Npa1p is an essen ial ans-ac ing ac o equi ed o
an ea ly s ep in he assembly o 60S ibosomal subuni s
in Saccha omyces ce e isiae
IVA
´N V. ROSADO and JESU
´S DE LA CRUZ
Depa amen o de Gene´ ica, Uni e sidad de Se illa, E-41012 Se illa, Spain
ABSTRACT
Ribosome biogenesis equi es >100 non ibosomal p o eins, which a e associa ed wi h di e en p e ibosomal pa icles. The
subs a es, he in e ac ing pa ne s, and he iming o ac ion o mos o hese p o eins a e la gely unknown. To elucida e he
unc ional en i onmen o he pu a i e ATP-dependen RNA helicase Dbp6p om Saccha omyces ce e isiae, which is equi ed
o 60S ibosomal subuni assembly, we ha e p e iously pe o med a syn he ic le hal sc een and he eby e ealed a gene ic
in e ac ion ne wo k be ween Dbp6p, Rpl3p, Nop8p, and he no el Rsa3p. In his epo , we ex ended he cha ac e iza ion o
his unc ional ne wo k by pe o ming a syn he ic le hal sc een wi h he sa3 null allele. This sc een iden i ied he so a
uncha ac e ized Npa1p (YKL014C). Polysome p o ile analysis indica es ha he e is a de ici o 60S ibosomal subuni s and an
accumula ion o hal -me polysomes in he slowly g owing npa1-1 mu an . No he n blo ing and p ime ex ension analysis
shows ha he npa1-1 mu a ion nega i ely a ec s p ocessing o all 27S p e- RNAs and he no mal accumula ion o bo h ma u e
25S and 5.8S RNAs. In addi ion, 27SA
2
p e- RNA is p ema u ely clea ed a si e C
2
. Mo eo e , GFP- agged Npa1p localizes
p edominan ly o he nucleolus and sedimen s wi h la ge complexes in suc ose g adien s, which mos likely co espond o
p e-60S ibosomal pa icles. We conclude ha Npa1p is equi ed o ibosome biogenesis and ope a es in he same unc ional
en i onmen o Rsa3p and Dbp6p du ing ea ly ma u a ion o 60S ibosomal subuni s.
Keywo ds: ibosome biogenesis; nucleolus; yeas gene ics; DEAD-box RNA helicase; p e ibosomal pa icles
INTRODUCTION
Ribosome syn hesis is a complex and highly ene gy-con-
suming p ocess in euka yo es (Wa ne e al. 2001). The e-
o e, he exp ession o he a ious ibosomal componen s is
subjec ed o a igh coo dina e con ol o ensu e hei p o-
duc ion a equimola amoun s (Plan a 1997; Lea y and
Huang 2001). In addi ion, ibosome con en luc ua es o
cope wi h he demand o p o ein syn hesis in esponse o
g ow h condi ions and s esses (Plan a e al. 1995; Wa ne
1999; Wa ne e al. 2001). Se e al p o eins in ol ed in i-
bosome biogenesis appea o ha e a unc ion in egula ing
he cell cycle (Pes o e al. 2001; Du and S illman 2002;
Oe inge and Tolle ey 2003), and de egula ion o ibo-
some biogenesis has been implica ed in uncon olled
g ow h and umo igenesis in mammalian cells (Rugge o
and Pandol i 2003).
In euka yo es, ibosome biogenesis akes place p ima ily
in he nucleolus (Olson e al. 2000; Pede son and Poli z
2000). The p ocess s a s wi h he ansc ip ion o he p i-
ma y ansc ip s (35S p e- RNA and p e-5S RNA in yeas )
by dis inc RNA polyme ases (Pol I and Pol III, espec-
i ely) and equi es he impo o mos ibosomal p o eins
( -p o eins) om he cy oplasm (Nomu a e al. 2004). The
35S p e- RNA is p ocessed a mul iple si es o gene a e he
ma u e 18S, 5.8S, and 25S RNA. The p e-5S RNA is only
p ocessed a i s 3⬘end o gene a e he ma u e 5S RNA (Fig.
1; Venema and Tolle ey 1999; Raue´ 2004). Concomi an o
he p e- RNA syn hesis and p ocessing eac ions, he p e-
RNA in e media es a e ex ensi ely modi ied (Deca u and
Fou nie 2002) and associa e wi h he -p o eins and a la ge
numbe o non ibosomal p o eins o o m p e ibosomal
pa icles (Fa ica and Tolle ey 2002; F omon -Racine e al.
2003; de la C uz e al. 2004a). Mos o ou cu en knowl-
edge on ibosome syn hesis comes om s udies wi h he
yeas Saccha omyces ce e isiae; howe e , he basic ou line o
he p ocess is highly conse ed h ough he euka yo ic king-
dom (Takahashi e al. 2003; Ge bi and Bo o jagin 2004).
The powe o yeas gene ics has led o he iden i ica ion o
Rep in eques s o: Jesu´s de la C uz, Depa amen o de Gene´ ica, Fac-
ul ad de Biologı´a, Uni e sidad de Se illa, A da. Reina Me cedes, 6,
E-41012 Se illa, Spain; e-mail [email p o ec ed].
A icle and publica ion a e a h p://www. najou nal.o g/cgi/doi/
10.1261/ na.7340404.
RNA (2004), 10:1073–1083. Published by Cold Sp ing Ha bo Labo a o y P ess. Copy igh © 2004 RNA Socie y. 1073
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many o he di e en non ibosomal ac o s in ol ed in i-
bosome syn hesis (K essle e al. 1999b; Kiss 2002). Recen
ad ances in he biochemical pu i ica ion o dis inc p e i-
bosomal complexes ha e acili a ed he iden i ica ion o
new non ibosomal ac o s and he loca ion o many o hem
wi hin he p e ibosomal pa icles (Milke ei e al. 2003).
