Molecular Biology of the Cell
Vol. 20, 2265–2275, April 15, 2009
Nuclear Shuttling of She2p Couples ASH1 mRNA
Localization to its Translational Repression by Recruiting
Loc1p and Puf6p
Zhifa Shen, Nicolas Paquin,* Ame ´lie Forget, and Pascal Chartrand
De ´partement de Biochimie, Universite ´ de Montre ´al, Montre ´al, QC, H3C 3J7 Canada
Submitted November 26, 2008; Revised February 10, 2009; Accepted February 12, 2009
Monitoring Editor: Marvin P. Wickens
The transport and localization of mRNAs results in the asymmetric synthesis of specific proteins. In yeast, the nucleo-
cytoplasmic shuttling protein She2 binds the ASH1 mRNA and targets it for localization at the bud tip by recruiting the
She3p–Myo4p complex. Although the cytoplasmic role of She2p in mRNA localization is well characterized, its nuclear
function is still unclear. Here, we show that She2p contains a nonclassical nuclear localization signal (NLS) that is
essential for its nuclear import via the importin ? Srp1p. Exclusion of She2p from the nucleus by mutagenesis of its NLS
leads to defective ASH1 mRNA localization and Ash1p sorting. Interestingly, these phenotypes mimic knockouts of LOC1
and PUF6, which encode for nuclear RNA-binding proteins that bind the ASH1 mRNA and control its translation. We find
that She2p interacts with both Loc1p and Puf6p and that excluding She2p from the nucleus decreases this interaction.
Absence of nuclear She2p disrupts the binding of Loc1p and Puf6p to the ASH1 mRNA, suggesting that nuclear import
of She2p is necessary to recruit both factors to the ASH1 transcript. This study reveals that a direct coupling between
localization and translation regulation factors in the nucleus is required for proper cytoplasmic localization of mRNAs.
The cytoplasmic transport and localization of mRNAs is
used by several eukaryotic organisms to control, in space
and time, the expression of proteins involved in cell fate
determination, cellular polarity, or asymmetric cell division
(St. Johnston, 2005; Du et al., 2007). With more than 30
mRNAs localized at the bud tip, the budding yeast Saccha-
romyces cerevisiae is an excellent model system for studying
the mechanisms behind mRNA transport and localization
(Chartrand et al., 2001; Darzacq et al., 2003). One of these
transcripts is the ASH1 mRNA, which is localized at the
distal tip of daughter cells during late anaphase (Long et al.,
1997; Takizawa et al., 1997). This localization promotes the
asymmetric sorting of Ash1p to the daughter cell nucleus
and results in the inhibition of mating-type switching in the
daughter cell (Jansen et al., 1996; Sil and Herskowitz, 1996).
The RNA-binding protein She2p is responsible for the rec-
ognition of bud-localized mRNAs, via its interaction with
specific localization elements in these transcripts (Olivier
et al., 2005). She2p also interacts with the transport machin-
ery, constituted of the type V myosin Myo4p, via the bridg-
ing protein She3p (Bohl et al., 2000; Long et al., 2000;
Takizawa and Vale, 2000). During its transport to the bud
tip, the ASH1 mRNA is translationally repressed by the
RNA-binding proteins Khd1p (Irie et al., 2002) and Puf6p
(Gu et al., 2004), and phosphorylation of these factors at the
bud tip promotes the local synthesis of the Ash1 protein
(Paquin et al., 2007; Deng et al., 2008).
Previous studies have shown that nuclear factors play an
important role in yeast mRNA localization. Puf6p and Loc1p
are predominantly nucleolar RNA-binding proteins that
bind directly the 3? untranslated region (UTR) of ASH1
mRNA in vitro and are involved in the translational repres-
sion of this transcript (Long et al., 2001; Gu et al., 2004; Komili
et al., 2007). Knockouts of these genes disrupt ASH1 mRNA
localization and Ash1p sorting to the daughter cell (Long
et al., 2001; Gu et al., 2004). She2p shuttles between the
cytoplasm and the nucleus (Kruse et al., 2002), and recent
evidence suggests that the nuclear transit of She2p is in-
volved in the translational regulation of ASH1 mRNA (Du
et al., 2008). However, the specific role of She2p in the
nucleus is still unclear.
