Biochem. J. (2011) 437, 269–278 (P in ed in G ea B i ain) doi:10.1042/BJ20110447 269
A plan p o on-pumping ino ganic py ophospha ase unc ionally
complemen s he acuola ATPase anspo ac i i y and con e s
ba ilomycin esis ance in yeas
Jos´
eR.P
´
EREZ-CASTI˜
NEIRA, Agus ´
ın HERN´
ANDEZ, Roc´
ıo DRAKE and Au elio SERRANO1
Ins i u o de Bioqu´
ımica Vege al y Fo os´
ın esis, Uni e sidad de Se illa-CSIC, A da. Ame ico Vespucio, 49, 41092 Se illa, Spain
V-ATPases ( acuola H+-ATPases) a e a speci ic class o mul i-
subuni pumps ha play an essen ial ole in he gene a ion
o p o on g adien s ac oss euka yo ic endomemb anes. Ano he
simple p o on pump ha co-localizes wi h he V-ATPase
occu s in plan s and many p o is s: he single-subuni H+-
PPase [H+- ansloca ing PPase (ino ganic py ophospha ase)].
Li le is known abou he ela i e con ibu ion o hese wo
p o eins o he acidi ica ion o in acellula compa men s. In
he p esen s udy, we show ha he exp ession o a chimae ic
de i a i e o he A abidopsis haliana H+-PPase AVP1, which is
p e e en ially a ge ed o in e nal memb anes o yeas , alle ia es
he pheno ypes associa ed wi h V-ATPase de iciency. Pheno ypic
complemen a ion was achie ed bo h wi h a yeas s ain wi h i s
V-ATPase speci ically inhibi ed by ba ilomycin A1 and wi h a
ma1-null mu an lacking a ca aly ic V-ATPase subuni . Cell
s aining wi h i al luo escen dyes showed ha AVP1 eco e ed
acuole acidi ica ion and no malized he endocy ic pa hway o he
ma mu an . Biochemical and immunochemical s udies u he
demons a ed ha a signi ican ac ion o he e ologous H+-
PPase is loca ed a he acuola memb ane. These esul s aise
he ques ion o he occu ence o dis inc p o on pumps in ce ain
single-memb ane o ganelles, such as plan acuoles, by p o ing
yeas V-ATPase ac i i y dispensabili y and he capabili y o H+-
PPase o gene a e, by i sel , physiologically sui able in e nal pH
g adien s. Also, hey sugges new ways o enginee ing mac olide
d ug ole ance and ou line an expe imen al sys em o es ing
al e na i e oles o ungal and animal V-ATPases, o he han he
me e acidi ica ion o subcellula o ganelles.
Key wo ds: chimae ic memb ane p o ein, g een luo escen
p o ein, he e ologous exp ession, p o on- ansloca ing ino ganic
py ophospha ase, Saccha omyces ce e isiae, acuola - ype
ATPase.
INTRODUCTION
The occu ence o an elec ochemical p o on g adien ac oss
he in e nal memb anes o euka yo ic cells is one o he known
equi emen s o he co ec unc ion o he endo- and exo-cy ic
pa hways [1]. Mo eo e , pH g adien s a e necessa y no only
o he co ec unc ioning o he esicle anspo machine y,
bu also o many o he o ganella unc ions, such as, among
o he s, dissocia ion o ligand– ecep o complexes in ecep o -
media ed endocy osis o glycosyla ion o p o eins in he Golgi
appa a us [2]. A speci ic class o p o on-pumping ATPases, V-
ATPases ( acuola ATPases o V- ype ATPases), play an essen ial
ole in he gene a ion o hese posi i e-inside p o on g adien s
[1,3]. V-ATPases we e o iginally desc ibed in he acuola
memb ane o plan s and ungi (hence hei name), he ea e hey
we e ound in many single-memb ane o ganelles o euka yo ic
cells. In addi ion, hey ha e also been epo ed o occu a
he plasma memb ane o ce ain mammalian cell ypes [2].
All in all, hese mul i-subuni p o eins ul il impo an oles in
di e se scena ios, bo h physiological and in disease, including
cance [3,4]. No su p isingly hen, he e is an ac i e sea ch
o speci ic inhibi o s o hese pumps ha could be used bo h
in basic esea ch and in he apeu ic applica ions. Ba ilomycin
A1 was he i s epo ed speci ic inhibi o o V-ATPases [5],
i consis s o a la ge mac ocyclic lac one ing which places i
in he mac olide an ibio ic g oup. Unlike o he euka yo es,
Saccha omyces ce e isiae cells can g ow ei he in he p esence o
V-ATPase inhibi o s [6] o when a null mu a ion occu s in one o
he genes encoding ce ain subuni s o he V-ATPase complex [1].
Howe e , hey show a ange o condi ional-le hal pheno ypes and
hus become e y sensi i e o neu al/alkaline pH alues [6,7],
high ex acellula calcium concen a ions [8] and ce ain me al
ca ions, such as zinc [9]; ac ually, he combina ion o sensi i i y
o high pH and ex acellula calcium is widely accep ed as he
mos cha ac e is ic Vma−pheno ype [1].
V-ATPases a e e y complex mul i-subuni pumps ha
con ain bo h memb ane-bound and pe iphe al componen s. This
pa adoxical complexi y, in compa ison wi h o he anspo e s,
has been jus i ied by p oposing moonligh ing unc ions o some
subuni s o e en whole domains o he V-ATPases [10,11].
Howe e , o s udy non- anspo unc ions o V-ATPases is a
complica ed ask i o ganelle acidi ica ion is no aken o e by
some o he p o ein(s). S ikingly, his si ua ion may be he na u al
scena io in plan cells and many p o is s whe e V-ATPases co-
exis wi h al e na i e p o on pumps. Howe e , o da e, no wo k
has p o en ha V-ATPases can be unambiguously subs i u ed by
any o he p o on pump in i o.
H+-PPases [p o on- ansloca ing PPases (ino ganic
py ophospha ases)] a e a ype o homo-oligome ic memb ane-
embedded p o eins con aining a single highly hyd ophobic
subuni ype ha couples he ene gy ob ained in he hyd olysis
o PPi (ino ganic py ophospha e) o he anspo o p o ons
Abb e ia ions used: ACMA, 9-amino-6-chlo o-2-me hoxy ac idine; DIC, di e en ial in e e ence con as ; DTT, di hio h ei ol; GFP, g een luo escen
p o ein; H+-PPase, H+- ansloca ing ino ganic py ophospha ase; PPase, ino ganic py ophospha ase; PPi, ino ganic py ophospha e; V-ATPase, acuola
H+-ATPase; yEGFP, yeas enhanced GFP.
1To whom co espondence should be add essed (email [email p o ec ed]).
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Biochemical Jou nal
270 J. R. P´
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nei a and o he s
ac oss biological memb anes [12–14]. They a e he s uc u ally
simples class o p ima y p o on pumps known o da e, and was
he las one es ablished, in he middle o he 1980s [12,13].
