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.
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