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Role o he yeas mul id ug anspo e Qd 2 in ca ion homeos asis and he oxida i e
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s ess esponse.
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Gabino RíosÁ, Ma c Cabedo, Bal asa Rull, Lynne Yenush, Ramón Se ano and José M.
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Mule .
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Ins i u o de Biología Molecula y Celula de Plan as (IBMCP), Uni e si a Poli ècnica
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de València-CSIC, ES-46022 Valencia, Spain.
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ÁP esen add ess: Ins i u o Valenciano de In es igaciones Ag a ias (IVIA), ca e e a
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Moncada-Náque a km 4.5, ES-46113 Moncada, Valencia, Spain.
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Co espondence: José M. Mule ,
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IBMCP
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Uni e si a Poli ècnica de València,
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Camino de Ve a S/N
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46022 Valencia (Spain)
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Tel.:+34 96 3877775; ax: +34 96 3877859;
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e-mail: [email p o ec ed]
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Keywo ds: Ion anspo , coppe homeos asis, oxida i e s ess.
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Running i le: Role o Qd 2 in coppe homeos asis and oxida i e s ess.
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2
1
Abs ac :
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We ha e iden i ied QDR2 in a sc eening o genes able o con e ole ance o sodium
4
and/o li hium s ess upon o e exp ession. Qd 2 is a mul id ug anspo e o he majo
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acili a o supe amily, o iginally desc ibed o i s abili y o anspo he an imala ial
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d ug quinidine and he he bicide ba ban. In o de o iden i y i s physiological subs a e,
7
we ha e sc eened o pheno ypes dependen on QDR2 and ound ha Qd 2 is able o
8
anspo mono alen and di alen ca ions wi h poo selec i i y, as shown by g ow h
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es s and he de e mina ion o in e nal ca ion con en . Mo eo e , s ains o e exp essing
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o lacking QDR2 also exhibi pheno ypes when eac i e oxygen species p oducing
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agen s, such as hyd ogen pe oxide o menadione, we e added o he g ow h medium.
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We ha e also ound ha he p esence o coppe and hyd ogen pe oxide ep ess he
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exp ession o QDR2. In addi ion, he coppe up ake o a qd 2 mu an s ain is simila o
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a wild ype, bu he ex usion is clea ly impai ed. Based on ou esul s, we p opose ha
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ee di alen coppe is he main physiological subs a e o Qd 2. As coppe is a
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subs a e o se e al edox eac ions ha occu wi hin he cy oplasm, his unc ion in
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coppe homeos asis explains i s ole in he oxida i e s ess esponse.
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3
In oduc ion:
1
2
The yeas o e exp ession app oach has been a powe ul echnique o iden i y he genes
3
de ining he molecula mechanisms unde lying ion homeos asis in yeas ( e iewed in
4
A ino e al., 2010). This echnique has allowed he iden i ica ion o he HAL genes, ha
5
comp ise egula o s o po assium anspo such as HAL1 (Gaxiola e al ., 1992) and
6
HAL3 (Fe ando e al., 1995), he HAL4 and HAL5 p o ein kinases (Mule e al., 1999),
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a ge s o ion oxici y as HAL2 (Mu guía e al., 1995) and he HAL6-10 ansc ip ion
8
ac o s (Mendizabal e al., 1998), among hem he calcineu in dependen ansc ip ion
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ac o CRZ1/HAL8/TCN1 (Ma heos e al., 1997; S a hopoulos and Cye , 1997). This
10
echnique has p o en o be e y powe ul o iden i y genes encoding o soluble
11
p o eins, bu has been less success ul in iden i ying genes encoding anspo e s o
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memb ane p o eins in gene al. This could be due o some echnical p oblems as genes
13
encoding memb ane p o eins a e usually unde - ep esen ed in cDNA o genomic
14
lib a ies (ou unpublished obse a ions). The main anspo e s de e mining ion
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homeos asis in Sa c c h a o my c es c e e i si a e a e he p o on pump ATPase Pma1 (Se ano
16
e al., 1986), esponsible o he c ea ion o he p o on g adien , and he high a ini y
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po assium anspo sys em encoded by he TRK1 and TRK2 genes (Gabe e al., 1988).