H+-PPases a e epo edly loca ed in cell memb ane esicles
and acidocalcisomes o p oka yo es and di e se acidic single-
memb ane o ganelles (acidocalcisomes, lysosomes, acuoles) o
ce ain euka yo es: p o is s and plan s [13,15–17]. H+-PPases
seem o co-localize wi h V-ATPases in hose euka yo es whe e
hey occu [18], hus, one poin o discussion is he physiological
signi icance o his appa en unc ional edundancy and whe he
H+-PPases con ibu e in a ying deg ees o he gene a ion o
p o on g adien s unde di e se physiological scena ios.
We ecen ly epo ed ha ansla ional usions wi h ce ain N-
e minal signal pep ides enhance he exp ession le els and/o al e
he subcellula dis ibu ion o di e se H+-PPases he e ologously
exp essed in S. ce e isiae. When his app oach was applied
o he acuola H+-PPase AVP1 om A abidopsis haliana,a
p o ein exp essed a mode a e le els in yeas in i s na u al o m
[19], i s subcellula dis ibu ion was al e ed; hus, chimae ic
p o eins cons uc ed by using he N- e minal signal sequence
o he H+-PPase om he p o is T ypanosoma c uzi [20] o
AVP1 we e shown o p e e en ially accumula e in in acellula
acidic esicles [21]. In he p esen s udy, we show ha he
exp ession o such an in e nal memb ane- a ge ed chimae ic
AVP1 de i a i e con e s yeas esis ance o he an ibio ic
mac olide ba ilomycin A1, a speci ic V-ATPase inhibi o . Fu he
expe imen s pe o med wi h a yeas ma mu an de ec i e in
i s V-ATPase ac i i y demons a ed ha he H+-PPase ac s by
unc ionally complemen ing he anspo unc ion o he o me
p o on pump. These esul s aise again he ques ion o he p esence
o bo h p o on pumps in ce ain o ganelles, such as he plan
acuole. Po en ial applica ions o his sys em in o de o elucida e
he oles o V-ATPases and/o H+-PPases a e also discussed.
EXPERIMENTAL
Yeas s ains
The YPC3 mu an s ain was gene a ed om he S. ce e isiae
haploid s ain W303-1A (MATa, ade2-1 can1-100 his3-11,15
leu2-3,112 p1-1,u a3-1) by he single-s ep ansplacemen
p ocedu e as desc ibed p e iously [21]. This mu an has he IPP1
gene coding o he cy osolic soluble PPase unde he con ol o
he yeas galac okinase (GAL1) p omo e . YPC4 was gene a ed
om S. ce e isiae mu an RS-1144 (W303-1A ma1::LEU2)
ollowing he same p ocedu e. Mu an RS-1144, p o ided by
P o esso Ram´
on Se ano (Uni e sidad Poli ´
ecnica de Valencia,
Valencia, Spain), was de i ed om s ain W303-1A by dis up ing
i s VMA1 (TFP1) gene wi h he yeas LEU2 casse e [22]. The
VMA1 gene o S.ce e isiae is ansla ed by p o ein splicing in o
wo di e en p o eins: he 69 kDa ca aly ic subuni o he acuola
H+-ATPase and a 50 kDa DNA endonuclease [23]. Vacuola
p o ease-de icien mu an BJ5457 was p o ided by P o esso
And ´
es Aguile a (Uni e sidad de Se illa, Se illa, Spain). This
mu an ca ies mu a ions bo h in he PEP4 gene and he PRB1
gene, coding o he yeas acuola p o eases P A (P o einase
A) and P B (P o einase B) espec i ely [24]. Yeas s ains used
in he p esen s udy a e desc ibed in Supplemen a y Table S1 a
h p://www.BiochemJ.o g/bj/437/bj4370269add.h m.
T ans o ma ion o YPC3 and YPC4
S. ce e isiae mu an s ains YPC3 and YPC4 we e ans o med
wi h plasmids pRS699b (nega i e con ol), pAVP1, pTcAVP1
and pTcGFPAVP1 by using he me hod desc ibed by Schies l and
Gie z [25]. All plasmids a e 2 μm-based de i a i es o he URA3-
con aining Esche ichia coli/S. ce e isiae shu le plasmid pRS699
[26], as desc ibed p e iously [21] (Supplemen a y Table S2
in [21]). These plasmids bea he yeas PMA1 p omo e o
cons i u i e exp ession o inse s [26]. Th ee di e en cons uc s
we e made using he sequence coding o he K+-dependen H+-
PPase om he highe plan A. haliana (AVP1): pAVP1, ca ying
an in on-less open eading ame cons uc o AVP1; pTcAVP1, a
5in- ame usion o he sequence coding o he N- e minal signal
pep ide o he T. c uzi H+-PPase (TcVP) wi h ha o AVP1; and
pTcGFPAVP1, an in- ame usion whe e he sequence coding o
yEGFP [yeas enhanced GFP (g een luo escen p o ein)] is placed
be ween he coding sequence o he TcVP N- e minal signal
pep ide and ha o AVP1. In addi ion o hese, plasmids pIPP1,
bea ing he whole open eading ame encoding he cy osolic
soluble PPase om yeas (IPP1), and pVMA1, bea ing he
genomic sequence o VMA1 coding o he A ca aly ic subuni o
he yeas V-ATPase, we e cons uc ed using he abo e men ioned
exp ession plasmid backbone (Supplemen a y Figu e S1 a
h p://www.BiochemJ.o g/bj/437/bj4370269add.h m). YPC3 and
YPC4 cells we e ini ially g own a 30◦C in galac ose-con aining
syn he ic medium [27] de oid o his idine (YPC3) o his idine and
leucine (YPC4); ans o man s we e selec ed by g owing cells on
2%aga pla es in cul u e medium wi hou his idine and u acil
(YPC3) o wi hou his idine, leucine and u acil (YPC4). So bi ol
(0.5 M) was added o he pla es whe e ans o man s o YPC4
we e selec ed as an osmo ic p o ec an .
Pheno ype complemen a ion es s
Complemen a ion s udies we e pe o med by p e-inocula ing 2 ml
o galac ose-con aining selec i e medium wi h ans o med cells
om he pla es and g owing o e nigh a 30◦C wi h agi a ion (200
e ./min). The ollowing day, 2 ml o glucose-con aining selec i e
medium o YPD medium [1%(w/ ) yeas ex ac /2%(w/ )
pep one/2%(w/ ) glucose] we e inocula ed wi h 20 μl (1:100
dilu ion) o cells p e iously g own on galac ose and hese we e
allowed o g ow as desc ibed abo e. This ea men is necessa y
o b ing down he PPase ac i i y associa ed wi h Ipp1p [28].
