosyn he ic) di ec ion [20]. Memb ane- bound, p o on- ansloca ing, ino ganic py ophospha a- ses (H + -PPases) belong o a ecen ly iden i®ed ca ego y In Mic obiol (2001) 4: 135±142 DOI 10.1007/s10123-001-0028-x J.R. Pe  ez-Cas in Äei a áR. Go Âmez-Ga cõÂa 1 R.L. Lo Âpez-Ma que ÂsáM. Losada áA. Se ano (&) Ins i u o de BioquõÂmica Vege al y Fo osõÂn esis, Cen o de In es igaciones Cien õ®cas ``Isla de la Ca uja'', Uni e sidad de Se illa±CSIC, A enida Ame ico Vespucio s/n, 41092 Se ille, Spain E-mail: [email p o ec ed] Tel.: +34-954489524 Fax: +34-954460065 P esen add ess: 1 Ins i u o de Pa asi ologõÂa y Biomedicina ``Lo Âpez-Ney a'', G anada, Spain o p o on pumps, dis inc om F-, P- and V-ATPases, which u ilize PPi hyd olysis as he d i ing o ce o H + mo emen ac oss biological memb anes [28]. H + -PPases ha e been iden i®ed and cha ac e ized bo h a bio- chemical and gene ic le els in highe plan s, some pho- osyn he ic bac e ia and, mo e ecen ly, in many bac e ia, a chaea and some pa asi ic p o is s [5, 9, 10, 12, 16, 18, 23, 25, 30, 31, 33]. Howe e , hey seem o be absen om animals, ungi and se e al ypes o bac e ia, including en e obac e ia. The sPPases o p o is s a e loca ed in cell o ganelles (plas ids, mi ochond ia) and ha e di e se molecula phylogeny So a , cy osolic sPPases ha e been ound and well cha ac e ized in bac e ia, ungi and animal cells. In hese euka yo es, a mi ochond ial sPPase was also ound as an ex insic p o ein o he inne memb ane and was ound o be essen ial o he unc ion o his o ganelle [22]. In yeas , bo h p o eins ha e he same molecula phylogeny, being euka yo e-like sPPases. The me abolic scena io conce ning sPPases is comple ely die en in pho osyn he ic p o is s (Fig. 2). P o is s [bo h pho o- syn he ic (mic oalgae) and he e o ophic (p o ozoa)] lack cy osolic sPPase, he physiological ole o his en- zyme p esumably being pe o med by a numbe o sol- uble and memb ane-bound p o eins in ol ed ei he in suga phospho yla ion o in ion anspo and homeo- s asis, espec i ely [13, 14, 15, 29]. Vi ually all sPPase ac i i y is loca ed in he cellula o ganelles ± namely, plas ids and mi ochond ia ± o hese lowe euka yo es (Fig. 2), as is also he case in highe plan pho osyn he ic issues [13, 14, 15, Go Âmez R (2001) PhD hesis, Uni- e si y o Se ille]. We ha e pu i®ed o homogenei y and cha ac e ized he sPPases o pho oau o ophic p o is s bea ing plas ids o die en ypes, om p imi i e cy- anobac e ia-like cyanelles o plan -like chlo oplas s (wi h wo en olding memb anes) and complex chlo o- plas s (wi h mo e han wo en olding memb anes). All a e monome ic p o eins wi h appa en molecula masses in he ange 32±40 kDa, as de e mined by SDS-PAGE and as -pe o mance gel pe mea ion ch oma og aphy; and hey esemble ungal and animal euka yo e-like sPPases, bo h in p o ein s uc u e and N- e minus se- quences [13, 14, 15, Go Âmez R e al., in p ep.]. Among o he s, he plas id sPPases om he glaucocys ophycean ¯agella e (wi h cyanelles) Cyanopho a pa adoxa (32 kDa), he he moacidophilic hodophycean mic o- alga Cyanidium calda ium (40 kDa), he euglenoid Euglena g acilis (38 kDa), he pho osyn he ic he e o- kon (ch omophy e) Och omonas danica (38 kDa) and he chlo ophycean mic oalga Chlamydomonas ein- ha d ii (37 kDa) ha e been cha ac e ized. The plas id sPPases a e he e o e euka yo e-like p o eins clea ly die en om he homohexame ic (20 kDa subuni ) sPPases o cyanobac e ia, which a e pho oau o ophic bac e ia esembling he ances al p oka yo ic endosym- bion ha ga e ise o hese o ganelles [13, 14, 15, Go Âmez R e al., in p ep.]