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This sys em is esponsible o main aining he in e nal con en o po assium a ound 100-
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200 mM independen ly o he po assium concen a ion in he medium, and he e o e is
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he main consume o he memb ane po en ial gene a ed by Pma1 (Mad id e al., 1998).
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Sodium and li hium a e oxic o Sa c c h a o my c es c e e i si a e. The main p o ein
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esponsible o ex usion o hese oxic ca ions om he cy oplasm is Ena1 (Ha o e al.,
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1991). In addi ion, he plasma memb ane sodium/p o on an ipo e Nha1 (P io e al.,
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1996; Kinclo a-Zimme manno a e al. 2006) pa icipa es in sodium ex usion a acidic
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pH and he sodium/p o on an ipo e Nhx1 localized in he p e acuola compa men is
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he majo anspo e in ol ed in sodium compa imen aliza ion (Nass and Rao, 1998).
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E en wi h his appa en nega i e selec ion agains memb ane p o eins, in a sc eening o
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yeas genes able o con e sal ole ance upon o e exp ession, we ha e iden i ied QDR2,
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a mul id ug esis ance gene belonging o he majo acili a o supe amily (MFS)
31
(Go eau e al., 1997). We isola ed QDR2 in ou sc eening based on i s abili y o
32
imp o e g ow h unde sodium s ess. MFS anspo e s a e ubiqui ously p esen in
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euka yo e and bac e ial genomes, and can unc ion as p o on-g adien coupled
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an ipo e s, unipo e s o sympo e s (Pao e al., 1998). In mos cases he mul id ug
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esis ance amily encodes anspo sys ems which d i e he ex usion o hyd ophobic
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molecules, mos o hem no p esen in he na u al en i onmen o he o ganism. The
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Qd 2 p o ein is localized in he plasma memb ane and sequence p edic ion indica es ha
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i con ains 12 ansmemb ane segmen s. The QDR2 gene belongs o he DHA1 amily
39
and is no conse ed in ela ed yeas s such as Ashbya gossipii o Kluy e omices lac is
40
(Gbelska e al., 2006). Qd 2 was o iginally iden i ied o i s abili y o con e ole ance
41
o he an imala ial d ug quinidine and he he bicide ba ban (Va gas e al., 2004). A la e
42
epo indica ed ha Qd 2 can also anspo he an icance agen s cispla in and
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bleomicin (Ten ei o e al., 2005). None o hese molecules a e p esen in he
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en i onmen , so he physiological unc ion o Qd 2 emains o be de e mined. I has
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been p oposed ha MFS anspo e s could also pa icipa e in ion homeos asis.
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Speci ically, i has been p oposed ha some MFS p o eins may con ibu e o sodium
47
ex usion (K ulwich e al., 2005). Qd 2 has also been p oposed o ha e a ole in
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po assium homeos asis (Va gas e al., 2007). In addi ion, he ou iden i ied subs a es
49
o Qd 2 a e posi i ely cha ged a physiological pH, sugges ing ha he physiological
50
4
ole o Qd 2 may be ela ed o ca ion homeos asis. In hese epo , we p esen e idence
1
ha Qd 2 is able o anspo mono alen and di alen ca ions, including ansi ion
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me als, among hem, coppe . In he en i onmen coppe is usually ound as Cu2+, owing
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mainly o he ac ha Cu+ is e y insoluble and is oxidized by O2, and hus, i s
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bioa alibili y is low. Ex acellula coppe is educed by he F 1/2 i on educ ase
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sys em, hen Cu+ is anspo ed o he cy oplasm by C 1 (Puig and Thiele, 2002).
6
Coppe is an essen ial mic onu ien o yeas , as i is inco po a ed in he me allic co e
7
o an ioxidan enzymes, such as Sod1, and is also p esen in some subuni s o he
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mi ochond ial cy och ome c oxidase ( e iewed in Bleackley and MacGilli ay, 2011).