A e o e nigh g ow h on glucose, 10- old se ial dilu ions o he
cul u es we e made in s e ile wa e and 5 μl d ops o each dilu ion
we espo edon oYPDandYPGal[1%(w/ ) yeas ex ac /2%
(w/ ) pep one/2%(w/ ) galac ose] aga pla es con aining 50 mM
T is/Mes adjus ed o pH 5.5, 7.5 o 8. CaCl2(100 mM) o ZnCl2
(4 mM) was added o he pla es a pH 5.5 whe e indica ed. Pla es
we e ypically g own a 30◦C o 2–5 days.
Fluo escence mic oscopy
Vacuola lumen acidi ica ion was assessed by luo escence
s aining wi h quinac ine as desc ibed p e iously [29]. Cells we e
isualized a e 10 min o luo opho e accumula ion wi h a ully
au oma ed Leica DM6000B mic oscope (Leica Mic osys ems)
wi h FITC g een luo escence il e s (exci a ion il e 480/40 nm,
dich oma ic mi o 505 nm, supp ession il e 527/30 nm), a
100×objec i e and equipped wi h a cooled CCD (cha ge-coupled
de ice) came a (ORCA-AG, Hamama su Pho onics). Vacuola
memb ane labelling was ca ied ou wi h he i al lipophilic dye
FM4-64 as desc ibed p e iously [29]. Cells we e isualized 30
min a e incuba ion wi h he dye as desc ibed abo e, excep
ha Texas Red il e s (exci a ion il e 560/40 nm, dich oma ic
mi o 595 nm, supp ession il e 645/75 nm) we e used. In all
cases, cells we e also iewed using DIC (di e en ial in e e ence
con as ) mic oscopy o obse e cell mo phology.
Isola ion o yeas acuola memb anes
Colonies o ans o med YPC4 cells we e collec ed om a pla e
and liquid-g own up o s a iona y phase in galac ose-con aining
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Func ional complemen a ion o yeas acuola H+-ATPase by a H+-PPase 271
selec i e medium; hen, 4 ml o s a iona y cul u es we e used
o inocula e 400 ml o YPD adjus ed o pH 5.5 wi h 50 mM
T is/Mes and g own o e nigh o ea ly exponen ial phase (A660
was app oxima ely 1). Yeas acuoles we e ob ained om hese
cul u es using he me hod desc ibed by Robe s e al. [30]
Isola ion o o al memb ane ac ions om yeas
P epa a ion o memb ane ac ions om YPC4 cells ans o med
wi h he di e en plasmids we e ob ained by a modi ica ion o a
me hod desc ibed p e iously [21]. Yeas colonies we e collec ed
om a pla e and liquid-g own up o s a iona y phase in galac ose-
con aining selec i e medium; hen, 4 ml o s a iona y cul u es
we e used o inocula e 400 ml o YPD. A e o e nigh g ow h,
cells we e sedimen ed by cen i uga ion a 700 g o 10 min,
washed ho oughly wi h wa e , esuspended in 5 ml o ice-cold
bu e A [25 mM T is/HCl (pH 8), 10%glyce ol, 4 mM 2-
me cap oe hanol, 2 mM DTT (di hio h ei ol), 2 mM EDTA, 10
mM MgCl2, 1 mM benzamidine, 2 mM ε-aminocap oic acid and
1 mM PMSF], and homogenized by igo ous shaking wi h glass
beads. The homogena e was dilu ed up o 25 ml wi h bu e
B [10 mM T is/HCl (pH 7.6), 10%glyce ol, 2 mM DTT and
1 mM EDTA] and cen i uged o 10 min a 700 g o emo e
beads and deb is. The esul ing supe na an was cen i uged o
30 min a 40000 e ./min (Beckman 60Ti o o ) hus yielding a
o al memb ane pelle . This pelle was homogenized in s ipping
bu e [60 mM T is/HCl (pH 8), 12%glyce ol, 0.72 M KCl and
1.2 mM CaCl2] [31] and cen i uged o 20 min a 40000 e ./min
(Beckman 60Ti o o ). This pelle was subsequen ly washed wi h
bu e B and cen i uged [40000 e ./min (Beckman 60Ti o o )
o 20 min]. The inal pelle s we e esuspended and homogenized
in1mlo bu e B.
PPase ac i i y and H+- ansloca ion assays
Memb ane-associa ed PPase ac i i y was measu ed by
spec opho ome ic de ec ion o eleased phospha e [32].
P o on- ansloca ion ac i i ies we e assayed by moni o ing he
luo escence quenching o ACMA (9-amino-6-chlo o-2-me hoxy
ac idine) [33]. The s a is ical signi icance o he di e ences
obse ed be ween se s o ac i i y da a was de e mined by
S uden ’s unpai ed es s using he applica ion a ailable a
h p://g aphpad.com/quickcalcs/ es 1.c m.
Wes e n blo ing and o al p o ein es ima ion
Wes e n blo analysis was ca ied ou a e SDS/PAGE as
desc ibed p e iously [34]. Fo immunode ec ion o H+-PPases,
an a ini y-pu i ied polyclonal an ibody aised in abbi agains he
memb ane-bound PPase (TVP) om he bac e ium The mo oga
ma i ima [35] was used a a 1:200 dilu ion. Fo Vma1p, GFP and
Pma1p immunode ec ion, comme cial monoclonal an ibody 8B1
(Molecula P obes), a polyclonal ‘Li ing Colo s’ an ibody om
BD Biosciences and a polyclonal an ibody p o ided by P o esso
Ram´
on Se ano (Uni e sidad Poli ´
ecnica de Valencia, Valencia,
Spain) we e used espec i ely, all o hem a a 1:2000 dilu ion.
The o al p o ein concen a ion was es ima ed using he B ad o d
me hod [36] wi h o albumin as a s anda d.
RESULTS
Chimae as o AVP1 bea ing he N- e minal domain o TcVP induce
ba ilomycin esis ance in YPC3 cells
YPC3 is a yeas mu an whose IPP1 gene coding o he essen ial
cy osolic PPase is unde he con ol o he galac okinase (GAL1)
Figu e 1 D op es s o yeas mu an YPC3 cells g own in he p esence o he
mac olide inhibi o ba ilomycin A1 a pH 5.5 and 8.0 (A) and in he p esence
o 100 mM CaCl2o 4 mM ZnCl2a pH 5.5 (B)
Cells we e ans o med wi h plasmids pRS699b (Con ol-), pIPP1, pAVP1, pTcAVP1 and
pTcGFPAVP1 and g own as desc ibed in he Expe imen al sec ion. Se ial dilu ions o he
cul u es we e made in s e ile wa e and spo ed on o YPGal and YPD pla es con aining 2 μM
ba ilomycin A1 and bu e ed a he indica ed pH alues wi h 50 mM T is/Mes. Pla es con aining
Ca2+and Zn2+sal s we e adjus ed o pH 5.5 wi h he same bu e . In (A) 1:10, 1:100 and
1:1000 se ial dilu ions a e shown. In (B) 1:10 and 1:100 dilu ions a e shown. G ow h was
eco ded a e 2 days a 30◦C in all cases, excep o Zn2+pla es which we e g own o
4 days. No sensi i i y o pH 8 o o Ca2+o Zn2+was obse ed o YPC3 cells in he absence
o ba ilomycin A1 ( esul s no shown). ba ., ba ilomycin A1; w/o, wi hou .