These analyses ha e also ede ined he model o he -sub-
uni assembly pa hway (Fa ica and Tolle ey 2002; F o-
mon -Racine e al. 2003; Tschochne and Hu 2003). In
his model, he 90S p e ibosomal pa icle con ains he com-
ple e machine y esponsible o he ea ly clea ages o 35S
p e- RNA as well as se e al la ge and small -p o eins, bu
lacks mos o he ac o s in ol ed in 60S -subuni o ma-
ion (D agon e al. 2002; Fa ica and Tolle ey 2002; G andi
e al. 2002; F omon -Racine e al. 2003;
de la C uz e al. 2004a). Following clea -
age a ea ly si es, he 90S pa icle gi es
ise o ea ly 43S and 66S p e ibosomal
pa icles, which con ain 20S and 27SA
p e- RNAs, espec i ely. I appea s ha
mos o he ac o s associa ed wi h he
35S p e- RNA, wi h a ew excep ions, a e
eleased a e he 35S p e- RNA is spli
in o 20S and 27SA p e- RNAs (G andi e
al. 2002; Nissan e al. 2002). The ea ly
43S p e ibosomal pa icle is apidly ex-
po ed o he cy oplasm, whe e he con-
e sion o 20S p e- RNA in o ma u e 18S
RNA and he las assembly eac ions
ake place (Udem and Wa ne 1973). The
s udy o he di e en pu i ied p e-40S
complexes indica es ha only ew ans-
ac ing ac o s a e needed o inish he
ma u a ion o 40S -subuni s om ea ly
43S p e ibosomal pa icles (Scha¨ e e al.
2002; Fa ica e al. 2003a; Van obays e al.
2003). Howe e , he s udy o he di e -
en pu i ied p e-60S complexes is consis-
en wi h he p esence o dis inc p e-60S
in e media es ha mo e om he
nucleolus o he nucleoplasm and om
he e o he cy oplasm. These in e medi-
a es a e e med, acco ding o hei posi-
ion in he pa hway, ea ly, medium, la e,
and cy oplasmic p e-60S -pa icles
(Fa ica and Tolle ey 2002; Nissan e al.
2002). The ea lies 66S p e ibosomal pa -
icle is likely he esul o he associa ion
o >50 non ibosomal p o eins and mos
la ge -p o eins wi h he 27SA p e- RNA
(Nissan e al. 2002). The complexi y o
he p e-60S -pa icles dec eases du ing
hei ma u a ion om he nucleolus o
he nucleoplasm (Nissan e al. 2002;
Sa eanu e al. 2003) and he expo -com-
pe en p e-60S pa icle has comple ed he p e- RNA p o-
cessing eac ions (Baßle e al. 2001; Nissan e al. 2002). As
o he p e-40S pa icles, las assembly eac ions occu in
he cy oplasm (Senge e al. 2001; Kalls om e al. 2003).
Mo e han 150 p o ein ans-ac ing ac o s a e so a
known o be in ol ed in ibosome biogenesis (F omon -
Racine e al. 2003; Milke ei e al. 2003). The oles o hese
ac o s ha e been in e ed on he basis o hei mu an
pheno ype wi h espec o p e- RNA me abolism o hei
associa ion wi h dis inc p e ibosomal pa icles ( o ex-
amples, see h p://www.expasy.o g/linde /p o eins.h ml).
Mos o hem lack conse ed enzyma ic domains and a e
e med gene ally -subuni assembly ac o s. Few a e p e-
dic ed o encode enzymes such as endo- and exonucleases,
FIGURE 1. (Legend on nex page)
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modi ying enzymes, AAA ATPases, ATP-dependen RNA
helicases, o GTPases (K essle e al. 1999b; de la C uz e al.
2004a). The pu a i e RNA helicases ep esen he la ges
class o enzymes in ol ed in ibosome biogenesis (K essle
e al. 1999b; de la C uz e al. 2004a). These enzymes seem o
unwind RNA duplex egions in an ATP-dependen manne
and a e supposed o assis he ex ensi e s uc u al ea -
angemen s ha a e expec ed o occu du ing he -subuni
assembly eac ions. So a , nei he p ecise subs a es no
clea in e ac ing pa ne s ha e been elucida ed o hese
p o eins. We ha e p e iously epo ed he unc ional cha -
ac e iza ion o one such pu a i e RNA helicase, Dbp6p,
which is an essen ial nucleola p o ein ha is equi ed o
60S -subuni assembly and has been p oposed o ac a an
ea ly s ep du ing his p ocess (K essle e al. 1998). In ag ee-
men wi h his, we ha e shown ha Dbp6p unc ionally
in e ac s wi h a se o ans-ac ing ac o s such as Dbp7p,
Dbp9p, Nop8p, and Rsa3p and he -p o ein Rpl3p, which
ha e also been p oposed o unc ion ea ly du ing 60S -
subuni assembly (de la C uz e al. 2004b). Rsa3p is a non-
essen ial p o ein (de la C uz e al. 2004b); he e o e, in
o de o gain mo e insigh in o he unc ional en i onmen
o Rsa3p and hence Dbp6p, we ha e pe o med a syn he ic
le hal (sl) sc een wi h he sa3 null allele. He e, we desc ibe
he iden i ica ion o he genes complemen ing he isola ed sl
mu an s and s udy u he he so a uncha ac e ized open
eading ame (ORF) YKL014C. We p o ide e idence ha
he essen ial YKL014Cp is a nucleola p o ein needed o
op imal biogenesis o 60S -subuni s. Ou esul s sugges
ha YKL014Cp is equi ed o 27S p e- RNA p ocessing
and o he no mal accumula ion o 25S and 5.8S ma u e
RNAs. YKL014Cp is associa ed wi h la ge complexes,
which mos likely ep esen p e-60S ibosomal pa icles. We
conclude ha YKL014Cp has an ea ly ole du ing 60S -
subuni assembly. While his wo k was in p og ess,
YKL014Cp, he ea e named Npa1p ( o Nucleola P e i-
bosomal Associa ed), was independen ly cha ac e ized and
iden i ied as a componen o a e y ea ly p e-60S -subuni
pa icle, which con ains he 27SA
2
p e- RNA and he ans-
ac ing ac o s Dbp6p, Dbp7p, Dbp9p, and Nop8p bu seems
o lack Rsa3p (Dez e al. 2004).
RESULTS
Syn he ic le hali y wi h he sa3 null allele
iden i ies Npa1p
Rsa3p is a nucleola nonessen ial p o ein equi ed o op-
imal biogenesis o 60S -subuni s (de la C uz e al. 2004b).
Se e al lines o expe imen al e idence sugges s ha Rsa3p
and Dbp6p unc ion oge he in i o (de la C uz e al.
2004b). To isola e no el ac o s belonging o he same unc-
ional en i onmen as Dbp6p and Rsa3p, we ca ied ou an
sl sc een wi h he sa3 null allele. To his end, s ain IVY252
(pHT4467-RSA3) was mu agenized by UV i adia ion and
abou 200,000 su i ing colonies we e sc eened o nonsec-
o ing and 5- luo oo o ic acid (5-FOA) sensi i i y pheno-
ype. Thi een posi i e candida es we e ob ained and sub-
sequen ans o ma ion wi h YCplac111-RSA3 escued
g ow h on 5-FOA-con aining medium, hus sugges ing ha
all candida es indeed ha bo bona ide genomic sl mu a-
ions. In addi ion, backc ossing he sl mu an s ains o
s ain IVY262, a MATa de i a i e o IVY252, demons a ed
ha he sl pheno ype o all mu an s is caused by a single
ecessi e mu a ion (see Ma e ials and Me hods).