9
Ano he ea u e o coppe is ha he edox pai o Cu+ and Cu2+, anging om +0,2 o
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+0,8 is ex emely use ul o biological eac ions (F aus o da Sil a and Williams, 2001)
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bu , on he o he hand, hese edox eac ions can lead o he o ma ion o hyd oxyl
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adicals h ough he Fen on eac ion (Valko e al., 2005). Coppe homeos asis should be
13
igh ly con olled, as i can be e y oxic due o unspeci ic binding o sulphu , oxygen
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and imidazole ligands (Culo a, 2010). Ou da a indica es ha Qd 2 ex udes di alen
15
coppe . This is, o da e, he i s desc ip ion o a yeas p o ein able o ex ude coppe .
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P e ious epo s ha e shown ha Qd 2 is able o anspo non-physiological subs a es,
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o po assium unde e y pa icula condi ions. He e we p opose ha coppe is he main
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physiological subs a e o Qd 2. As coppe is a subs a e o some dele e ious edox
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eac ions ha can occu inside he cell, his ole in coppe homeos asis also ela es Qd 2
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o edox homeos asis.
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5
1
Ma e ials and Me hods:
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3
Yeas s ains and cul u e condi ions:
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S anda d me hods o yeas cul u e and manipula ion we e used (Gu h ie and Fink,
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1991). The BY4741 s ains lacking QDR2 o QDR1 we e ob ained om he Eu osca
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collec ion (F ank u , Ge many). YPD medium con ained 2% glucose, 2% pep one, and
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1% yeas ex ac . SD medium (syn he ic minimal medium) con ained 2% glucose, 0.7%
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yeas ni ogen base (Di co) wi hou amino acids, 50 mM succinic acid adjus ed o pH
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5.5 wi h T is, and he amino acids, pu ine and py imidine bases equi ed by he s ains.
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G ow h assays we e pe o med on solid media by spo ing se ial dilu ions o sa u a ed
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cul u es on o pla es wi h he indica ed composi ion. The indica ed sal s we e added a
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he indica ed concen a ion in each case, wi h he excep ion o H2O2, and menadione,
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ha we e added a e au ocla ing.
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15
Isola ion o QDR2 and plasmid cons uc ion:
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The sc een o ole ance o sodium and li hium has been desc ibed p e iously (Mule e
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al., 1999). QDR2 was isola ed om he genomic clone PM54 as a Bgl II agmen ha
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con ained he ull ORF YIL121w, comp ising 1107 bp be o e he s a codon and 299
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bp a e he s op codon, and subcloned in o he Bam HI si e o YEp351 (2 µm o igin,
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LEU2 ma ke ) (Hill e al., 1986), p o isionally named HAL11, bu enamed QDR2 a e
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he publica ion o (Va gas e al., 2005).
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23
Fo analysis o QDR2 exp ession using he Lac-Z epo e gene, we ampli ied 611 bp o
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he p omo e egion o QDR2 wi h P ime P om QDR2D XSVWUHDP¶-CTC AAG CTT
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TCC CAC ATG ACG TGC AG; Hind III si e unde lined) and P ime P om QDR2R
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GRZQVWUHDP¶-CCC AAG CTT GCC ATC GTT GCA GTAC; EcoR I si e unde lined),
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diges ed and liga ed in o he Hind III si e o plasmid pYIp355 (ampicillin esis ance in
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bac e ia and URA3 complemen a ion in yeas ; Mye s e al, 1986). The esul ing plasmid
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was named JM214.
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Measu emen o In acellula ca ion concen a ions:
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Cells we e g own in YPD o an abso bance a 660 nm o 0.6 o 0.7, cen i uged o 5
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min a 1.900 X g, esuspended a he same concen a ion in YPD con aining he
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indica ed chemical a he indica ed concen a ion and incuba ed a 30 ºC o 90 minu es.