p omo e , he e o e hese cells a e non- iable when glucose is
p esen as a ca bon sou ce. I has been shown p e iously ha
plan and bac e ial memb ane-bound H+-PPases can unc ionally
complemen he yeas cy osolic sPPase (Ipp1p), especially when
used o ce ain N- e minal signal pep ides [21,28]. Addi ion
o mac olide ba ilomycin A1 o YPC3 cells ans o med wi h
di e en plasmids bea ing cons i u i ely exp essed genes coding
o soluble o memb ane-bound PPases esul ed in sensi i i y o
neu al/alkaline pH and o he di alen ca ions Ca2+and Zn2+,
when g own on galac ose (Figu e 1), ha is, when Ipp1p is
exp essed. In con as , when cells we e g own on glucose in
he p esence o ba ilomycin, hose exp essing di e en chimae ic
de i a i es o AVP1, he K+-dependen H+-PPase om he highe
plan A. haliana, eco e he capaci y o g ow a pH 7.5–8.0
(Figu e 1A). In he p esence o 100 mM calcium a pH 5.6,
some g ow h was obse ed in YPC3 cells o e exp essing Ipp1p,
al hough hey became signi ican ly sensi i e o his ca ion. Cells
exp essing he di e en AVP1 chimae as showed a somewha
smalle sensi i i y o his CaCl2(Figu e 1B). In he p esence o
Zn2+, only hose cells ans o med wi h plasmids pTcAVP1 and
pTcGFPAVP1 exp essing in e nal memb ane- a ge ed chimae as
could g ow a e 4 days, pa icula ly he la e (Figu e 1B).
Chimae as o AVP1 bea ing he N- e minal domain o TcVP e e se
he sensi i i y o YPC4 Vma1−cells o neu al/alkaline pH, Ca2+
and Zn2+
In o de o ully asce ain whe he he obse ed ba ilomycin
A1 ole ance was ac ually b ough abou by complemen a ion
o V-ATPase p o on- anspo ac i i y, simila expe imen s o
hose desc ibed abo e we e pe o med wi h YPC4 cells. This
mu an also has IPP1 unde he con ol o he GAL1 p omo e
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Figu e 2 D op es s o yeas mu an YPC4 cells g own a pH 5.5, 7.5 and
8.0 (A) and in he p esence o 100 mM CaCl2o 4 mM ZnCl2a pH 5.5 (B)
Cells we e ans o med wi h plasmids pRS699b (Con ol-), pIPP1, pAVP1, pTcAVP1,
pTcGFPAVP1 and pVMA1 and g own as desc ibed in Expe imen al sec ion. Se ial dilu ions
o he cul u es we e made in s e ile wa e and spo ed on o YPGal and YPD pla es bu e ed a
he indica ed pH alues wi h 50 mM T is/Mes. Pla es con aining Ca2+and Zn2+sal s we e
adjus ed o pH 5.5 wi h he same bu e . In (A) 1:10, 1:100 and 1:1000 se ial dilu ions a e shown.
In (B) 1:10 and 1:100 dilu ions a e shown. G ow h was eco ded a e 4 days in all cases, excep
o hose a pH 8 which we e g own o 7 days. ba ., ba ilomycin A; w/o, wi hou .
bu , addi ionally, i has he gene coding o he ca aly ic subuni
VMA1 o he acuola H+-ATPase dis up ed. YPC4, like YPC3,
is unable o g ow on glucose, bu , as expec ed, when g own
on galac ose i exhibi s he ypical ange o Vma−pheno ypes:
e y high sensi i i y o neu al/alkaline pH alues and o Ca2+
and Zn2+ca ions (Figu es 2A and 2B). Howe e , when YPC4
cells we e ans o med wi h AVP1 and i s chimae ic de i a i es,
hey eco e ed he capaci y o g ow on glucose a pH 5.5 and
7.5, especially in he case o he TcGFPAVP1 chimae a. In he
p esence o Ca2+, only cells ans o med wi h plasmids pTcAVP1
and pTcGFPAVP1 we e able o g ow, whe eas only he la e
showed g ow h in he p esence o Zn2+.Again,asin hecaseo
YPC3 wi h ba ilomycin A1, he cons uc bea ing he gene coding
o Ipp1p could only suppo g ow h on glucose a pH 5.6 and in
he absence o di alen ca ions. On he o he hand, ans o ma ion
o YPC4 wi h VMA1 esul ed in he e e sal o he pheno ypes
associa ed wi h he dis up ion o his gene only in he p esence o
galac ose (Figu es 2A and 2B).
Exp ession o TcGFPAVP1 allows acidi ica ion o acuoles in YPC4
cells
To e i y ha AVP1 and i s de i a i es we e able o
p omo e acidi ica ion o in e nal compa men s, we analysed
he accumula ion o quinac ine, a i al dye ha concen a es
in acidic lumina. Figu e 3 shows ypical pa e ns o quinac ine
accumula ion in acuoles o YPC4 cells ans o med wi h
he di e en cons uc s es ed. No in e nal acidi ica ion could
be obse ed in cells exp essing he soluble cy osolic PPase
Ipp1p, whe eas cells exp essing AVP1 and TcAVP1 showed a
punc a e pa e n o g een luo escence sugges ing acidi ica ion
o p e acuola /endosomal compa men s. In con as wi h his,
cells ans o med wi h he plasmid pTcGFPAVP1 showed a
di e en luo escence pa e n, wi h a clea acuola accumula ion
Figu e 3 Vacuola acidi ica ion o ans o med YPC4 cells s udied by
accumula ion o quinac ine
Cells we e ans o med wi h he indica ed plasmids and g own as desc ibed in he Expe imen al
sec ion. Vacuole acidi ica ion was assessed by accumula ion o he luo escen p obequinac ine
as desc ibed p e iously [28]. Cells we e isualized in a Leica DM6000B mic oscope. The
le -hand column shows DIC mic oscopy o a ypical ield o ans o med YPC4 cells, he middle
column shows he pa e n o quinac ine accumula ion (obse ed wi h a FITC g een il e ) in he
same cells, and he igh -hand column shows he o e lap o bo h.
o quinac ine jus like YPC4 cells ans o med wi h plasmid
pVMA1 g own in galac ose. The o me cells showed a punc a e
g een luo escence pa e n, co esponding o he TcGFPAVP1
polypep ide, when isualized in he absence o quinac ine (see
below). I is impo an o poin ou ha some o he cells
ans o med wi h pTcGFPAVP1 exhibi ed clus e s o smalle
acuoles a he han he single la ge acuole usually obse ed
in cells exp essing Vma1p (see Figu e 3). These esul s a e in
ag eemen wi h he p e iously p esen ed d op es s (compa e wi h
Figu es 1 and 2), which show compa a i ely highe g ow h o
TcGFPAVP1-exp essing clones a neu al/alkaline pH alues and
in he p esence o Ca2+/Zn2+, hese ole ance pheno ypes being
s ic ly dependen on physiologically compe en acidic acuoles.