In ag eemen wi h he p e iously e ealed syn he ic en-
hancemen in e ac ions (de la C uz e al.
2004b), one mu an was complemen ed
by DBP6 and ano he one by NOP8 (see
Ma e ials and Me hods). In e es ingly,
se en mu an s we e complemen ed by
RPL3 and one by DBP9 (see Ma e ials
and Me hods). These likely ep esen al-
lele-speci ic syn he ic le hal ela ionships
because he sa3 null mu an is no syn-
he ically enhancing he g ow h pheno-
ype o p e iously isola ed pl3 o dbp9
alleles (de la C uz e al. 2004b). One o
he sl mu an s ains (sl1-4) is he subjec
o his s udy and he emaining wo a e
nei he complemen ed by any o he ea -
lie -men ioned genes no by NPA1 and
a e s ill being cha ac e ized. To clone he
gene complemen ing he sl mu a ion
p esen in he sl1-4 s ain, we ook ad-
an age o i s ecessi e slow-g ow h (sg)
pheno ype. This s ain was ans o med
wi h a yeas genomic lib a y and he li-
FIGURE 1. P e- RNA p ocessing in S. ce e isiae.(A) S uc u e and p ocessing si es o he 35S
p e- RNA. This p ecu so con ains he sequences o he ma u e 18S, 5.8S, and 25S RNAs
ha a e sepa a ed by wo in e nal ansc ibed space sequences, ITS1 and ITS2, and lanked
by wo ex e nal ansc ibed space sequences, 5⬘ETS and 3⬘ETS. The ma u e RNA species
a e shown as ba s and he ansc ibed space sequences as lines. The p ocessing si es and hei
loca ions as well as he a ious p obes used a e indica ed. (B) P e- RNA p ocessing pa hway.
The p ima y RNA Pol I ansc ip unde goes co alen modi ica ions (no shown), and i is
clea ed a i s 3⬘end o yield he 35S p e- RNA, which is he longes de ec able p ecu so . The
35S p e- RNA is clea ed a si e A
0
o gene a e he 33S p e- RNA. This molecule is subse-
quen ly p ocessed a si es A
1
and A
2
, esul ing in he sepa a ion o he p e- RNAs des ined o
he small and la ge ibosomal subuni s. The inal ma u a ion o he 20S p ecu so akes place
in he cy oplasm, whe e clea age a si e D yields he ma u e 18S RNA. The 27SA
2
p ecu so
is p ocessed by wo al e na i e pa hways ha bo h lead o he o ma ion o ma u e 5.8S and
25S RNAs. In he majo pa hway, he 27SA
2
p ecu so is i s clea ed a si e A
3
and hen he
27SA
3
p ecu so is exonucleoly ically diges ed 5⬘→3⬘up o si e B
1S
o yield he 27SB
S
p e-
cu so . A mino pa hway p ocesses he 27SA
2
o he 27SA
3
molecule a si e B
1L
by an as ye
unknown mechanism, p oducing he 27SB
L
p e- RNA. While p ocessing a si es B
1S
and B
1L
is being comple ed, he 3⬘end o ma u e 25S RNA is gene a ed by 3⬘→5⬘ imming o si e
B
2
. The subsequen p ocessing o bo h 27SB species appea s o be iden ical. Clea age a si e
C
2
gene a es he 25.5S and 7S p e- RNAs. The 7S p e- RNA is 3⬘→5⬘ immed o he 3⬘end
o he ma u e 5.8S RNA. The 25.5S species is 5⬘→3⬘diges ed o he ma u e 25S RNA. The
p ima y RNA Pol III ansc ip is immed o he 3⬘end o he ma u e 5S RNA. The da a
p esen ed in his s udy sugges ha Npa1p is equi ed o e icien p ocessing o he 27S
p e- RNAs. Fo e iews on p e- RNA p ocessing and he known p ocessing enzymes, see
Venema and Tolle ey (1999) and Raue´ (2004).
Npa1p is equi ed o 60S biogenesis
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b a y plasmid was eco e ed om h ee colonies ha g ew
o he wild- ype ex en a 37°C. Sequence and subcloning
analysis o he inse demons a ed ha he uncha ac e ized
essen ial ORF, YKL014C, which was named NPA1, is su i-
cien o es o e wild- ype g ow h (see Ma e ials and Me h-
ods). Fu he mo e, his ORF also es o es ed/whi e sec o -
ing and g ow h on 5-FOA pla es o he sl1-4 s ain.
To isola e he sl mu a ion om he sl1-4 s ain, he ea e
named npa1-1, we c ossed his s ain o an isogenic wild-
ype s ain, W303-1A, spo ula ed he esul ing diploid, and
dissec ed he e ads. In all comple e e ads, wo sg spo e
clones we e ob ained. Fo u he analyses, he meio ic seg-
egan IVY325 (see Table 1 o i s ele an geno ype) was
selec ed. The npa1-1mu a ion, which causes by i sel he sg
pheno ype (doubling ime o 4hinliquid YPD medium a
30°C,5ha 37°C),iscomplemen ed o he wild- ype ex en
by a cen ome ic plasmid ha bo ing he NPA1 gene a bo h
30°C and 37°C (Fig. 2A; da a no shown). The npa1-1mu-
an is, as expec ed, sl wi h he sa3 null allele (Fig. 2B). To
es o linkage be ween he npa1-1mu a ion and he ORF
YKL014C, we c ossed IVY325 wi h IVY507 (YCplac33-
NPA1), a haploid s ain ha ha bo s an npa1⬋kanMX4
dis up ion a he ch omosomal locus and con ains as a sole
NPA1 allele he cen ome ic plasmid YCplac33-NPA1. A e
spo ula ion and e ad analysis, all comple e e ads showed
a2
+
:2
−
seg ega ion o cell iabili y on pla es con aining
5-FOA, wi h all he iable spo e clones ha ing an sg phe-
no ype (da a no shown). This esul indica ed ha he
npa1-1mu a ion is linked o he NPA1 gene.