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Aliquo s we e aken, cen i uged in plas ic ubes o 5 min a 2.000 pm and 4 ºC and
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washed wice wi h 10 ml o ice cold solu ion o 20 mM MgCl2. The cell pelle s we e
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esuspended in 0.5 ml o 20 mM MgCl2. Ions we e ex ac ed by hea ing he cells o 15
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min a 95 ºC. A e cen i uga ion, aliquo s o he supe na an we e analyzed wi h an
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a omic abso p ion spec ome e (SensAA) in lame emission mode. Fo he coppe
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ex usion assays s ains we e incuba ed wi h he indica ed amoun s o coppe o 120
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minu es. A ha poin aliquo s we e aken o de e mine he coppe con en a ime 0 and
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he es o he cul u e was washed wice wi h 20 mM MgCl2 and ans e ed o esh
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YPD medium. Aliquo s we e aken a he indica ed imes and ea ed as explained
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p e iously. Coppe was measu ed in a plasma emission spec opho ome e (Shimadzu).
45
46
47
ȕ-Galac osidase assays
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Plasmid JM214, diges ed wi h Nco I, was in eg a ed by homologous ecombina ion in
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he URA3 locus o he BY4741 yeas s ain. Th ee independen colonies we e used o
50

6
analysis. Cul u es we e incuba ed o 1.5 h a e addi ion o he men ioned chemical. ȕ -
1
Galac osidase ac i i y was measu ed in pe mea ed cells as desc ibed p e iously (Rios e
2
al.,1997). Uni s o ac i i y we e no malized o cell densi y.
3
4
5
6
7
7
1
Resul s:
2
3
Qd 2 con e s ole ance o sodium and li hium
4
5
We ha e sc eened o yeas genes able o con e ole ance upon o e exp ession. In he
6
pas , his s a egy has been use ul o iden i y de e minan s o ion homeos asis, such as
7
he HAL genes (A ino e al., 2010, and e e ences wi hin). This echnique has also been
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use ul o sc een o genes om o he o ganisms, such as plan s (Mule e al., 2004;
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Se ano e al., 2003). Despi e he amoun o published da a, some iden i ied genes
10
emain uncha ac e ized. We sc eened 200.000 independen colonies o yeas
11
ans o med wi h an episomal plasmid con aining Sa c c h a omy c es c e e i si a e genomic
12
agmen s. A agmen con aining QDR2 was isola ed om ou independen clones o
13
i s abili y o con e ole ance o li hium and sodium. Only QDR2 (YIL121w) was
14
comple e in he 4 di e en clones. This gene sha es 70% homology wi h QDR1
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(YIL120w), which was also p esen in some o he isola ed clones. The e o e, we
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subcloned bo h and compa ed hei abili y o con e ole ance o sodium o li hium upon
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o e exp ession. Only QDR2 was esponsible o he sal ole ance pheno ype, as
18
o e exp ession o QDR1 did no con e sodium o li hium ole ance (Fig. 1). The
19
o iginal sc eening was pe o med in he RS16 gene ic backg ound (Gaxiola e al.,
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1992). In o de o assess whe he he pheno ype was ep oducible in di e en gene ic
21
backg ounds, we ans o med di e en yeas s ains wi h he plasmid o e exp essing
22
QDR2. We could ep oduce he obse ed ole ance o sodium and li hium in W303-1A
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(da a no shown) and in BY4741 (B achmann e al., 1998) (Fig. 2). The P- ype ATPase
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ENA1 is he main anspo e esponsible o sodium and li hium ex usion om he
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cy oplasm in S. c e e i si a e (Ha o e al., 1991). This gene belongs o a amily composed
26
by h ee o ou membe s (depending on he s ain) loca ed in andem in he yeas
27
genome. In o de o de e mine whe he he obse ed sodium and li hium ole ance could
28
be due o an indi ec e ec on ENA1, we ans o med a SKY697 s ain (Fe ando e al.,
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1995) which has a comple e dele ion o he ou ENA genes. We could also obse e
30
ole ance in his gene ic backg ound (da a no shown). In addi ion o e exp ession o
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QDR2 had no e ec on ENA1 exp ession unde no mal condi ions o a e induc ion
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wi h sodium o li hium (da a no shown), so he sodium and li hium pheno ype is
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independen o he main ex usion pump o sodium and li hium, Ena1.