Exp ession o all AVP1 de i a i es allows no mal in e naliza ion o
he i al dye FM4-64
The i al luo escen dye FM4-64 binds o he plasma memb ane
and ollows he endocy ic pa hway o each he acuole [29].
FM4-64 in e naliza ion has been shown o be se e ely impai ed
in ma mu an s [37]. Figu e 4 shows labelling o acuole
memb anes in YPC4 cells a e incuba ion wi h FM4-64 o
30 min. I can be obse ed ha cells exp essing he AVP1
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Figu e 4 Endocy ic in e naliza ion o he luo escen ma ke FM4-64 in ans o med YPC4 cells
The a e o endocy osis was s udied by incuba ing ans o med YPC4 cells g own o ea ly exponen ial phase wi h FM4-64 o 15 min, as desc ibed p e iously [28]. Cells we e hen ans e ed o YPD
and isualized as desc ibed in Figu e 3 a e 30 min o incuba ion wi h he luo escen dye. In (A), he le -hand column shows DIC mic oscopy o a ypical ield o ans o med YPC4 cells, he middle
column shows he pa e n o FM4-64 labelling in he same cells, and he igh -hand column shows he o e lap o bo h. (B) YPC4 cells ans o med wi h plasmid pTcGFPAVP1. F om le - o- igh :
isualiza ion o he cells by DIC mic oscopy, wi h ed (Texas Red) and g een (FITC) il e s, o e lap o ed and g een il e mic opho og aphs, and o e all o e lap o he ee o me mic opho og aphs.
and TcGFPAVP1 p o eins ha e hei acuoles clea ly labelled,
whe eas cells exp essing TcAVP1 showed labelling o acuoles
and o he smalle esicles. Con ol cells exp essing Vma1p
also showed a clea pa e n o acuola memb ane s aining. No
in e nal labelling was ob ained in cells ans o med wi h pIPP1.
Cells ans o med wi h pTcGFPAVP1 we e also isualized wi h
a g een (FITC) luo escence il e showing a punc a e pa e n
ha sugges ed ei he a p e e en ial localiza ion o TcGFPAVP1
in in e nal memb anes o he han he acuola memb ane, o a
deg ada ion o he GFP moie y by acuola p o eases (Figu e 4B),
a possibili y ha was subsequen ly s udied by using a acuola
p o ease-de icien mu an (see below). In some o hese cells,
clus e s o small acidic esicles we e also obse ed. This e ec
had p e iously been desc ibed wi h YPC3 cells [21].
Memb ane-bound H+-PPases a e p esen in YPC4 acuoles
In o de o check whe he he di e en na u al and chimae ic
plan H+-PPases exp essed in YPC4 cells we e localized in
he acuoles, ac i i y assays and immunode ec ion expe imen s
we e ca ied ou wi h pu i ied acuole p epa a ions ob ained
om YPC4 cells ans o med wi h he di e en plasmids
desc ibed abo e. Cells ans o med wi h pTcGFPAVP1 showed
a cha ac e is ic ace o ACMA luo escence quenching ha
was dependen on he addi ion o PPi, his quenching was
e e sed by he addi ion o he H+/K+ionopho e nige icin.
Smalle quenching a es we e ob ained wi h cells ans o med
wi h pTcAVP1 and pAVP1 (Figu e 5A). Vacuoles om cells
ans o med wi h pVMA1 did no show PPi-dependen ACMA
quenching, jus like cells ans o med wi h plasmids pRS699b
and pIPP1 ( esul s no shown). When ATP-dependen p o on
anspo was eco ded, only acuoles isola ed om cells
ans o med wi h plasmid pVMA1 exhibi ed ACMA quenching
(Figu e 5A). This quenching was sensi i e o ba ilomycin
A1 ( esul s no shown). Memb ane-associa ed PPase ac i i y
le els de e mined o acuola memb anes ob ained om he
di e en YPC4 ans o man s we e consis en wi h he H+-
ansloca ion esul s (Figu e 5B), hus no PPase ac i i y was
de ec ed in samples ob ained om cells ans o med wi h
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Figu e 5 PPi- and ATP-dependen p o on-pumping ac i i y measu ed as quenching o ACMA luo escence (A), memb ane-associa ed PPihyd olysis ac i i y
(B) and Wes e n blo analyses (C) o acuola memb ane p epa a ions ob ained om ans o med YPC4 cells
(A) P o on- ansloca ion ac i i y was measu ed by eco ding he PPi-dependen (le -hand panels) and ATP-dependen ( igh -hand panels) ACMA luo escence quenching. The uppe panels show
ypical aces ob ained in hese expe imen s. The lowe panels show ini ial a es o luo escence quenching o he di e en p epa a ions. Values a e means +
−S.E.M. o h ee independen expe imen s
(
n
=3) and as e isks indica e di e ences ha a e s a is ically signi ican (P<0.005). n.d., no de ec ed. App oxima ely 30 μg o p o ein was used o hese assays. (B) Speci ic PPihyd olysis
ac i i y associa ed wi h acuola memb anes ob ained om YPC4 cells ans o med wi h he di e en plasmids es ed. Ac i i y alues shown a e means +
−S.E.M. (
n
=3) using p epa a ions ob ained
om YPC4 cells ans o med wi h pRS699b as a e e ence. As e isks indica e di e ences ha a e s a is ically signi ican (P <0.005). Open and closed ba s ep esen ac i i ies in he absence and
p esence o po assium chlo ide espec i ely. n.d., no de ec ed ac i i y. (C) Immunode ec ion o AVP1 (uppe panel) and yeas Vma1p (middle panel) in acuola memb anes o ans o med YPC4
cells. App oxima ely 30 μg o p o ein was loaded pe lane. As e isks indica e bands associa ed wi h chimae ic p o ein TcGFPAVP1 (app oxima ely 120 kDa) and AVP1 (72 kDa).
plasmids pRS699b, pIPP1 and pVMA1. Samples om cells
exp essing he di e en AVP1 chimae as did show PPase ac i i y
ha was inc eased by he addi ion o K+, a ea u e o AVP1
[19]. Immunode ec ion in Wes e n blo s o acuola memb anes
o he di e se na i e and chimae ic plan H+-PPases used
in he p esen s udy (Figu e 5C) ga e somewha unp edic ed
esul s despi e he use o p o ease inhibi o cock ails du ing he
isola ion p ocedu e, hus a polypep ide o he expec ed size o
he na i e AVP1 p o ein (app oxima ely 72 kDa) was ob ained
in he case o cells ans o med wi h pAVP1, pTcAVP1 and
pTcGFPAVP1; addi ionally, ano he band o app oxima ely 64
kDa was p esen in hese p epa a ions. The ela i e amoun s
o H+-PPases immunode e c ed in hese p epa a ions we e,
howe e , in acco dance wi h he es ima ed ac i i y alues. A
mino band o app oxima ely 120 kDa, he size expec ed o
TcGFPAVP1, could also be de ec ed only in samples ob ained
om cells ans o med wi h plasmid pTcGFPAVP1 (compa e wi h
Figu e 5C).