. A ca e ul analysis o he sPPase p epa a ions pu i®ed om he chlo ophycean mic oalga C. einha d ii e- ealed he p esence o wo polypep ides o sligh ly di - e en molecula mass, bo h o hem wi h PPase ac i i y: a majo euka yo e-like enzyme named sPPase1 (37 kDa, SDS-PAGE) and a mino one named sPPase2 (32 kDa, SDS±PAGE) [14, 15, Go Âmez R e al., in p ep.]. Mono- speci®c polyclonal an ibodies aised in abbi s agains hese wo p o eins did no c oss- eac , indica ing ha hey should be s uc u ally die en p o eins. Wes e n blo analyses wi h he an i-sPPase1 an ibody immuno- de ec ed a single polypep ide (32±40 kDa), which co - esponded o he plas id sPPase in cell- ee c ude ex ac s o o he Chlo ophyceae, euglenoids, Glauco- cys ophyceae, Rhodophyceae, pho osyn he ic he e o- kon s (Ch omophyceae, dia oms) and plan pho osyn he ic issues (Table 1), bu no o ha in plan oo issues and bac e ia. No e ha , in ag eemen wi h biochemical da a, his an ibody also immunode ec ed yeas cy osolic sPPase. Subcellula ac iona ion using Pe coll g adien VCX analysis localized sPPase1 in he chlo oplas ac ion and sPPase2 in he mi ochond ial one [15, Go Âmez R (2001) PhD hesis, Uni e si y o Se ille]. An A abidopsis haliana cDNA ha encodes he p ecu so polypep ide o an euka yo e-like sPPase wi h a N- e minal chlo oplas ansi pep ide has been iden- i®ed by BLAST homology sea ches. This plan ppa gene has been cloned (accession numbe AJ252210) and he - e ologously o e exp essed in Esche ichia coli, whe e he p o ein was p ocessed o he ma u e ac i e o m; and i was ecien ly inmunode ec ed by he an ibody an i- sPPase1 o C. einha d ii, bo h ecombinan -plan and na u al-alga ma u e p o eins ha ing i ually iden ical Fig. 1 Me abolic scheme showing he in acellula phospha e cycle o ene giza ion±deene giza ion in which ino ganic py ophospha a- ses (PPases) play a key ole. The me abolic scena io ep esen s a p oka yo ic cell bu may be applied also o p o is cellula o ganelles. NTP Nucleosides iphospha e, PolyP ino ganic polyphospha es 136 molecula masses. To ou knowledge, his is he ® s ppa gene encoding a p ecu so polypep ide wi h a ``chlo- oplas ansi pep ide'' desc ibed so a [15, Go Âmez R e al., in p ep.]. In ag eemen wi h i s p edic ed cellula localiza ion, No he n blo expe imen s showed ha his gene is exp essed in g een issues (lea es, shoo s), bu no in oo s. In acco dance wi h he biochemical da a, BLAST homology sea ches on EST da abases allowed us o iden i y wo po en ially encoding sPPase cDNAs o he mic oalga C. einha d ii. Bo h algal ppa genes ha e been cloned (accession numbe s AJ298231 o ppaI gene coding o sPPase1, AJ298232 o ppaII gene coding o sPPase2) and ound o be exp essed in pho oau o ophic C. einha d ii cells, om which he wo sPPase p o eins we e pu i®ed. One o he cDNAs possesses a chlo oplas ansi pep ide and encodes he polypep ide p ecu so o an euka yo e-like sPPase ( he chlo oplas sPPase1); and he o he encodes a smalle bac e ial-like sPPase (p e- sumably he mi ochond ial sPPase2). The e o e, he sPPases se o C. einha d ii is o med by wo p o eins o dis inc molecula phylogeny ha a e loca ed in die en cellula o ganelles [14, 15, Go Âmez R e al., in p ep.]