Npa1p encodes a p o ein o 1764 amino acids wi h a
p edic ed molecula mass o 203.3 kDa. The p o ein seems
o be neu al (pI = 7.31) and p obably exis s in low in a-
cellula abundance on he basis o a codon adap a ion index
o 0.158. A da abase sea ch e ealed no ob ious p o ein
mo i s o Npa1p, bu i s i s 693 amino acids seem o ha e
a p edic ed s uc u e ela ed o p o eins o he HEAT- epea
amily such as he PR65A subuni o p o ein phospha ase
2A (Yeas Resou ce Cen e In o ma ics Pla o m, h p://
www.yeas c.o g; Hazbun e al. 2003) and a low homology
wi h Tpd3p, he yeas PR65A p o ein. We could also iden-
i y po en ial homologs o yeas Npa1p in human, D o-
sophila,A abidopsis, and o he ungi (Sequence Simila i y
Que y Tool, Saccha omyces Genome Da abase; Ch is ie e al.
2004). The s ong sequence conse a ion sugges s ha
Npa1p ca ies ou an impo an , e olu iona ily conse ed
unc ion in all euka yo es. This unc ion migh be ela ed o
60S -subuni syn hesis, as sugges ed by he gene ic in e -
ac ion be ween Npa1p and Rsa3p. Mo eo e , sea ches in
he p e ibosomal ne wo k (h p://www.p e- ibosome.de;
Milke ei e al. 2003) and he YEAST p o ein complex da-
abase (h p://yeas .cellzome.com) ound Npa1p associa ed
wi h 90S p e ibosomal and ea ly p e-60S pa icles. In he
nex sec ions, we in es iga ed he possible in ol emen o
Npa1p in 60S -subuni biogenesis.
Npa1p is a nucleola p o ein
As a i s s ep in he unc ional cha ac e iza ion o Npa1p,
we s udied he subcellula localiza ion o Npa1p using an
NPA1-eGFP usion allele (see Ma e ials and Me hods). This
allele, which is exp essed om he NPA1 p omo e , is ully
unc ional because i complemen ed he npa1 null allele o
he wild- ype ex en a 30°C and 37°C (da a no shown).
Wes e n blo analysis wi h an i-GFP an ibodies de ec ed a
single p o ein ha mig a ed a he expec ed molecula mass
o abou 220–230 kDa (da a no shown; see Fig. 8). In he
s ain IVY507 (YCplac33-NPA1-eGFP pUN100-DsRed-
NOP1), he g een luo escence is de ec ed in he nucleolus
(Fig. 3B). The nucleola localiza ion is con i med by co-
localiza ion o he usion DsRed-Nop1p p o ein and exclu-
sion o he nucleoplasm, which was isual-
ized by s aining DNA wi h 4⬘,6-diamidino-
2-phenylindole dihyd ochlo ide (DAPI;
Fig. 3E–F). Ou esul s a e in ull ag ee-
men wi h hose desc ibed in a ecen sys-
ema ic p o ein localiza ion s udy (Huh e
al. 2003). The p edominan localiza ion o
Npa1-eGFPp in he nucleolus sugges s
a speci ic ole o Npa1p in ibosome bio-
genesis.
Npa1p is equi ed o 60S
-subuni accumula ion
To assess he unc ion o Npa1p in ibo-
some biogenesis, we used he npa1-1mu-
an s ain. We i s checked whe he his
mu a ion causes a de iciency in -subuni s
by polysome p o ile analysis. Compa ed
wi h he isogenic wild- ype s ain, he e
TABLE 1. Yeas s ains used in his s udy
Name
a
Rele an geno ype Sou ce
W303-1A MATa
W303-1B MAT␣
YDK11-5A MAT␣ade3::kanMX4 K essle e al. 1999a
YMD3-1A MAT␣ sa3::HIS3MX6 de la C uz e al. 2004b
YMD3-2D MATa sa3::HIS3MX6 de la C uz e al. 2004b
IVY252 MAT␣ sa3::kanMX4 ade3::kanMX4
[pHT4467-RSA3]
This s udy
IVY262 MATa sa3::kanMX4 ade3::kanMX4
[pHT4467-RSA3]
This s udy
IVY325 MAT␣npa1-1 This s udy
IVY507
b
MAT␣LYS2 MET15 npa1::kanMX4
[YCplac111-NPA1]
This s udy
a
All s ains used in his s udy, excep he IVY507 s ain, a e de i a i es o he diploid
s ain W303. IVY507 is a de i a i e o he diploid s ain Y24863 (see Ma e ials and
Me hods o o he geno ypic ma ke s).
b
Depending on he expe imen al condi ions, he o iginal YCplac111-NPA1 was e-
placed by o he NPA1-con aining plasmids.
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was a educ ion o ee 60S -subuni s ela i e o 40S -
subuni s and also a dec ease in 80S and polysomes in he
npa1-1s ain a bo h 30°C and 37°C (Fig. 4A,B; da a no
shown). As a esul o he sho age o 60S -subuni s, he e
was also an accumula ion o hal -me polysomes (Fig. 4B).
The ela i e educ ion o he 60S -subuni s was u he
analyzed by comple ely dissocia ing mono- and polysomes
in o 40S and 60S -subuni s by using low Mg
2+
suc ose
g adien s. Indeed, in he npa1-1s ain, we obse ed a 30%
educ ion in he o e all quan i y o 60S -subuni s ela i e
o he wild- ype s ain (A
254
a io ∼1.8 in he wild- ype
s ain and A
254
∼1.2 in he npa1-1s ain a 30°C, da a no
shown). These esul s indica e ha he pa ial loss o unc-
ion o Npa1p by he npa1-1mu a ion causes a de ici in
60S -subuni s. In ag eemen wi h his de ici , he npa1-1
mu an s ain is hype sensi i e o an ibio ics such as cyclo-
heximide and neomycin ha in e e e wi h he ansla ion
p ocess (Fig. 4C; da a no shown). Toge he wi h he
nucleola localiza ion o Npa1p, hese esul s s ongly sug-
ges ha Npa1p is a ans-ac ing ac o in ol ed in 60S
-subuni biogenesis.