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35
Qd 2 anspo s li hium, bu is no essen ial o mono alen ca ion homeos asis.
36
37
A e con i ming ha he sal ole ance pheno ype was ep oducible in di e en s ains
38
and independen o ENA1, we in es iga ed whe he QDR2 unc ion was essen ial o
39
ole ance o mono alen oxic ca ions by in es iga ing he pheno ypes o a qd 2 s ain.
40
A qd 2 s ain showed a e y weak sensi i i y pheno ype when g own in he p esence o
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mono alen oxic ca ions (Fig. 2A). We also analyzed he qd 1 mu an s ain unde he
42
same condi ions, bu g ow h was simila o he wild ype con ol s ain in all condi ions
43
assayed (da a no shown). We also ied o unde s and he mechanism o ole ance
44
de e mined by QDR2. The mos ob ious explana ion o he obse ed ole ance is ha
45
Qd 2 is anspo ing oxic ca ions ou side he cell. We g ew di e en s ains in medium
46
con aining LiCl and ou esul s indica e ha cells lacking QDR2 accumula e mo e
47
li hium and cells o e exp essing QDR2 accumula e less han con ol cells, indica ing
48
ha Qd 2 is anspo ing li hium (Fig. 2B).
49
50
8
Qd 2 has a ole in di alen ca ion homeos asis.
1
2
In o de o in es iga e he spec um o ca ions anspo ed by Qd 2, we es ed o he
3
oxic ca ions and we ound pheno ypes ela ed o ansi ion me als such as nickel,
4
manganese and coppe . O e exp ession o QDR2 con e s ole ance o Ni2+ and Mn2+
5
(Fig. 3A), al hough we could no obse e any sensi i i y in he mu an s ain. Ion
6
con en analysis showed small di e ences (da a no shown). We could no obse e any
7
clea pheno ype upon o e exp ession o QDR2 in coppe con aining medium, bu he
8
qd 2 mu an s ain was e y sensi i e o his ca ion. This esul sugges s ha Qd 2 has a
9
ole in di alen ca ion ex usion (Fig. 3A). We also in es iga ed coppe con en a e a
10
¶LQFXEDWLRQ,QWHUQDOFRQWHQWEHWZHHQZLOGW SHDQGWKHVWUDLQRYHUH[SUHVVLQJ QDR2
11
was simila , con i ming he obse ed pheno ype ha o e exp ession o QDR2 does no
12
con e ole ance, bu he mu an s ain accumula ed abou 50% mo e han coppe han
13
he wild ype (Fig. 2B).
14
15
16
We u he in es iga ed whe he Qd 2 could con ibu e o homeos asis o essen ial
17
di alen ca ions, such as calcium o magnesium. We did no obse e any di e ence in
18
g ow h in he p esence o excess magnesium o calcium, o di e ences in in e nal
19
con en (da a no shown). These esul s do no disca d ha Qd 2 could ha e a ole in
20
condi ions wi h limi ing calcium o magnesium. Fo his pu pose, we compa ed he
21
g ow h o di e en s ains in he p esence o he di alen ca ion chela o s E hylene
22
diamine e a-ace ic acid (EDTA) o e hylene glycol e a-ace ic acid (EGTA). Unde
23
hese condi ions he mu an s ains showed be e g ow h ha wild ype o s ains
24
o e exp essing QDR2, sugges ing ha Qd 2 could ake pa in calcium o magnesium
25
ex usion (Fig 3C).
26
27
Qd 2 can anspo di alen ca ions inside he cell.