. The ecen ly comple ed A. haliana genome p ojec has added new in e es ing in o ma ion ha con® med ou esul s. Thus, a single ppa gene loca ed in ch omo- some 5 ( he same one we cloned and expe imen ally alida ed) encodes a chlo oplas euka yo e-like sPPase; and a se o ® e pa alogous, highly-simila ppa genes loca ed in die en ch omosomes encode a amily o bac e ia-like sPPases. One o hese genes co esponds o a mi ochond ial polypep ide p ecu so and should be equi alen o he sPPase2 o C. einha d ii. The o he ou bac e ial sPPase genes exhibi a e y high homol- ogy, e en a he DNA le el and a e clea ly equi alen o he o hologous ppa gene ha encodes he cy osolic sPPase ound in po a o ube [11], so hey should be exp essed in non-pho osyn he ic issues ( oo s). The high simila i y ound among he bac e ia-like sPPases o A. haliana sugges s ha hey p obably o igina ed by qui e ecen gene duplica ion e en s om a common ances o , pe haps simila o he mic oalgal sPPase2 gene [15, Go Âmez R (2001) PhD hesis, Uni e si y o Se ille]. The esul s desc ibed abo e ha e cla i®ed he mo- lecula phylogeny o he sPPases o pho osyn he ic eu- ka yo es (mic oalgae, plan s). All pho osyn he ic plas ids con ain a nuclea -encoded euka yo e-like sPPase; and his ®nding sugges s ha he homologous bac e ial enzyme o he ances al p oka yo ic endo- symbion was los e y ea ly du ing he e olu iona y p ocesses ha ga e ise o pho osyn he ic plas ids and was unc ionally subs i u ed by he nuclea -encoded sPPase o he euka yo ic hos cell (Fig. 3). In con as , he mi ochond ial sPPases o mic oalgae and plan s a e nuclea -encoded bac e ia-like p o eins, as should also be he case o he cy osolic sPPases o non-pho osyn he ic issues [13, 14, 15, Go Âmez R e al., in p ep.]. The sce- na io ound o he ungal and animal lineages is in his espec qui e die en , since hey ha e euka yo e-like cy osolic and mi ochond ial sPPases [22]. Recen da a sugges ha his may also be he case o he o ganella (mi ochond ial, apicoplas , kine oplas ) sPPases o a numbe o p o ozoan s ains ( ypanosoma ids, api- complexa) ha seem o be euka yo e-like sPPases (see Table 1; Go Âmez R (2001) PhD hesis, Uni e si y o Fig. 2 Schema ic ep esen a ion o he me abolic scena io con- ce ning he h ee PPases ha occu in die en cell compa men s o pho osyn he ic p o is s and hei ela ionships wi h he cy osolic o hophospha e (Pi) pool. Cellula o ganelles, plas ids and mi o- chod ia, con ain espec i ely he soluble PPases (sPPases)cand m ha hyd olyse he ino ganic py ophospha e (PPi) gene a ed in anabolic eac ions, whe eas he p o on- ansloca ing PPase (H + - PPase) o he in acellula memb anes o acuoles o lysosomes do he same job in he cy osol. In his case, howe e , he chemical ene gy o he phospha e bond is used o gene a e an elec ochem- ical p o on g adien ha is used o d i e a numbe o sympo and an ipo memb ane sys ems. A simila scena io is p oposed o p o ozoa, excep ha no pho osyn he ic plas ids a e p esen in his case 137 Se ille). Summa izing, al hough many p o is s ha e sPPases only in cellula o ganelles (plas ids, mi ochon- d ia), he pho osyn he ic euka yo es (mic oalgae, plan s) a e, so a , he only g oup o o ganisms in which wo sPPases wi h die en molecula phylogeny and dis inc cellula localiza ion occu . H + -PPases occu in endocellula memb anes ( acuoles, lysosomes, acidocalcisomes) o a b oad ange o e olu iona ily di e se p o is s The memb ane-bound H + -PPase was ® s desc ibed in ch oma opho es om he pho o ophic bac e ium Rhodospi illum ub um and shown o be able o bo h syn hesize and hyd olyse PPi [1, 3]. Euka yo ic H + - PPases we e o iginally iden i®ed in highe plan s and epo ed o be loca ed in he acuola memb ane ( on- oplas ), ca