Npa1p is equi ed o no mal p e- RNA p ocessing
P e- RNA p ocessing is in ima ely linked o he -subuni
assembly p ocess (see In oduc ion). The e o e, o s udy in
mo e de ail he unc ion o Npa1p in he biogenesis o 60S
-subuni s, we assessed he s eady-s a e le els o ma u e
RNA and p ecu so s by No he n blo hyb idiza ion and
p ime ex ension analyses in he npa1-1s ain and com-
pa ed hem o hose in he isogenic null sa3 and wild- ype
s ain. S ains IVY325 (npa1-1), YMD3-1A (⌬ sa3), and
W303-1B (wild ype) we e i s g own a 30°C in liquid YPD
medium and hen shi ed o 37°C o up o 9 h. Cells we e
main ained in mid-log phase (OD
600
o ∼0.8) and ha es ed
a di e en ime poin s and o al RNA was ex ac ed and
FIGURE 2. The npa1-1mu a ion con e s a slow-g ow h pheno ype
and i is syn he ically le hal wi h he sa3 null allele. (A) The s ain
IVY325 (npa1-1) was ans o med wi h ei he YCplac111 o he
complemen ing plasmid YCplac111-NPA1. As con ols, s ains W303-
1B (wild ype) and YMD3-1A (⌬ sa3) we e ans o med wi h YC-
plac111. T ans o man s we e g own on SD-Leu pla es o 4da 30°C.
(B) The s ains IVY325 (npa1-1), ca ying he plasmid YCplac33-
NPA1, and YMD3-2D (⌬ sa3) we e c ossed, he esul ing diploid was
spo ula ed, and he e ads we e dissec ed. Comple e e ads we e
s eaked on YPD pla es and es eaked on 5-FOA-con aining pla es o
coun e selec YCplac33-NPA1. A ep esen a i e e a ype e ad is
shown on a YPD pla e ( op hal ) o on a 5-FOA con aining pla e
(bo om hal ). Pla es we e incuba ed o 4da 30°C.
FIGURE 3. Npa1p localized p edominan ly o he nucleolus. Fluo-
escence mic oscopy was pe o med wi h exponen ially g owing cells
exp essing he Npa1-eGFPp and DsRed-Nop1p om hei cogna e
p omo e s. (A) Phase con as acquisi ion (Noma ski) o he same
ield as in B–F.(B) Npa1-eGFPp was de ec ed by he GFP luo escence.
(C) Ch oma in DNA was s ained wi h DAPI. (D) DsRed-Nop1p was
de ec ed by he DsRed luo escence. (E) Colocaliza ion o Npa1-eG-
FPp and ch oma in DNA. (F) Colocaliza ion o Npa1-eGFPp and
DsRed-Nop1p.
Npa1p is equi ed o 60S biogenesis
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analyzed. As shown in Figu e 5, a mild inc ease o he 35S
p e- RNA was de ec ed in all s ains a e ans e o 37°C.
This can be due o he e ec s o he hea shock, al hough 9
h a 37°C is likely enough ime o adap a ion. Howe e , in
he npa1-1s ain, his inc ease was accompanied by a e-
duc ion o 20S p e- RNA and an accumula ion o an abe -
an 23S p e- RNA and, o a lesse ex en , o an abe an
21S p e- RNA. This sugges s ha p e- RNA p ocessing a
ea ly si es is delayed in he npa1-1s ain a 37°C. The ap-
pea ance o 21S p e- RNA sugges s ha clea age a si e A
2
is mo e a ec ed han clea age a si es A
0
and A
1
. In good
ag eemen wi h his, le els o ma u e 18S RNA we e
sligh ly dec eased in he npa1-1s ain a 37°C. Mos im-
po an , he e was a signi ican dec ease in he 27SA and
27SB p e- RNAs in he npa1-1mu an a 37°C. The de-
c ease o he 27SB p e- RNA is e en obse ed when npa1-1
is g own a 30°C. Consis en ly, he e was a educ ion o he
s eady-s a e le els o 25S RNA (Fig. 5). In gene al, he
le els o p e- RNAs and ma u e RNAs in he sa3 null
s ain we e simila o hose o he wild- ype s ain, al hough
we could de ec some dec ease in he 27SB p e- RNAs a
37°C (Fig. 5).
Analysis o low-molecula -weigh RNA species showed
ha al hough le els o ma u e 5S RNA emained una -
ec ed, bo h he ma u e 5.8S
S
and 5.8S
L
RNAs sligh ly un-
de accumula ed in he npa1-1s ain a 37°C (Fig. 6). This
educ ion is due o dec eased le els o 7S
S
and 7S
L
p e-
RNAs and i is ully consis en wi h he dec eased le els o
he 27SB p e- RNAs (Fig. 6). In addi ion, a agmen ha
ex ends om A
2
o C
2
was accumula ed (Fig. 6). This sug-
ges s ha inac i a ion o Npa1p allows a p ema u e clea age
o 27SA
2
a si e C
2
ha educes he con e sion o 27SA
2
in o
27SB and 7S p e- RNAs.
In o de o esol e he 27SA
2
and 27SA
3
p e- RNAs and
dis inguish be ween he 27SB
L
and 27SB
S
p e- RNA, we
pe o med p ime ex ension analyses. As shown in Figu e 7,
he signal om he p ime ex ension s op a si es B
1L
and
B
1S
and, o a lesse ex en , a si es A
3
and A
2
was ound o
be signi ican ly dec eased in he npa1-1s ain compa ed
wi h he sa3 null and he wild- ype s ain. In addi ion,
p ime ex ension analysis h ough si e C
2
showed a clea
educ ion in he le el o he 25.5S p e- RNA (Fig. 7). Fi-
nally, hese analyses con i med ha le els o 27SB p e-
RNAs we e also sligh ly dec eased in he sa3 null s ain
(Fig. 7).
FIGURE 4. The npa1-1mu an has a de ici in ee 60S -subuni s,
accumula es hal -me polysomes, and is hype sensi i e o cyclohexi-
mide. (A) W303-1B (wild ype) and (B) IVY325 (npa1-1) we e g own
in YPD a 30°C. Cells we e ha es ed a an OD
600
o 0.8, and cell
ex ac s we e esol ed in 7%–50% suc ose g adien s. The A
254
was
con inuously measu ed. Sedimen a ion is om le o igh . The peaks
o ee 40S and 60S ibosomal subuni s, 80S ee couples/monosomes,
and polysomes a e indica ed. Hal -me s a e indica ed by a ows. (C)
D ug sensi i i y assay. Di e en dilu ions o log-phase cul u es om
W303-1B (wild ype), YMD3-1A (⌬ sa3), and IVY325 (npa1-1) we e
es ed o hei sensi i i ies o wo di e en concen a ion o cyclo-
heximide (Cyh).