In this study, we show that She2p contains a nonclassical
nuclear localization signal (NLS) that is essential for its
nuclear import by the importin ? Srp1p. Exclusion of She2p
from the nucleus by mutagenesis of its NLS leads to defec-
tive mRNA localization and Ash1p sorting. We find that
nuclear She2p is associated with Puf6p and Loc1p indepen-
dently of their interaction with RNA. Exclusion of She2p
from the nucleus decreases its interaction with Loc1p and
Puf6p and disrupts the binding of these factors to the ASH1
mRNA. This study leads us to suggest a mechanism where
She2p interacts with the translation regulation factors Puf6p
and Loc1p in the nucleus and recruits these factors to the
ASH1 mRNA, thereby promoting the localization of this
transcript at the bud tip. This coordinated recruitment of a
localization factor and translational repressors to ASH1 tran-
scripts in the nucleus suggests that mRNA transport and
translational control machineries are coupled and that this
coupling in the nucleus is required for proper cytoplasmic
localization and local translation of this transcript.
This article was published online ahead of print in MBC in Press
on February 25, 2009.
* Present address: Department of Biology, MIT, Cambridge, MA 02139.
Abbreviations used: NLS: nuclear localization signal.
© 2009 by The American Society for Cell Biology2265
MATERIALS AND METHODS
Growth Media and Yeast Strains
Yeast cells were grown in either synthetic growth media lacking the nutrients
indicated or rich media (Rose et al., 1990). Transformation was performed
according to the protocol of Schiestl and Giestz (1989). Yeast gene disruption
cassette was created by PCR amplification of the loxP-KAN-loxP construct in
plasmid pUG6 and primers specifics for the gene of interest (Guldener et al.,
1996). Specific disruption was confirmed by PCR analysis of genomic DNA.
Yeasts strains and plasmids used in this study are described in Supplemen-
tary Tables S1 and S2, respectively.
Immunoprecipitation and Reverse Transcription-PCR
Fifty milliliters of yeast cells were grown to early log phase (OD600? 1) at
30°C in the appropriate medium. Formaldehyde was added to a final con-
centration of 1%, and cells were incubated at room temperature (RT) for 20
min. Glycine was added to a final concentration of 300 mM. Cells were
washed twice in 1? PBS, harvested by centrifugation, and resuspended at an
OD600of 100 in the extraction buffer (25 mM HEPES-KOH, pH 7.5, 150 mM
KCl, 2 mM MgCl2, 0.1% IGEPALCA-630, 1 mM dithiothreitol, 87.5 ?g/ml
phenylmethylsulfonyl fluoride, 0.5 ?g/ml pepstatin, 0.5 ?g/ml leupeptin, 0.5
?g/ml aprotinin, and 23 U/ml RNAguard). The cells were broken with glass
beads, vortexed five times for 30 s, on ice with a 1-min pause between each
vortex. The supernatant was used for immunoprecipitation and Western blot.
For the immunoprecipitation of myc-tagged She2p, 10 ?g of anti-myc anti-
body (9E10) was added to 500 ?l of supernatant and incubated at 4°C with
agitation for 1 h; 50 ?l of protein A-Sepharose beads was then added, and the
incubation at 4°C was continued for 2 h. For immunoprecipitation of tandem
affinity purification (TAP)-tagged proteins, 50 ?l of IgG-agarose beads was
added to 500 ?l of supernatant. The beads were washed four times for 3 min
at 4°C with a wash buffer (25 mM HEPES-KOH, pH 7.5, 150 mM KCl, and 2
mM MgCl2). The RNA was eluted from the beads with 200 ?l of 50 mM
Tris-HCl, pH 8.0, 100 mM NaCl, 10 mM EDTA, and 1% SDS by incubating 10
min at 65°C, followed by a phenol-chloroform extraction and ethanol precip-
itation. For the reverse transcription, 2 ?l of RNA was incubated at 70°C for
5 min in the presence of 0.5 ?g of pd(N)6 and quickly chilled on ice. The
reverse transcription reaction was performed according to indications in a 1?
buffer (50 mM Tris-HCl, pH 8.3, 50 mM KCl, 4 mM MgCl2, and 10 mM
dithiothreitol) containing 10 mM dNTPs and 20 U of RNAguard, with 100 U
reverse transcriptase for 1 h at 42°C. The cDNAs were then amplified by PCR
using primers in the ASH1 sequence.
Fluorescence In Situ Hybridization and
Yeast cells were processed for fluorescence in situ hybridization (FISH) and
immunofluorescence according to the protocols described in Chartrand et al.