alysing elec ogenic H + - ansloca ion om he cy osol o he acuole lumen, hus gene a ing an elec ochemical g adien , which can be u ilized o couple di e se seconda y anspo p ocesses (Fig. 2). This elec ochemical g adien has simila o g ea e magni- ude han ha c ea ed by he o he p o on pump p esen in he onoplas : he acuola H + -ATPase [28]. Vacuo- la H + -PPases ha e a subuni size o abou 66 kDa and adia ion inac i a ion analyses ha e shown ha he p o ein seems o be a dime in i o [17]. The biochemical cha ac e iza ion o H + -PPases om die en sou ces has sugges ed he occu ence o wo ypes o p o ein, depending on hei equi emen o Table 1 P o is s in which soluble py ophospha ase (sPPase) p o- eins loca ed in cellula o ganelles (plas ids, mi ochond ia, api- coplas s) and/o genes encoding hei p ecu so s ha e been iden i®ed. E idence om expe imen s (in he au ho s' labo a o y, ei he by p o ein pu i®ca ion, subcellula ac iona ion, Wes e n blo analysis, o gene cloning) and/o om bioin o ma ic analyses o genome da abases (g). MMi ochond ial sPPase, M/A mi o- chond ial o apicoplas sPPase, Pplas id sPPase, ponly pa ial sequences wi h no N- e minal egions a e known and/o no cellula localiza ion da a a e ye a ailable. The wo sPPase genes o Chlamydomonas einha d ii and he one o Leishmania majo ha e been cloned and expe imen ally alida ed He e o ophic p o is s (p o ozoa) Pho osyn he ic p o is s (mic oalgae) T ypanosoma idae Chlo ophyceae Leishmania majo (M, g) Chlamydomonas einha d ii (M, P, g) T ypanosoma b ucei (M, g) Chlo ella usca (P) Dunaliella salina (P) Apicomplexa Mono aphidium b aunii (P) Plasmodium alcipa um (M/A, g) P. be ghei (p, g) Rhodophyceae C yp ospo idium pa um (p, g) Cyanidium calda ium (P) He e o ophic euglenoids Euglenophyceae As asia longa (p) Euglena g acilis (P) En amoebidae Glaucocys ophyceae En amoeba his oly ica (p, g) Cyanopho a pa adoxa (P) Dic yos eliida Pho osyn he ic he e okon s (S amenopiles) Dic yos elium discoideum (p, g) Ch ysophyceae Och omonas danica (P) Non-pho osyn he ic he e okon s Bacilla iophyceae (dia oms) (S amenopiles) Na icula pelliculosa (P) Oomyce es Phaeodac ylum ico nu um (P) Phy oph ho a sojae (p, g) Fig. 3 The e olu iona y ela ionships be ween he sPPases o cyanobac e ia and plas ids. Cyanelles a e p imi i e plas ids, e y simila o cyano- bac e ial cells, ound in glauco- cys ophycean p o is s. They ha e euka yo e-like sPPases, sugges ing ha he unc ional subs i u ion o he ances al enzyme o he cyanobac e ia- like endosymbion was an ea ly e en du ing plas id e olu ion 138 po assium o ull ac i i y. Thus, highe plan H + -PPases we e shown o ha e a nea -obliga o y equi emen o millimola concen a ions o po assium o ac i i y [6], whe eas R. ub um H + -PPase was epo ed o be insen- si i e o mono alen ca ions [25]. Mo e ecen ly, he gene encoding he A. haliana H + -PPase was isola ed and sequenced; and he p o ein was shown o be a po assium- dependen p o on pump, by exp essing he gene in he yeas Saccha omyces ce e isiae (which only has soluble PPases) and cha ac e izing he PPi-hyd olysis ac i i y and he PPi-dependen H + - ansloca ion ac i i y asso- cia ed wi h a acuola memb ane-en iched ac ion o he ans o med yeas cells [18]. This app oach was subsequen ly ollowed by die en g oups, hus cha ac- e izing a numbe o H + -PPases [4, 9, 16]. Ini ially, po- assium-sensi i e p o eins we e ound only in euka yo es, namely highe plan s [18, 28] and pa asi ic p o is s [16, 23, 30, 31, 33], whe eas H + -PPases