28
29
In e es ingly, when we in es iga ed he spec um o di alen ca ions anspo ed by
30
Qd 2, we ound ha some ansi ion me als p oduced di e en esul s. We ound ha
31
s ains de ec i e o qd 2 we e sligh ly ole an o cadmium and cobal . We did no
32
obse e any pheno ype upon o e exp ession o QDR2 (Fig. 4A). We pe o med mos o
33
ou expe imen s in ich media (YPD) ha does no selec o he plasmid. Unde no mal
34
condi ions YEp351, a 2 mic on de i a i e yeas episomal plasmid used in his s udy is
35
e y s able (Hill e al., 1986). Howe e , when his plasmid con ains a gene whose
36
exp ession has some dele e ious e ec , a nega i e selec ion can occu , such ha s ains
37
ha ha e los he plasmid o exp ess less o he inse ed gene a e selec ed. To es
38
whe he he lack o pheno ype in s ains o e exp essing QDR2 was due o a nega i e
39
selec ion, we used minimal SD media wi hou leucine, o p e en he g ow h o yeas
40
colonies wi hou plasmid. Unde hese condi ions s ains o e exp essing QDR2 g ow
41
less han con ol s ains in he p esence o cobal , indica ing ha QDR2 o e exp ession
42
is dele e ious unde hese g ow h condi ions (Fig. 4B). To asses whe he his e ec
43
could be ela ed o anspo o whe he i is an indi ec e ec , we measu ed he
44
accumula ion o his ca ion in cells g own in he p esence o cobal . We obse ed ha
45
he qd 2 mu an accumula es less cobal ha wild ype con ol cells (Fig. 4C).
46
47
48
9
QDR2 exp ession is ep essed by coppe and by hyd ogen pe oxide
1
2
In o de o in es iga e he egula ion o QDR2, we cons uc ed a plasmid con aining he
3
Lac-Z epo e gene (Mye s e al., 1986) exp essed unde he con ol o he QDR2
4
p omo e . We ha e shown ha Qd 2 is in ol ed in mono alen and di alen ca ion
5
homeos asis, wi h poo selec i i y. So i s we es ed changes in exp ession a e
6
ea men s wi h di e en oxic ca ions, bu we did no obse e any signi ican
7
di e ences, wi h he excep ion o coppe , whe e we could obse e an app oxima ely 10
8
old ep ession (Fig. 5B). This obse a ion sugges s ha he physiological ole o QDR2
9
is dele e ious in he p esence o coppe , an appa en disc epancy wi h he ac ha a
10
qd 2 mu an s ain is e y sensi i e o coppe . Mono alen coppe is insoluble, so
11
coppe is p esen in he medium as a di alen ca ion. Di alen coppe is educed in he
12
ex acellula ma ix by he Cu-Fe educ ase F e1. Mono alen coppe is hen anspo ed
13
inside he cell by he high a ini y anspo e s C 1 and C 2 (Dancis e al., 1994).
14
Besides being a mic onu ien , in acellula Cu+ pools mus be igh ly con olled, as an
15
excess o his ca ion can lead o oxici y h ough he o ma ion o oxygen adicals ia de
16
Fen on eac ion (Valko e al., 2005). Speci ically, Cu+ can eac wi h hyd ogen pe oxide
17
o p oduce he hyd oxyl adical and Cu2+. Al e na i ely, Cu2+ could eac wi h he
18
supe oxide anion ia he Habe -Weiss eac ion o o m molecula oxygen and Cu+. The
19
in ol emen o coppe ca ions in hese classical bioino ganic chemis y eac ions could
20
p o ide a hin o unde s and he physiological ole o Qd 2. In he p esence o hyd ogen
21
pe oxide Cu+ will p oduce hyd oxyl adicals, dele e ious o he cell, and hus
22
comp omising H2O2 de oxi ica ion by ca alases o glu a hione pe oxidases. I Qd 2 is
23
ex uding Cu2+ om he cell, his could inc ease he a e o Fen on eac ion by
24
elimina ing one o he p oduc s. I his hypo hesis is co ec , we would p edic ha Qd 2
25
would be dele e ious in he p esence o H2O2. As indica ed in Fig. 5A, qd 2 s ain
26
g ows be e han he wild ype con ol s ain and QDR2 exp ession is ep essed in he
27
p esence o H2O2 (Fig. 5B). In acellula Cu2+ can also induce he o ma ion o
28
molecula oxygen ia he Habe -Weiss eac ion, using he supe oxide anion as a
29
subs a e. I Qd 2 is ex uding Cu2+ om he cy oplasm, he dele e ious e ec o Habe -
30
Weiss eac ion will be diminished, as Qd 2 will elimina e he subs a e om he
31
cy oplasm. We used menadione as a supe oxide gene a o (Cas o e al., 2008) and
32
ound ha o e exp ession o QDR2 con e s ole ance o menadione (Fig. 5A), and we
33
did no obse e a signi ican dec ease in exp ession o QDR2 upon a ea men wi h
34
menadione (Fig. 5B).