(2000). For in situ hybridization, yeast spheroplasts were hybridized with a
pool of Cy3-conjugated ASH1 DNA oligonucleotide probes. For immunoflu-
orescence, a 1:50 dilution of a mouse anti-myc 9E10 antibody (Oncogene
Science, Cambridge, MA) was used as primary antibody. For the secondary
antibody, a 1:1000 dilution of a anti-mouse Cy3-conjugated antibody (Jackson
ImmunoResearch Laboratories, West Grove, PA) was used.
Protein Expression and Purification
Recombinant protein GST-She2, GST-She2-M2, and GST-Srp1 were overpro-
duced in Escherichia coli BL21 transformed with pGEX-6P1-She2, pGEX-6P1-
She2-M2, and pGEX-5x3-Srp1. The cells were harvested 3 h after induction
with 1 mM IPTG at 30°C, resuspended in 0.1% PBS-Triton X-100, 1 M NaCl,
and 1 mg/ml lysozyme and antiproteases cocktail (PMSF ? pepstatin ?
leupeptin ? aprotinin) for 30 min on ice and sonicated. The lysate was cleared
by centrifugation for 15 min at 15,000 ? g at 4°C, to yield the supernatant with
the overexpressed soluble protein. The glutathione S-transferase (GST) fusion
proteins were purified by affinity chromatography with glutathione-Sepha-
rose 4B (GE Healthcare, Waukesha, WI) and eluted with 10 mM reduced
glutathione in PBS. The recombinant protein fractions were dialyzed over-
night in PBS and concentrated using a 10-kDa molecular-weight cutoff filter
unit (Centricon-Millipore, Bedford, MA). For the elution of Srp1p, the GST tag
was cleaved with Factor Xa overnight at room temperature.
GST Pulldown Assays
For the recombinant protein interactions of Srp1p with She2p and She2-M2,
purified Srp1p was incubated with 5 ?g of GST-She2p (wild type or mutant)
bound to glutathione-Sepharose 4B (GE Healthcare). The binding was per-
formed at room temperature for 3 h in 500 ?l of binding buffer (50 mM
HEPES-KOH, pH 7.3, 20 mM potassium acetate, 2 mM EDTA, 0.1% Triton
X-100, and 5% glycerol). The matrix was recovered by centrifugation and
washed four times with 500 ?l of binding buffer. The bound proteins were
eluted by boiling in Laemmli buffer and separated on a 10% SDS-PAGE. For
the interactions between recombinant GST-Srp1p and endogenous She2p-
myc, She2-M2-myc, She2-M2-M5A-myc, and She2-M5A-myc, 5 ?g of recom-
binant GST-Srp1p was bound to glutathione-Sepharose 4B and incubated
with yeast extract for 2.5 h at 18°C. The matrix was recovered by centrifuga-
tion and washed four times with 500 ?l of binding buffer. The bound proteins
were eluted with preheated SDS sample buffer (50 mM Tris-HCl, pH 6.8, 2%
SDS, 10% glycerol, 1% ?-mercaptoethanol, 12.5 mM EDTA, and 0.02% bro-
mophenol blue). Eluted proteins were analyzed by Western blot.
Monomeric She2p Interacts Directly with the Importin ?
Srp1p in Order to Enter the Nucleus
Previous work has shown that She2p transits through the
nucleus (Kruse et al., 2002; Du et al., 2008). Because of its size
(26 kDa), which is below the nuclear pore diffusion limit (40
kDa in yeast), a She2p monomer may enter passively
through the nuclear pores. However, because the functional
structure of She2p is that of a homodimer of more than 50
kDa (Niessing et al., 2004), it is possible that active nuclear
import is required. To determine if She2p shuttles between
the nucleus and the cytoplasm actively or passively, a yeast
genetic assay was used (Rhee et al., 2000). In this assay, the
protein of interest is fused to a chimera made of a modified
LexA protein (mLexA), containing a disrupted NLS, and of
the GAL4 activation domain (mLexA-GAL4AD). If the pro-
tein of interest contains a NLS, it promotes the nuclear
import of the mLexA-GAL4AD chimera, which activates the
expression of reporter genes (LacZ and HIS3). In this assay,
a fusion of She2p with the mLexA-GAL4AD resulted in the
activation of LacZ, whereas the mLexA-GAL4AD itself in-
duced little ?-galactosidase activity, suggesting that She2p
promotes the nuclear import of the fusion protein (Figure
1A). It was possible that the She2p-mLexA-GAL4AD fusion
protein could diffuse passively through the nuclear pores.