om p oka yo ic sou ces we e insensi i e o mono alen ca ions [5, 25]. Howe e , du ing he cou se o he A. haliana genome p ojec , ano he gene coding o a pu a i e H + -PPase was iden i®ed; his gene showed a highe homology wi h bac e ial genes han wi h he o he A. haliana H + -PPase gene iden i®ed a ha ime. Consis en ly, he new gene was shown o code o a po assium-insensi i e H + -PPase by he e ologous exp ession in S. ce e isiae and was also epo ed o be exp essed in se e al plan issues [10]. The p esence o bo h ypes o H + -PPase in A. haliana sug- ges s die en physiological oles and e en die en subcellula localiza ion o he wo ypes o p o ein, howe e , hese aspec s emain o be s udied. This e i- dence also demons a ed ha he occu ence o he wo ypes o H + -PPase was a om clea . The si ua ion has ecei ed a new wis wi h he wo k ca ied ou in ou labo a o y wi h he H + -PPase om he hype he mo- philic eubac e ium, The mo oga ma i ima. We ha e bio- chemically cha ac e ized he he e ologously exp essed p o ein in yeas and ound i o be s imula ed mo e han six- old by po assium [26]. This was he ® s epo o a po assium-sensi i e H + -PPase in a p oka yo e. Mo e- o e , since T. ma i ima is a membe o he o de The mo ogales, one o he deepes and mos slowly e ol ing lineages in bac e ia, his ®nding also has an impo an e olu iona y signi®cance. Thus, po assium- s imula ed H + -PPases seem o be as ancien as hei po assium-insensi i e coun e pa s, which, as men ioned be o e, a e dis ibu ed among euka yo es (including p o is s), a chaea and bac e ia. Al hough, as desc ibed abo e, plen y o e idence is al eady a ailable abou H + -PPases, many impo an is- sues abou hese p o eins emain o be sol ed. These issues can be summa ized in h ee majo poin s: (a) he occu - ence o memb ane-bound H + -PPases, (b) hei physio- logical ole and (c) he eason o he exis ence o p o eins wi h die en sensi i i ies o mono alen ca ions. Acidocalcisomes a e acidic, calcium-s o age o gan- elles ound in se e al p o is s, al hough hey we e ® s de®ned in ypanosoma ids [7, 32]. Since acidocalci- somes we e ini ially hough o be ela ed o he acuoles o plan s, he p esence o a H + -PPase in hese o ganelles was in es iga ed and ound in Leishmania dono ani [30], T ypanosoma b ucei [31, 34], T. c uzi [32, 33], Plasmo- dium be ghei,Plasmodium alcipa um and Toxoplasma gondii [8, 30]. The genes coding o hese p o eins in P. alcipa um [8, genome p ojec ], T. c uzi [16] and T. gondii [8] ha e been cloned and sequenced. As a as pho osyn he ic p o is s a e conce ned, he si ua ion is less clea . Thus a , H + -PPases ha e only been iden i®ed in he mac oscopic unicellula alga Ace abula ia medi e anea and in he allophy ic alga Cha a co allina [24]. In any case, he in o ma ion con- ce ning he occu ence o PPase genes in p o is s is sca ce and non-sys ema ic. H + -PPases do no seem o be p esen in animals and, hus, hey migh be po en ial a ge s o accines and d ugs agains pa asi ic p o is s. The e o e, s udies on he possible implica ion o hese p o eins in he de elopmen o he diseases caused by hese o ganisms migh be o g ea impo ance. Majo s eps owa ds his goal a e o ®nd ou : (a) whe he H + - PPase genes a e p esen in he genome o o he pa asi ic p o is s, (b) he condi ions unde which hey become exp essed and (c) he s uc u al cha ac e is ics o he p o eins hey encode. A molecula app oach could gi e us aluable ools o accomplish hese objec i es. In ad- di ion, he s udy o he occu ence o he die en H + - PPase genes in such a he e ogeneous g oup o o ganisms will gi e mos aluable in o ma ion abou he e olu ion and phylogeny o his unique class o p o on pumps and hei physiological oles in li ing cells. By using polyclonal an ibodies agains conse ed amino acid egions [9, 10, 28], we ha e ound immuno- eac i e bands o he expec ed molecula mass in Wes e n blo s o memb ane p epa a ions om a numbe o pho- osyn he ic p o is s ha ing simple and complex plas ids (Bacilla iophyceae, Chlo ophyceae, Ch omophyceae, C yp ophyceae, Euglenophyceae, Glaucocys ophyceae, Rhodophyceae, o he pho osyn he ic he e okon s; [27], Pe  ez-Cas in Äei a e al., in p ep.; Table 2). Also, by using a PCR app oach wi h degene a e oligonucleo ides de- signed om amino acid domains common o H + -PPases o highe plan s and Rhodospi illum ub um, we ha e pe o med a sys ema ic sea ch o genes coding o H + - PPases in pa asi ic and ee-li ing p o ozoa and euka - yo ic mic oalgae (Table 2). We ha e usually ampli®ed a DNA agmen (ca. 0.6 kb) o he 3¢ egion o he gene ( om he cy osolic loop V o he C- e minus egion), co esponding o ca. 25±30% o he o al coding egion ha was demons a ed o be a good molecula ma ke o phylogene ic s udies. Sou he n blo analysis u he con® med he p esence o hese genes in he espec i e genomes o he o ganisms es ed. Compu e analysis o he sequences ob ained allowed us o analyse he some- imes su p ising phylogene ic ela ionships amongs he H + -PPases om e olu iona ily dis an o ganisms. Using his molecula gene ics s a egy, we ound plan -like H + -PPases genes ( e y simila o hose o human pa asi ic ypanosomes) in a ange o pa asi ic, mos ly non-pa hogenic ypanosoma ids o insec s and 139 o he lowe animals (C i hidia,Endo ypanum,He pe- omonas,Lep omonas) and plan s (Phy omonas), hus indica ing ha he p esence o his p o ein is no nec- essa ily associa ed wi h he pa hogenic cha ac e o hese p o ozoa. I is in e es ing in his espec ha we ha e also iden i®ed plan -like H + -PPase genes in a numbe o ee-li ing, non-pa asi ic p o ozoa o o he phylogene ic g oups: bo h in cilia es [such as hymenos oma ids (Te - ahymena,Pa amecium), pe i ichous (Vo icella) and hypo ichous (His iculus) s ains (ha ing he peculia ea u es o genes o his g oup, namely in- ame s op codons coding o Gln and, depending o he s ain, e y sho , 20±30 bp-long in ons)] and in he e o ophic euglenoids (As asia longa). In addi ion, in ag eemen wi h immunochemical da a, ou g oup has iden i®ed and cloned H + -PPase genes om ep esen a i e s ains o he main phylogene ic g oups o pho osyn he ic p o is s: Chlo ophyceae (Chlamydomonas,Chlo ella,Nannochl- o is,Te aselmis), Rhodophyceae (Cyanidium), pho o- syn he ic he e okon s [Och omonas (Ch omophyceae), Nannochlo opsis (Eus igma ophyceae), Phaeodac ylum, Skele omonas (Bacilla iophyceae, dia oms)], C yp o- phyceae (C yp omonas,Rhodomonas), Hap ophyceae (Isoch ysis) and Dynophyceae (He e ocapsa; Table 2; [27] Pe  ez-Cas in Äei a e al., in p ep.). No e ha he PCR s a egy desc ibed abo e allowed us o iden i y bo h po assium-s imula ed and po assium- independen H + -PPase genes in all o he main g oups o pho osyn he ic p o is s: Chlo ophyceae, C yp ophyceae, Dynophyceae, Euglenophyceae, Hap ophyceae, He e o- kon s (Bacilla iophyceae, Eus igma ophyceae) and Rhodophyceae ± mos o which do no belong o he plan e olu iona y lineage ± and in ee-li ing p o ozoa (cilia es, euglenoids). In e es ingly, phylogene ic s udies including hese sequence da a and o he