Vacuola p o eases a e in ol ed in he p ocessing o na i e AVP1
and i s chimae ic de i a i es a ge ed o he acuola memb ane
The possibili y ha AVP1 and i s chimae as migh be subjec ed
o p o eoly ic p ocessing by acuola p o eases in i o was
in es iga ed by using he acuola -p o ease-de icien yeas mu an
BJ5457 [24]. Immunode ec ion analyses ca ied ou wi h o al
memb ane p epa a ions ob ained wi h mu an s YPC4 and BJ5457
ans o med wi h plasmids pAVP1, pTcAVP1 and pTcGFPAVP1
using an an ibody agains a bac e ial memb ane-bound PPase
(an i-TVP) showed ema kable esul s (Figu e 6A). Thus, in
he case o YPC4, a polypep ide o app oxima ely 72 kDa
was de ec ed in all cases, al hough in cells ans o med wi h
pTcGFPAVP1, wo mo e bands o app oxima ely 120 kDa and 64
kDa we e clea ly ecognized by he an i-TVP an ibody. Pep ide
mass inge p in analysis o he 120 kDa polypep ide by MALDI–
TOF (ma ix-assis ed lase -deso p ion ioniza ion– ime-o - ligh )
MS showed ha i ac ually co esponded o a usion be ween
GFP and AVP1 ( esul s no shown). Consis en ly, an an ibody
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Figu e 6 Wes e n blo analysis (A) and memb ane-associa ed PPihyd olysis ac i i y (B) o o al memb ane p epa a ions ob ained om ans o med YPC4
and BJ5457 cells, and luo escence mic oscopy isualiza ion o YPC4 and BJ5457 cells ans o med wi h pTcGFPAVP1 (C)
(A) Immunode ec ion o AVP1 and de i a i es wi h an ibodies agains a memb ane-bound PPase (an i-TVP, uppe panel) and GFP (an i-GFP) in o al memb anes o YPC4 (le -hand panels) and
BJ5457 ( igh -hand panels) cells ans o med wi h plasmids pRS699b, pIPP1, pAVP1, pTcAVP1 and pTcGFPAVP1. App oxima ely 70 μg o p o ein was loaded pe lane. As e isks indica e bands
associa ed wi h chimae ic p o ein TcGFPAVP1 (app oxima ely 120 kDa) and AVP1 (72 kDa). (B) Speci ic PPihyd olysis ac i i y associa ed wi h o al memb anes ob ained om YPC4 and BJ5457
cells ans o med wi h he di e en plasmids es ed. Ac i i y alues shown a e means +
−S.E.M. (
n
=3) using p epa a ions ob ained om cells ans o med wi h pRS699b as a e e ence. As e isks
indica e di e ences ha a e s a is ically signi ican (P<0.005). Open and closed ba s ep esen ac i i ies in he absence and p esence o po assium chlo ide espec i ely. n.d., no de ec ed. (C) YPC4
and BJ5457 cells we e ans o med wi h plasmid pTcGFPAVP1 and isualized in a Leica DM6000B mic oscope. Le -hand column shows DIC mic oscopy o a ypical ield o cells, he middle column
shows he pa e n o g een luo escence dis ibu ion (FITC g een il e ) in he same cells, and he igh -hand column shows he o e lap o bo h. Whi e a owheads indica e acuola memb anes.
agains GFP (an i-GFP) only ecognized he 120 kDa band. In
he case o mu an BJ5457, he an i-TVP an ibody ecognized
polypep ides o app oxima ely 72 kDa only in p epa a ions
ob ained wi h cells ans o med wi h plasmids pAVP1 and
pTcAVP1, whe eas a single polypep ide o app oxima ely 120
kDa was de ec ed in memb anes o cells ans o med wi h plasmid
pTcGFPAVP1. The an i-GFP an ibody could only de ec he la e
(Figu e 6A).
Analysis o memb ane-associa ed PPihyd oly ic ac i i y
showed ha , in YPC4, he p esence o GFP a he N- e minus
o AVP1 inc eased he speci ic ac i i y o he esul ing p o ein
3–4- old wi h espec o AVP1 and TcAVP1. In mu an BJ5457,
he speci ic PPase ac i i ies associa ed wi h AVP1, TcAVP1 and
TcGFPAVP1 we e signi ican ly inc eased wi h espec o he
same p o eins exp essed in YPC4. The inc eases we e much
highe in he cases o AVP1 (app ox. 6- old) and TcAVP1
(app ox. 4- old) han in he case o TcGFPAVP1 (app ox. 1.7- old)
(Figu e 6B).
Fluo escen mic oscopy isualiza ion o YPC4 and BJ5457
cells ans o med wi h plasmid pTcGFPAVP1 ema kably showed
di e en pa e ns o g een luo escence dis ibu ion, hus, in YPC4
cells, a punc a e pa e n was obse ed, sugges ing a p e e en ial
accumula ion o g een luo escence in in e nal memb ane sys ems
o he han he acuola memb ane. In con as , g een luo escence
mos ly associa ed wi h he la e was clea ly obse ed in BJ5457
cells (Figu e 6C).