FIGURE 5. E ec s o he npa1-1mu a ion on s eady-s a e le els o
high-molecula -weigh p e- RNA and ma u e RNA species. RNA was
ex ac ed om W303-1B (wild ype), YMD3-1A (⌬ sa3), and IVY325
(npa1-1) ollowing g ow h in YPD a 30°C o a a ious ime poin s
a e a shi o 37°C. RNA co esponding o an equi alen numbe o
cells was esol ed on a 1.2% aga ose- o maldehyde gel and ans e ed
o a nylon memb ane o No he n hyb idiza ion. The same il e was
hyb idized consecu i ely wi h di e en p obes. P obe names a e in-
dica ed in pa en heses on he igh (see Fig. 1A o hei loca ion in he
35S p e- RNA).
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Toge he , hese da a s ongly sugges ha Npa1p is
mainly equi ed o p ope ma u a ion o bo h 25S and 5.8S
RNAs. The inac i a ion o Npa1p by he npa1-1mu a ion
has a majo impac on p ocessing o all 27S p e- RNAs,
al hough he e is also an e ec on he clea age e iciency a
si e A
2
and o a lesse ex en a si es A
0
and A
1
. Finally, he
appea ance o he abe an A
2
-C
2
agmen molecule indi-
ca es ha Npa1p is also equi ed o main ain he no mal
o de o p ocessing o he 27SA
2
p e- RNA. Ou esul s
wi h he npa1-1s ain a e simila o hose epo ed o a
GAL⬋NPA1 s ain on in i o deple ion o Npa1p (Dez e al.
2004).
Npa1p is associa ed o
high-molecula -weigh pa icles
Recen epo s ha e iden i ied Npa1p in 90S pa icles as well
as in se e al p e-60S pa icles (see ea lie ). In o de o de-
e mine he s eady-s a e le els o Npa1p associa ed wi h
la ge p e ibosomal pa icles, we analyzed he sedimen a ion
beha io o he Npa1-eGFPp usion p o ein in suc ose den-
si y g adien s. Fo his pu pose, o al ex ac s we e p epa ed
om he IVY507 (YCplac33-NPA1-eGFP) s ain and sub-
jec ed o low Mg
2+
suc ose g adien ul acen i uga ion.
Then, ac ions we e collec ed and analyzed by Wes e n blo
o de ec Npa1-eGFPp. As shown in Figu e 8, a peak o
Npa1-eGFPp was ound associa ed wi h high-molecula -
weigh pa icles. The maximum o his peak sedimen s
sligh ly lowe han ma u e 60S -subuni s (Fig. 8, ac ion
14 e sus ac ion 12). I has been p e iously desc ibed ha
di e en p e-60S -pa icles mig a e o his egion o he
suc ose g adien (de la C uz e al. 1998a, 2004b). No p o ein
was de ec ed a he posi ions expec ed o he ee p o eins
(Fig. 8, ac ion 1–3). Taking also in o accoun he nucleola
localiza ion o Npa1-eGFP (see Fig. 3), we conclude ha he
majo i y o Npa1p is s ably associa ed wi h p e-60S -pa -
icles.
DISCUSSION
Sl sc eens ha e p o en o be e y help ul ools in iden i ying
componen s o mul ip o ein complexes, such as he nuclea
po e complex o p e ibosomal pa icles (Doye and Hu
1995; Venema and Tolle ey 1999). We ha e p e iously
desc ibed he unc ion o Dbp6p, a pu a i e RNA helicase
ha is equi ed o an ea ly s ep du ing he assembly o 60S
-subuni s (K essle e al. 1998). We ha e elucida ed he
unc ional en i onmen o Dbp6p by es ablishing a gene ic
in e ac ion ne wo k wi h he ans-ac ing ac o s Dbp7p,
Dbp9p, Nop8p, Rsa1p, and Rsa3p and he 60S -p o ein
Rpl3p (K essle e al. 1999a; de la C uz e al. 2004b). All o
hese p o eins ha e been implica ed in simila ea ly nucleo-
la s eps du ing he assembly o 60S -subuni s, wi h he
excep ion o Rsa1p, which localizes o he nucleoplasm.
Rpl3p belongs o a g oup o -p o eins ha associa e ea ly
wi h p e ibosomal pa icles (K uiswijk e al. 1978). In o de
o lea n mo e abou he unc ional en i onmen o Rsa3p
and hence Dbp6p, we ha e pe o med a sl sc een wi h he
sa3 null allele. We ound mu a ions in Rpl3p, Dbp6p, Dbp9p,
and Nop8p. We also iden i ied a mu a ion in he so a
uncha ac e ized ORF YKL014C/NPA1. In e es ingly, his
FIGURE 7. E ec s o he npa1-1mu a ion on s eady-s a e le els o
27S and 25.5S p e- RNA species. The same RNA samples desc ibed in
he legend o Figu e 5 we e used o p ime ex ension analysis. P obe
g (see Fig. 1A o i s loca ion in he 35S p e- RNA) was labeled and
used o he eac ions. No e ha his p obe allows de ec ion o 27SA
2
(as he s op a si e A
2
), 27SA
3
(as he s op a si e A
3
), bo h 27SB (as
s ops a si es B
1L
and B
1S
), and 25.5S (as he s op a si e C
2
).
FIGURE 6. E ec s o he npa1-1mu a ion on s eady-s a e le els o
low-molecula -weigh p e- RNA and ma u e RNA species. The same
RNA samples desc ibed in he legend o Figu e 5 we e sepa a ed on a
7% polyac ylamide-u ea gel, ans e ed o a nylon memb ane, and
hyb idized consecu i ely wi h di e en p obes. P obe names, excep
he one used o de ec ma u e 5S RNA, a e indica ed in pa en heses
on he igh (see Fig. 1A o hei loca ion in he 35S p e- RNA).
Npa1p is equi ed o 60S biogenesis
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mu a ion, npa1-1, is syn he ically le hal no only wi h he
sa3 null allele bu also wi h di e en dbp6,dbp9,nop8, and
pl3 alleles and he dbp7 null mu an (I.V. Rosado, unpubl.).
In his wo k, we show ha Npa1p is a ans-ac ing ac o
ha , like Dbp6p, Dbp7p, Dbp9p, Nop8p, and Rsa3p, is
equi ed o an ea ly s ep in he pa hway o 60S -subuni
syn hesis. Npa1p is, like mos ac o s in ol ed in ibosome
biogenesis (F omon -Racine e al. 2003), localized p edomi-
nan ly in he nucleolus in exponen ially g owing cells. The
npa1-1mu a ion esul ed in he de ici o 60S -subuni and
he appea ance o hal -me polysomes. Mos likely, as a
consequence o his de ici , he npa1-1s ain is hype sen-
si i e o an ibio ics ha in e e e wi h he ansla ion p o-
cess. Simila esul s ha e been desc ibed o many mu an s
de ec i e in 60S -p o eins o ac o s in ol ed in 60S -
subuni biogenesis ( o examples, see Hess e al. 1994; Be-
na d e al. 1998; de la C uz e al. 1998b).