35
36
37
Ex usion o coppe depends on QDR2
38
39
We ha e ound ha coppe is he only ca ion ha egula es QDR2 exp ession and ha a
40
qd 2 mu an s ain is sensi i e o coppe . These esul s sugges ha cy oplasmic Cu2+
41
should be he mos ele an physiological subs a e o Qd 2. To con i m his hypo hesis
42
we ha e measu ed coppe up ake and coppe ex usion in qd 2 mu an s. A qd 2 mu an
43
accumula es mo e coppe han i s pa en al wild ype, bu up ake a sho imes is
44
undis inguishable, sugges ing ha up ake a e is simila and he di e ence is he
45
ex usion a e (Fig. 6A). To con i m his hypo hesis we e alua ed he coppe ex usion
46
in qd 2 cells. We incuba ed wild ype cells wi h 12,5 mM CuSO4 and qd 2 mu an wi h
47
10 mM in o de o a ain a simila le el o in acellula coppe a ime 0. Wild ype cells
48
could ex ude coppe , bu his ex usion was impai ed in qd 2 cells (Fig. 6B).
49
50
51
16
Gbelska, Y., K ijge , J.J. & B eunig, K.D. (2006) E olu ion o gene amilies: he
1
mul id ug esis ance anspo e genes in i e ela ed yeas species. FEMS Yeas
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Res. 6: 345-355.
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and phylogene ic cha ac e iza ion o yeas open eading ames wi h he majo
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an ipo e Nha1 in luences he plasma memb ane po en ial o Sa c c h a o my c es
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3458.
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mechanism o ion homeos asis and sal ole ance in yeas : he Hal4 and Hal5
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Mule , J.M., Alejand o, S., Rome o, C. & Se ano, R. (2004) The ehalose pa hway and
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in acellula glucose phospha es as modula o s o po assium anspo and
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Mule , J.M., Alemany, B., Ros, R., Cal e e, J.J. & Se ano, R. (2004) Exp ession o a
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plan se ine O-ace yl ans e ase in Saccha omyces ce e isiae con e s osmo ic
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17
ole ance and c ea es an al e na i e pa hway o cys eine biosyn hesis. Yeas 21:
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2
Mu guia, J.R., Belles, J.M. & Se ano, R. (1995) A sal -sensi i e 3'(2'),5'-bisphospha e
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nucleo idase in ol ed in sul a e ac i a ion. Science 267: 232-234.
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P io , C., Po ie , S., Soucie , J.L. & Sych o a, H. (1996) Cha ac e iza ion o he NHA1
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Rad, M.R, Ki ch a h, L. & Hollenbe g, C.P. (1994) A pu a i e P- ype Cu 2+ -
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Rios, G., Fe ando, A. & Se ano, R. (1997) Mechanisms o sal ole ance con e ed by
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gene, CAD2, which is a mu a ed pu a i e coppe - anspo e gene (PCA1),
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de e minan o esis ance o quinidine, ba ban, cispla in, and bleomycin.
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17