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DISCUSSION
In p e ious s udies, we emula ed in yeas he cy osolic PPi
me abolism o he plan cell, i.e. absence o a soluble cy osolic
PPase, so ha he PPigene a ed by he anabolism was e icien ly
hyd olysed by a memb ane-embedded PPi-dependen p o on
pump [21,28]. In he p esen s udy, we ha e aken one s ep o wa d
using a me abolic backg ound in which he ATP-dependen p o on
pump o yeas in e nal memb anes, he V-ATPase, has been ei he
chemically inhibi ed o unc ionally impai ed by mu a ion o a
gene encoding an essen ial ca aly ic subuni . The esul s ob ained
s ongly sugges ha a dis inc ype o PPi-dependen p o on
pump can e icien ly subs i u e o he V-ATPase o S. ce e isiae
in di e se physiological scena ios. Thus o e exp ession o he
K+-dependen H+-PPase om he highe plan A. haliana,a
p o ein ha occu s along wi h a V-ATPase in plan onoplas
[18], alle ia es he ange o pheno ypes associa ed wi h he lack
o he la e . The p esence o an app op ia e N- e minal domain
p o ed o be impo an in o de o obse e he op imal pheno ypic
complemen a ion in ou sys em. The N- e minal domain wi h
signal sequence ea u es o a p o is an H+-PPase, when used
o AVP1, al e s he subcellula dis ibu ion o he la e so ha
i becomes p e e en ially loca ed in in e nal memb anes o yeas
[21]. No su p isingly, as shown in he p esen s udy, chimae ic
p o eins o AVP1, cons uc ed wi h ha signal pep ide (TcAVP1
and TcGFPAVP1), unc ionally subs i u ed o he V-ATPase mo e
e icien ly han he na i e AVP1 p o ein wi h no ex a N- e minal
domain. The di e ence in a ou o TcGFPAVP1 compa ed wi h
TcAVP1 is p obably due o he highe o e all exp ession le els
a ained by he o me (see below). Ac ually, unde non- es ic i e
condi ions owa ds Vma−de ec s, TcGFPAVP1 suppo s g ow h
o YPC4 mo e e icien ly han TcAVP1 (Figu e 2A) which means
ha he o me is mo e e icien a complemen ing he pheno ype
associa ed wi h he absence o Ipp1p. Addi ion o GFP a he N-
e minus o some memb ane p o eins has been p e iously epo ed
ei he because i s abilizes p o eins exp essed in he e ologous
sys ems o because he p esence o GFP a he C- e minus yields
polypep ides wi h no ac i i y [21,38,39]. In ac , bo h assump ions
a e ue o heplan H
+-PPase AVP1 exp essed in yeas , as he
p esen s udy and ou p e ious s udy [21] demons a es.
The esul s wi h bo h ba ilomycin- ea ed YPC3 and YPC4
cells also showed ha no e sion o he e ologously exp essed
AVP1 was able o complemen Vma−pheno ypes in galac ose,
e en hough he co esponding genes a e unde he con ol o he
cons i u i e PMA1 p omo e . Simila ly, when he pa en al s ains
(ba ilomycin- ea ed W303-1A and RS-1114) we e ans o med
wi h he same plasmids no complemen a ion o he V-ATPase
by AVP1 was obse ed ei he on galac ose o on glucose
( esul s no shown). These esul s sugges ed ha AVP1 canno
pump p o ons when he soluble PPase is p esen in he cy osol,
p obably due o he compe i ion o he subs a e, he e o e
i was necessa y o b ing down he ac i i y o he la e o
negligible le els in o de o ha e he o me e icien ly wo king
in i o. YPC3 and YPC4 we e designed and gene a ed so
ha his could be accomplished by ans e ing he cells om
galac ose o glucose and allowing hem o g ow o se e al
hou s. Ac ually, his e lec s he me abolic scena io in cells
o plan g een issues, whe e sPPases a e es ic ed o ene gy-
linked o ganelles (mi ochond ia and chlo oplas s) and, p obably,
nuclei [17], al hough i mus be men ioned ha o he PPi-
u ilizing enzymes in ol ed in cen al ca bon me abolism, such
as a PPi-dependen phospho uc okinase, a e also p esen in
he plan cell cy osol [40]. All in all, hese esul s sugges ha he
p esence o ac i e soluble PPases is incompa ible wi h H+-PPase
unc ions, and helps o explain why he o me a e no ound
in he cy osol o euka yo ic cells i H+-PPases a e p esen in
endomemb anes.
Acidi ica ion o in e nal compa men s o YPC4 cells was
s udied wi h he luo escen p obe quinac ine, which accumula es
in acidic acuoles. TcGFPAVP1 was able o acidi y he acuole in
YPC4 cells, al hough i also seemed o induce some agmen a ion
o his compa men . This has al eady been epo ed o YPC3
cells and may be ela ed o he accumula ion o his he e ologous
chimae ic p o ein in he in e nal compa men s o yeas cells
[21]. I should be no ed in his espec ha a mo e o less
se e e acuola agmen a ion akes place in yeas unde se e al,
and no necessa ily pa hological, scena ios. In con as , cells
ans o med wi h AVP1 and TcAVP1 showed a punc a e pa e n
o quinac ine accumula ion which p obably means acidi ica ion o
p e acuola /endosomal esicles. This pa e n was mo e
signi ican in he case o TcAVP1, p obably due o he e ec
o he addi ion o he N- e minal signal domain added o he la e
ha a ou s accumula ion o his chimae ic p o ein in in e nal
memb anes o yeas [21]. In he p esence o quinac ine, cells
exp essing TcGFPAVP1 only exhibi ed he g een luo escence
associa ed wi h he dye; howe e , hese cells we e also expec ed
o show g een luo escence due o he GFP. Consequen ly, hey
we e also isualized in he absence o quinac ine showing, in
his case, a punc a e pa e n o luo escence (see below). This
sugges s ha quinac ine luo escence masks ha o GFP in ou
sys em when bo h a e moni o ed simul aneously.
Resul s wi h FM4-64 a e consis en wi h hose ob ained wi h
quinac ine in he case o TcGFPAVP1; howe e , hey u he
sugges ha ull acidi ica ion o he acuole may no be absolu ely
essen ial o ha e he endocy ic sys em ope a ional a he igh pace,
acidi ica ion o he p e acuola /endosomal compa men s being
su icien . The ac ha he g een luo escence obse ed in YPC4
cells ans o med wi h plasmid pTcGFPAVP1 does no necessa ily
co-localize wi h he ed luo escence associa ed wi h acidic
esicles u he sugges s ha ei he TcGFPAVP1 polypep ide
is no signi ican ly ansloca ed o he acuola memb ane o
he GFP moie y o his chimae a has been los , p esumably by
p o eolysis.