When p e- RNA p ocessing was examined in npa1-1
cells, we ound ha he mos p onounced pheno ype was
he d as ic educ ion in he s eady-s a e le els o all 27S
p e- RNA a 37°C, especially bo h 27SB p e- RNAs. Con-
sis en ly, lowe le els o bo h 7S p e- RNAs we e de ec ed.
P ime ex ension e eals ha 25.5S le els we e also educed.
As a consequence o all hese p e- RNA
p ocessing pheno ypes, he e is a de ec-
i e syn hesis o ma u e 25S and 5.8S
RNAs. In ag eemen wi h he ac ha
5S RNA o ms a s able RNP wi h he
60S -p o ein Rpl5p (Deshmukh e al.
1993), le els o 5S RNA we e almos
una ec ed in he npa1-1s ain. Simila
p e- RNA p ocessing de ec s ha e been
desc ibed on mu a ion in o deple ion
o se e al ac o s in ol ed in 60S -sub-
uni syn hesis, among hem Dbp6p,
Dbp7p, Dbp9p, and Nop8p (Dauge on
and Linde 1998; K essle e al. 1998;
Zanchin and Gold a b 1999; Dauge on
e al. 2001). P e- RNA p ocessing analy-
sis also sugges s ha he sa3 null allele
leads o a mild educ ion in bo h 27SB
p e- RNAs. In addi ion, we ound ha
he npa1-1mu a ion a ec s he le els o
ma u e 18S RNA and i s 20S p ecu so
a 37°C. This is likely due o delayed
p ocessing a si e A
2
and o a lesse ex-
en a si es A
0
and A
1
. As a conse-
quence, bo h a 21S and, mo e abun-
dan ly, a 23S abe an p e- RNA accu-
mula ed. This ype o de ec in 18S
RNA syn hesis is a gene al ea u e o
mu a ions ha in e e e wi h he syn-
hesis o ma u e 25S and 5.8S RNAs
(Venema and Tolle ey 1999). In e es -
ingly, an abe an A
2
-C
2
agmen also
accumula ed in he npa1-1s ain a 37°C bu no in he sa3
null mu an . This agmen has been p e iously obse ed in
he Ss 1p-deple ed s ain and he empe a u e-sensi i e
cic1-2/nsa3 mu an a 37°C (Fa ica e al. 2002, 2003b). I
seems possible ha imp ope ea ly assembly in he pa hway
o 60S -subuni s allows p ema u e p ocessing o he 27SA
2
p e- RNA a si e C
2
, leading o educed le els o 27S and 7S
p e- RNAs and des abiliza ion o 25.5S p e- RNA (Fa ica e
al. 2002). I would be pe inen o assess he accumula ion
o he A
2
-C
2
agmen on inac i a ion o o he ac o s such
as Dbp6p, Dbp7p, Dbp9p, o Nop8p, whose mu an s show
p e- RNA p ocessing de ec s e y simila o he ones ound
in he npa1-1s ain. We conclude ha Npa1p is equi ed
o ea ly p e- RNA p ocessing e en s in he pa hway o 25S
and 5.8S RNA syn hesis. In con as o o he ac o s, such
as Cic1p (Fa ica e al. 2003b), Npa1p seems no o be e-
qui ed o nuclea expo o 60S -subuni s because we did
no ind nuclea accumula ion o he Rpl25p-eGFP la ge
subuni epo e in he npa1-1mu an a 30°C o ollowing
ans e o 37°C o 9 h (da a no shown).
We ound mos o Npa1p associa ed wi h la ge com-
plexes ha a e la ge han ma u e 60S -subuni s and could
co espond o p e-60S -pa icles. Consis en wi h his pos-
FIGURE 8. Analysis o he sedimen a ion o Npa1-eGFPp in suc ose g adien s. To al cell
ex ac s we e done om IVY507 (YCplac33-NPA1-eGFP) cells ollowing g ow h a 30°C and
esol ed in 7%–50% suc ose g adien s con aining a low concen a ion o Mg
2+
o dissocia e
ibosomes in o subuni s. The A
254
was con inuously measu ed. The p o ile ob ained is shown;
sedimen a ion is om le o igh . The peaks o 40S and 60S ibosomal subuni s a e indica ed.
F ac ions we e collec ed om he g adien s, p o eins we e ex ac ed om each ac ion, and
equal olumes we e esol ed on 7% SDS-polyac ylamide gels and subjec ed o Wes e n blo -
ing. T s ands o o al ex ac and numbe s co espond o ac ion numbe . A blo deco a ed
wi h an i-GFP an ibody is shown and he posi ion o Npa1-eGFP is indica ed.
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sibili y, Dez e al. (2004) ha e pu i ied a p e ibosomal com-
plex con aining Npa1p. In e es ingly, hese au ho s demon-
s a ed ha Npa1p is p edominan ly associa ed wi h he
27SA
2
p e- RNA, sugges ing ha his complex co esponds
o a e y ea ly p e-60S -pa icle. In ag eemen wi h bo h
his ac and ou gene ic da a, Dbp6p, Dbp7p, Dbp9p, and
Nop8p a e also s ably associa ed wi h he Npa1p-con aining
complex (Dez e al. 2004).
In conclusion, we ha e iden i ied an addi ional ac o
equi ed o ea ly assembly o 60S -subuni s. Howe e ,
because Npa1p lacks known enzyma ic ac i i y o ob ious
p o ein domains, we clea ly s ill do no unde s and i s
mechanis ic unc ion in his pa hway. S ikingly, Npa1p has
been desc ibed o in e ac in he yeas wo-hyb id sys em
wi h Cdc28p, which is he main cell cycle cyclin-dependen
p o ein kinase (Ue z e al. 2000). Se e al p o eins ha e been
implica ed in bo h ibosome syn hesis and cell cycle p o-
g ession (see In oduc ion). Howe e , ou esul s sugges
ha he e is no signi ican block o npa1-1cells a any s age
o he cell cycle a 30°C o 37°C (I.V. Rosado, unpubl.).
Fu he wo k is needed o unde s and he ole o Npa1p in
60S -subuni s and o cla i y i s possible link o cell cycle
p og ession.