sequences pub- lished o iden i®ed by sea ching in mic obial genome da abases (NCBI, TIGR websi es, see Tables 1, 2) show ha euka yo ic H + -PPases appea o be polyphyle ic. Two e olu iona y lineages we e ound o bo h po assi- um-s imula ed and po assium-independen H + -PPases: one o hem co esponds o he p o o ypic V-PPase se o plan s and is ound in apicomplexans, cilia es, g een al- gae, ypanosoma ids and some he e okon s (bo h pho- osyn he ic and he e o ophic), whe eas ano he se o H + -PPases mo e ela ed o p o eobac e ial p o eins (and he e o e mo e p imi i e) is ound mos ly in pho o ophic p o is s wi h complex plas ids (C yp ophyceae, Dyno- phyceae, Hap ophyceae, dia oms, some euglenoids) and in some ma ine ( ed, g een) algae. These esul s sugges die en e olu iona y his o ies and/o ho izon al gene ans e e en s [27, Pe  ez-Cas in Äei a e al., in p ep.]. Summa izing, he b oad dis ibu ion o bo h po as- sium-s imula ed and po assium-independen H + -PPases among bac e ia and p o is s ± ei he pho osyn he ic/ he e o ophic, pa asi ic o ee-li ing ± sugges s an an- ces al o igin and he occu ence o die en e olu ion- a y lineages o hese p o on pumps and/o he occu ence o ho izon al gene ans e e en s be ween phylogene ically di e se species. In his espec , i may be ele an ha we ound plan -like, po assium-depen- den H + -PPase genes in plan endopa asi ic mic o- o ganisms, bo h euka yo ic ( he ypanosoma id Phy omonas) and p oka yo ic (Ag obac e ium ume ac- iens) [27, Pe  ez-Cas in Äei a e al., in p ep.]. Finally, a possible pho osyn he ic ances y o he po assium-s im- ula ed,plan -likeH + -PPaseso pa asi ic ypanosoma ids and ee-li ing cilia es dese es conside a ion, since i has been ecen ly epo ed ha se e al enzymes o he oxi- da i e pen ose phospha e pa hway o T ypanosoma b ucei ha e cyanobac e ial and/o plan phylogenies [19], sugges ing ha he ypanosoma id lineage may be seconda ily non-pho osyn he ic. No e ha many Euglenoids ± a sis e g oup phylogene ically e y close o Table 2 P o is s in which H + -PPase p o eins o hei genes ha e been iden i®ed. E idence om expe imen s (ei he Wes e n blo s o memb ane p epa a ions o PCR ampli®ca ion and sequencing o gene egions, using degene a e oligonucleo ides) and/o om bio- in o ma ic sea ches on genome da abases (g). Unpublished DNA sequences ob ained in he au ho s' labo a o y ha e been submi ed o da abases and mos o hem a e unde con®den ial s a us un il publica ion He e o ophic p o is s (p o ozoa) Pho osyn he ic p o is s (mic oalgae) T ypanosoma idae Chlo ophyceae Ch i idia ascicula a Chlamydomonas einha d ii (g) He pe omonas musca um C. eu iale Endo ypanum schaudinni Chlo ella usca Lep omonas c enocephali C. salina Phy omonas sp. Dunaliella salina Leishmania majo Mono aphidium b aunii L. dono ani Te aselmis chuii T ypanosoma c uzi T. bal ica T. b ucei (g) Rhodophyceae He e o ophic euglenoids Cyanidium calda ium As asia longa Po phy idium pu pu eum Euglenophyceae Dic yos eliida Euglena g acilis Dic yos elium discoideum Glaucocys ophyceae Al eola es Cyanopho a pa adoxa Apicomplexa C yp ophyceae Plasmodium alcipa um (g) C yp omonas sp. P. be ghei Rhodomonas bal ica Plasmodium sp. Toxoplasma gondii (g) Hap ophyceae Ciliopho a (cilia es) Isoch ysis galbana Hymenos oma ids Pa amecium e au elia Al eola es Te ahymena py i o mis Dynophyceae Pe i ichous He e ocapsa sp. Vo icella mic os oma Hypo ichous Pho osyn he ic he e okon s His iculus ca icola (S amenopiles) Eus igma ophyceae Non-pho osyn