Biochemical analysis o acuola memb ane p epa a ions om
YPC4 ans o med wi h he di e en plasmids es ed showed ha
AVP1 and all o i s chimae ic de i a i es we e he e ologously
exp essed and loca ed, a leas pa ially, in he acuoles,
as demons a ed by he da a o memb ane-associa ed H+-
ansloca ing and PPase ac i i ies, as well as by immunode ec ion
analysis o acuola memb ane p epa a ions. Ac i i y da a we e
consis en wi h he pheno ypes obse ed, hus TcGFPAVP1
showed highe PPase and H+- ansloca ing ac i i ies han
TcAVP1 which, in u n, p oduced highe ac i ies han he na i e
AVP1. Immunode ec ion wi h he an i-TVP an ibody e ealed ha
TcAVP1 and TcGFPAVP1 ga e highe exp ession le els in his
compa men han AVP1. This is consis en wi h p e ious epo s
ega ding he ole o he N- e minal signal domain o T. c uzi
H+-PPase on he subcellula dis ibu ion o AVP1 [21]. On he
o he hand, a signi ican p ocessing o all o he p o eins examined
was also ound; ac ually, only a iny amoun o a polypep ide o
app oxima ely 120 kDa, co esponding o a usion o GFP and
AVP1, was de ec ed, and ano he band o app oxima ely 64 kDa
was consis en ly obse ed in all cases. This e idence sugges ed
ha acuola p o eases may be in ol ed in he clea age o AVP1
and i s de i a i es, wi h a concomi an deg ada ion o he GFP in
he case o TcGFPAVP1. In o de o s udy his si ua ion, he
acuola -de icien yeas mu an BJ5457 [24] was ans o med
wi h plasmids pAVP1, pTcAVP1 and pTcGFPAVP1 and o al
memb ane p epa a ions ob ained wi h he esul ing s ains we e
analysed and compa ed wi h p epa a ions ob ained wi h YPC4
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Func ional complemen a ion o yeas acuola H+-ATPase by a H+-PPase 277
cells ans o med wi h he same plasmids. Immunochemical
analysis s ongly sugges ed ha acuola p o eases a e in ol ed
in he p ocessing o AVP1 and chimae ic de i a i es obse ed in
mu an YPC4. These esul s, along wi h he memb ane-associa ed
PPase ac i i y da a, u he suppo he idea ha , al hough AVP1
and TcAVP1 can be ound in YPC4 acuoles, a high p opo ion
o hese polypep ides is subjec ed o p o eolysis. This e ec is
much smalle in he case o TcGFPAVP1, hus sugges ing ha
he combina ion o he T. c uzi H+-PPase N- e minal signal
pep ide and yEGFP is mo e e icien han he signal pep ide
alone a al e ing he subcellula dis ibu ion o he esul ing
chimae a. Fluo escence mic oscopy suppo ed he da a ob ained
by immunode ec ion and ac i i y assays, hus, unlike in mu an
BJ5457, he g een luo escence co esponding o TcGFPAVP1 in
mu an YPC4 showed a pa e n no clea ly associa ed wi h he
acuola memb ane. The e a e wo con ibu ions o his e ec
acco ding o ou esul s: on he one hand, a signi ican ac ion
o TcGFPAVP1 is a ge ed o o he in e nal memb anes and, on
he o he hand, he polypep ide loca ed in he acuola memb ane
is subjec ed o p o eolysis so ha mos o he GFP moie y is
deg aded. The e o e he ole o acuola p o eases p o ides an
explana ion o why he luo escence associa ed wi h GFP is no
loca ed in he acuola memb ane and u he sugges s ha he
p o on pump esponsible o he acidi ica ion o he acuole in
YPC4 cells ans o med wi h pTcGFPAVP1 is ac ually a na i e-
like AVP1 p o ein and no he chimae ic de i a i e.
Taken oge he ou esul s show ha he PPi-dependen p o on
pump AVP1 can alle ia e he mos cha ac e is ic pheno ypes
associa ed o he lack o V-ATPase in yeas , especially when
he o me is p e e en ially a ge ed o in e nal memb anes by
a aching an app op ia e signal pep ide o i s N- e minus [21].
In he opinion o he au ho s, his is he i s di ec p oo ha
aH
+- ansloca ing PPase can sus ain g ow h and signi ican
acidi ica ion o in acellula lumina in he absence o any V-
ATPase ac i i y.
These esul s aise ano he ques ion ela ed o he occu ence
o wo p o on pumps in he same subcellula loca ion in ce ain
o ganisms, one o hem being an ex emely complex molecula
machine ( he V-ATPase), and he o he a much simple memb ane
pump. The e a e nume ous ecen da a suppo ing ha V-
ATPases may play mo e oles han he acidi ica ion o in e nal
compa men s, such as he pa icipa ion o he V0domain in
memb ane usion [41] and o he s [42]. On he o he hand,
he V-ATPase complex may o e many mo e possibili ies o
ac i i y modula ion and, hus, may be mo e inely uned han
he compa a i ely simple H+-PPase. Addi ionally, epo s o
co- egula ion and physical in e ac ions be ween V-ATPases and
H+-PPases in plan onoplas may complica e u he he ask
o disc imina ing he espec i e oles o bo h pumps [18]. A
yeas exp ession sys em like he one in he p esen s udy can help
asce ain he impo ance o di e en V-ATPase polypep ides o
whole domains in moonligh ing unc ions independen o lumen
acidi ica ion.
In any case, he H+-PPases p obably imply an adap i e
ad an age o he o ganisms whe e hey occu because hey o e
an al e na i e pump o gene a e p o on g adien s in in e nal
o ganelles by using an abundan by-p oduc o anabolism, PPi.
[43]. This is likely o be a c i ical issue unde condi ions ha
equi e he op imiza ion o cellula ene gy usage. In his espec ,
i is wo hwhile poin ing ou ha he H+-PPase seems o be he
main onoplas pump in o he plan sys ems, such as g ape be y
cells [44].
The yeas sys em desc ibed in he p esen wo k may ha e
bio echnological applica ions, hus i is a good expe imen al
sys em o accomplish s udies o andom mu agenesis aimed a
ob aining enhanced e sions o AVP1 and o he PPi-dependen
ion pumps. On he o he hand, YPC4 ans o med wi h di e se
H+-PPases migh be a use ul sys em o check o possible oles
o he han he acidi ica ion o in e nal o ganelles p oposed o
ungal and animal V-ATPases, and i may be a sui able sys em o
s udy he e ec o H+-PPases inhibi o s in i o, as hese p o on
pumps occu in mic o-o ganisms esponsible o diseases such
as mala ia, leishmaniasis, Chagas disease and sleeping sickness
[15,45]. Finally, his expe imen al sys em en isages no el ways
o enginee ing cell ole ance o mac olides, a class o d ugs o
inc easing ele ance in he apeu ics o a ange o diseases in
which cellula p o on homoeos asis may play c ucial oles, such
as cance .
AUTHOR CONTRIBUTION
Jos´
eP
´
e ez-Cas i˜
nei a ca ied ou mos o he expe imen al wo k and w o e he pape ;
Agus ´
ın He n´
andez gene a ed he YPC4 mu an and co ec ed he pape ; Roc´
ıo D ake
pe o med p elimina y s udies; and Au elio Se ano supe ised he wo k and w o e he
inal e sion o he pape .
ACKNOWLEDGEMENTS
We hank P o esso Ram´
on Se ano o p o iding he an i-Pma1p an ibody and yeas
mu an s ain RS-1144, and P o esso And ´
es Aguile a o p o iding yeas s ain BJ5457.
We also hank D Alicia O ea o excellen echnical assis ance wi h luo escence
mic oscopy and Ms Isabel Jim´
enez o yeas cul u ing.
FUNDING
This wo k was suppo ed by he Regional Andalusian Go e men and he Spanish
Minis e io de Ciencia e Inno aci´
on inancial suppo o PAIDI g oup BIO-261 [g an
numbe s P07-CVI-03082, BFU2007-61887 and BFU2010-15622], pa ially unded by he
EU FEDER (Fondo Eu opeo de Desa ollo Regional) p og amme.
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c
The Au ho s Jou nal compila ion c
2011 Biochemical Socie y