MATERIALS AND METHODS
S ains, media, and gene ic manipula ions
All yeas s ains used in his s udy a e de i a i es o s ain W303
(MATa/MAT␣ade2-1/ade2-1 his3-11,15/his3-11,15 leu2-3,112/
leu2-3,112 p1-1/ p1-1 u a3-1/u a3-1) wi h he excep ion o he
de i a i es o he s ain Y24863, which is in he BY4743 back-
g ound (MATa/MAT␣his3⌬1/his3-⌬1 leu2⌬0/leu2⌬0 lys2⌬0/
LYS2 me 15⌬0/MET15 u a3⌬0/u a3⌬0 YKL014C⬋kanMX4/
YKL014C) and was pu chased om EUROSCARF (Table 1). The
s a ing s ain o he sl sc een, IVY252, was cons uc ed by c oss-
ing YDK11-5A and YMD3-2D. The esul ing diploid was spo u-
la ed and he e ads we e dissec ed. Two spo e clones o opposi e
ma ing ypes, IVY252 and IVY262, ca ying he ele an ma ke s
we e selec ed and ans o med wi h pHT4467-RSA3. RSA3 was
cloned in pHT4467 (Venema and Tolle ey 1996) as a 1.5-kb XbaI
agmen om pFL44-RSA3 (de la C uz e al. 2004b). G ow h and
handling o yeas we e acco ding o s anda d p ocedu es (Bu ke e
al. 2000). An ibio ic-con aining pla es we e p epa ed by adding
he d ugs om s ock solu ions in o YPD be o e pou ing he pla es.
E ec i e concen a ions ha ing no appa en e ec s on he g ow h
o W303-1B we e 1 mg/mL pa omomycin, 5 mg/mL neomycin,
and 0.1 µg/mL cycloheximide. Esche ichia coli s ain DH5␣was
used o cloning and p opaga ion o plasmids.
Isola ion o sl mu an s
S ain IVY252 (pHT4467-RSA3) was g own in YPD liquid me-
dium un il mid-log phase, and ∼500,000 cells we e sp ead on YPD
pla es a he densi y o abou 800 cells/pla e. The pla es we e hen
UV i adia ed, esul ing in a 40%–50% su i al, and incuba ed o
5 d a 30°C in he da k. Red colonies we e s eaked wice on o
esh YPD pla es o iden i y he nonsec o ing ones. Then hese
we e s eaked on pla es con aining 5-FOA and incuba ed o 5da
30°C. Eigh een candida es we e unable o g ow on 5-FOA pla es.
To con i m ha his pheno ype was no due o genomic in eg a-
ion o he plasmid pHT4467-RSA3, we ans o med candida es
wi h pHAC111-RSA3 and pHAC111 (de la C uz e al. 2004b).
Thi een candida es, named sl1-1, sl1-3, sl1-4, sl1-5, sl1-7,
sl1-8, sl2-2, sl2-3, sl3-3, sl3-4, sl4-1, sl4-2, and sl4-3, showed e-
s o ed sec o ing and 5-FOA esis ance on ans o ma ion wi h
pHAC111-RSA3 bu no wi h pHAC111. Candida es we e c ossed
o IVY262, he esul ing diploid s ain was spo ula ed, and he
e ads we e dissec ed. Two sl mu an seg egan s we e eco e ed
om 10 comple e e ads o each diploid s ain analyzed, indica -
ing he monogenic cha ac e o all sl mu a ions. The esul ing
diploid s ains we e also s eaked on YPD and 5-FOA-con aining
pla es. All diploids showed es o ed sec o ing and 5-FOA esis-
ance, indica ing he ecessi e cha ac e o all sl mu a ions. The
g ow h pheno ype o he di e en sl mu an s was assessed on YPD
pla es a 30°C, 18°C, and 37°C.
Cloning o NPA1
All sl mu an s we e ans o med wi h plasmids YCplac111-DBP6,
YCplac111-DBP7, YCplac111-DBP9, YCplac111-NOP8, YCplac111-
RPL3, YCplac111-RSA1, and YCplac111 (de la C uz e al. 2004b)
and ans o man s we e selec ed on SD pla es lacking leucine (SD-
Leu). T ans o man s we e s eaked on SD-Leu pla es o iden i y
sec o ing colonies and hen on 5-FOA-con aining pla es. The sl
pheno ype o he sl4-1 mu an was complemen ed by he DBP6
gene. The sl pheno ype o he sl4-2 mu an was complemen ed by
he DBP9 gene. The sl pheno ype o he sl1-8 mu an was comple-
men ed by he NOP8 gene. The sl pheno ype o he sl1-1, sl1-3,
sl1-5, sl1-7, sl2-2, sl3-3, and sl4-3 mu an s was complemen ed by
he RPL3 gene. S ain sl1-4, which has an sg pheno ype a any
empe a u e es ed, was ans o med wi h a YCplac111-based yeas
genomic lib a y (K essle e al. 1999a) and abou 15,000 ans o -
man s we e sc eened o wild- ype g ow h on SD-Leu pla es a
37°C. One plasmid, pIV222, con aining an 8.4-kb inse , comple-
men ed bo h he sg and sl pheno ypes o he sl1-4 mu an . The
sequence o he e minal egions shows ha he lib a y inse
con ained YKL013C (ARC19) and YKL014C as sole comple e
ORFs. Fu he subcloning o YKL014C as a 5.9-kb ApaI-NsiI ag-
men , which was blun ended, in o SmaI- es ic ed YCplac111
( he ea e named YCplac111-NPA1) and YCplac33 ( he ea e
named YCplac33-NPA1) con i med ha YKL014C was su icien
o complemen he sg and sl pheno ype o he sl1-4 mu an .
Fluo escence mic oscopy
S ain Y24863 was i s ans o med wi h YCplac111-NPA1, i was
subsequen ly spo ula ed, and he e ads we e dissec ed. A
npa1⬋KanMX4 haploid seg egan ha bo ing YCplac111-NPA1
(IVY507) was i s ans o med wi h YCplac33-NPA1-eGFP and
subjec ed o plasmid seg ega ion on SD-U a pla es. Plasmid YC-
plac33-NPA1-eGFP was cons uc ed as ollows: a 5.9-kb ApaI-NsiI
agmen om pIV222 was blun ended and cloned in o SmaI-
es ic ed YCplac33-yeGFP/TCYC1 (a gi om M. Hall). One
candida e in he app op ia e o ien a ion, pIV236, was selec ed.
Then, a PCR was pe o med using pIV222 as a empla e and he
Npa1p is equi ed o 60S biogenesis
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