he ic he e okon s Nannochlo opsis gadi ana (S amenopiles) N. ocula a Oomyce es Ch ysophyceae Phy oph ho a in es ans (g) Och omonas danica Bacilla iophyceae (dia oms) Na icula pelliculosa Phaeodac ylum ico nu um Skele omonas cos a a 140 ypanosoma ids ± a e pho osyn he ic, ha ing plas ids o seconda y symbio ic o igin; and some pa asi ic p o is s, mos no ably he apicomplexans ( ha , like cilia es, belong o he al eola es), a e known o be seconda ily non-pho- osyn he ic, al hough hey s ill possess a plas id, he api- coplas (bo h g oups ha e plan -like H + -PPases, see Table 2). The possibili y ha he ances al ypanosoma- id lineage had plas ids ha we e subsequen ly los de- se es conside a ion, since se e al g oups o non- pho osyn he ic euka yo es a e belie ed o ha e los hei plas ids seconda ily, mos no ably he oomyce es, a g oup o mos ly pa asi ic ungus-like he e okon s ha sha e many biochemical and s uc u al cha ac e is ics wi h plan s and also possess a plan -like H + -PPase (see Ta- ble 2).The e o e, heH + -PPasesmaybeuse ul ma ke s o elucida e he e olu iona y pas o he die en euka yo ic lineages and pe haps also o cla i y he in iguing ela- ionships be ween pho osyn hesis and pa asi ism in he p o is wo ld. We a e cu en ly pe o ming molecula physiology s udies wi h he Chlo ophyceae Chlamydomonas ein- ha d ii and Chlo ella usca, wo model pho osyn he ic p o is s whose H + -PPase genes ha e been cloned in ou labo a o y (accession numbe s AJ304836 and AJ251470, espec i ely). Due o hei e y e sa ile me abolisms and suscep ibili y o gene ic manipula ion, hese p o is s a e ideal sys ems o cla i y he physiological ole(s) o his p o on pump. Bo h mRNA and p o ein le el analyses show a clea induc ion o he mic oalgal H + -PPase unde a numbe o ionic and osmo ic s ess condi ions [21]. These le els a e also aec ed in esponse o changes in ophic condi ions (pho o-, mixo-, he e o ophy). I seems, he e o e, ha p o is H + -PPases a e igh ly eg- ula ed in esponse o a numbe o en i onmen al condi- ions ( ophic le el, s ess) ha p esumably may aec he ene ge ics s a us o he cell [21, Lo Âpez-Ma que Âs e al., in p ep.]. The ®ne egula ion o he genes encoding hese ionic pumps, also ound in pho osyn he ic bac e ia [21], sugges s ha hey mus play an impo an physiological ole in he adap i e esponses o all hese o ganisms. Acknowledgemen s This wo k was suppo ed by g an PB 97-1135 om DGICYT (MCYT, Spain) and by G upo PAI CVI-0261 (Jun a de AndalucõÂa). Wo k on PPases o pa asi ic p o is s has been pe o med in collabo a ion wi h he g oups o D s. L.M. Ruiz-Pe  ez and D. Gonza Âlez-Pacanowska (Ins i u o de Pa asi o- logõÂa y Biomedicina ``Lo Âpez-Ney a'', CSIC, G anada, Spain) and D . Jo ge Al a (Cen o Nacional de Mic obiologõÂa, Ins i u o de Salud Ca los III, Mad id, Spain). Thanks a e due o D . A. To es (Uni e si y o Se ille) o DNA samples o some cilia es. Some p elimina y sequence da a we e ob ained om he mic obial ge- nome websi es o NCBI and TIGR. Gene sequences ob ained in he au ho s' labo a o y ha e been submi ed o da abases and mos o hem a e unde con®den ial s a us un il publica ion. Se e al pub- lica ions on he opics desc ibed he e a e in p epa a ion. Re e ences 1. Bal schesky M (1967) Ino ganic py ophospha e and ATP as ene gy dono s in ch oma opho es om Rhodospi illum ub um. Na u e 216:241±243 2. Bal schesky M, Ny e Ân P (1984) The syn hesis and u iliza ion o ino ganic py ophospha e. 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