A plant proton-pumping inorganic pyrophosphatase functionally complements the vacuolar ATPase transport activity and confers bafilomycin resistance in yeast

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]). c The Au ho s Jou nal compila ion c 2011 Biochemical Socie y www.biochemj.o g Biochemical Jou nal 270 J. R. P´ e ez-Cas i˜ 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 c The Au ho s Jou nal compila ion c 2011 Biochemical Socie y 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 c The Au ho s Jou nal compila ion c 2011 Biochemical Socie y 272 J. R. P´ e ez-Cas i˜ nei a and o he s 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 c The Au ho s Jou nal compila ion c 2011 Biochemical Socie y Func ional complemen a ion o yeas acuola H+-ATPase by a H+-PPase 273 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 c The Au ho s Jou nal compila ion c 2011 Biochemical Socie y 274 J. R. P´ e ez-Cas i˜ nei a and o he s 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 c The Au ho s Jou nal compila ion c 2011 Biochemical Socie y Func ional complemen a ion o yeas acuola H+-ATPase by a H+-PPase 275 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). c The Au ho s Jou nal compila ion c 2011 Biochemical Socie y 276 J. R. P´ e ez-Cas i˜ nei a and o he s 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 c The Au ho s Jou nal compila ion c 2011 Biochemical Socie y 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. REFERENCES 1 Kane, P. M. (2006) The whe e, when, and how o o ganelle acidi ica ion by he yeas acuola H+-ATPase. Mic obiol. Mol. Biol. Re . 70, 177–191 2 Nishi, T. and Fo gac, M. (2002) The acuola H+-ATPases: na u e’s mos e sa ile p o on pumps. Na . Re . Mol. Cell Biol. 3, 94–103 3 Fo gac, M. (2007) Vacuola ATPases: o a y p o on pumps in physiology and pa hophysiology. Na . Re . Mol. 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