To eliminate this possibility, the 70-kDa protein VirE2, from
Agrobacterium tumefaciens, was added to the She2p-mLexA-
GAL4AD in order to increase the size of the fusion protein
(Rhee et al., 2000). Even this large fusion protein was actively
imported in the nucleus in a She2p-dependent manner (Fig-
ure 1A), suggesting that She2p contains an active NLS.
The main nuclear import pathway in yeast depends on the
importin ? Srp1p (Lange et al., 2007). A previous large scale
two-hybrid screen in yeast found that Srp1p interacts with
She2p (Ito et al., 2001), suggesting that nuclear import of
She2p may depend on Srp1p. To explore this possibility, the
interaction between She2p and Srp1p was tested in a yeast
two-hybrid assay. However, no interaction between these
two proteins could be detected in this assay (Figure 1B).
Although She2p forms a homodimer and dimerization is im-
portant for its RNA-binding capacity (Niessing et al., 2004), it is
possible that Srp1p interacts only with the She2p monomer.
Two mutants of She2p that have been shown to disrupt She2p
dimerization, She2p-M1 and She2p-M2, containing the muta-
tions Cys683Tyr and Ser1203Tyr, respectively, were gener-
ated (Niessing et al., 2004; and data not shown). When these
monomeric mutants were tested in the yeast two-hybrid assay,
both interacted strongly with Srp1p (Figure 1B), suggesting
that it is indeed the She2p monomer which interacts with
Srp1p. To confirm these results, recombinant Srp1p, GST-
She2p, and GST-She2p-M2 were purified from bacteria and the
direct interaction between Srp1p and the She2p variants was
tested by GST pulldown. As shown in Figure 1C, although the
GST-She2 protein was unable to pull down Srp1p, GST-
She2p-M2 and Srp1p did bind in this assay, suggesting a direct
interaction between Srp1p and a She2p monomer.
The observation that only monomeric She2p interacted
with Srp1p raised the possibility that a fraction of endoge-
nous yeast She2p may be able to interact with Srp1p. To
investigate this question, wild-type She2p and mutant M2
Z. Shen et al.
Molecular Biology of the Cell2266
Oleynikov, Y., and Singer, R. H. (2003). Real-time visualization of ZBP1
association with ?-actin mRNA during transcription and localization. Curr.
Biol. 13, 199–207.
Olivier, C., Poirier, G., Gendron, P., Boisgontier, A., Major, F., and Chartrand,
P. (2005). Identification of a conserved RNA motif essential for She2p recog-
nition and mRNA localization to the yeast bud. Mol. Cell. Biol. 25, 4752–4766.
Pan, F., Huttelmaier, S., Singer, R. H., and Gu, W. (2007). ZBP2 facilitates
binding of ZBP1 to ?-actin mRNA during transcription. Mol. Cell. Biol. 27,
Paquin, N., Me ´nade, M., Poirier, G., Donato, D., Drouet, E., and Chartrand, P.
(2007). Local activation of yeast ASH1 mRNA translation through phosphor-
ylation of Khd1p by the casein kinase Yck1p. Mol. Cell 26, 795–809.
Rhee, Y., Gurel, F., Gafni, Y., Dingwall, C., and Citovsky, V. (2000). A genetic
system for detection of protein nuclear import and export. Nat. Biotech. 18,
Rose, M. D., Winston, F., and Hieter, P. (1990). Methods in Yeast Genetics. A
Laboratory Course Manual, Cold Spring Harbor, NY: Cold Spring Harbor
Schiestl, R., and Gietz, R. D. (1989). High efficiency transformation of intact
yeast cells using single stranded nucleic acids as a carrier. Curr. Genet. 16,
Sil, A., and Herskowitz, I. (1996). Identification of asymmetrically localized
determinant, Ash1p, required for lineage-specific transcription of the yeast
HO gene. Cell 84, 711–722.
St. Johnston, D. (2005). Moving messages: the intracellular localization of
mRNAs. Nat. Rev. Mol. Cell Biol. 6, 363–375.
Takizawa, P. A., Sil, A., Swedlow, J. R., Herskowitz, I., and Vale, R. D. (1997).
Actin-dependent localization of an mRNA encoding a cell-fate determinant in
yeast. Nature 389, 90–93.
Takizawa, P. A., and Vale, R. D. (2000). The myosin motor, Myo4p, binds
Ash1 mRNA via the adapter protein, She3p. Proc. Natl. Acad. Sci. USA 97,
She2p Links Puf6p and Loc1p to ASH1 mRNA
Vol. 20, April 15, 20092275