652 EMBO reports vol. 3 | no. 7 | pp 652–659 | 2002© 2002 European Molecular Biology Organization
Intracellular trafficking of yeast
M. Teresa Teixeira, Klaus Förstemann, Susan M. Gasser1& Joachim Lingner+
Swiss Institute for Experimental Cancer Research (ISREC), CH-1066 Epalinges and1Department of Molecular Biology, Sciences II, University of Geneva,
CH-1211 Geneva 4, Switzerland
Received March 13, 2002; revised May 10, 2002; accepted May 21, 2002
Telomerase uses an internal RNA moiety as template for the
synthesis of telomere repeats. In Saccharomyces cerevisiae,
the telomerase holoenzyme contains the telomerase reverse
transcriptase subunit Est2p, the telomerase RNA moiety TLC1,
the telomerase associated proteins Est1p and Est3p, and Sm
proteins. Here we assess telomerase assembly by determining
the localization of telomerase components. We found that
Est1p, Est2p and TLC1 can migrate independently of each
other to the nucleus. With limiting amounts of TLC1, over-
expressed Est1p and Est2p accumulated in the nucleolus,
whereas enzymatically active Est2p–TLC1 complexes are
distributed over the entire nucleus. The distribution to the
nucleoplasm depended on the specific interaction between
Est2p and TLC1 but was independent of Est1p and Est3p.
Altogether, our results suggest a role of the nucleolus in telo-
merase biogenesis. We also describe experiments that support
a transient cytoplasmic localization of TLC1 RNA.
Telomerase is a ribonucleoprotein (RNP) polymerase that uses
an internal RNA moiety as template for the synthesis of telomere
repeats (Greider and Blackburn, 1989; Lingner et al., 1997b). In
Saccharomyces cerevisiae, telomerase assembly involves
association of three Ever Shorter Telomeres (EST1–3) gene prod-
ucts and presumably seven Sm proteins with the telomerase
RNA moiety TLC1 (Lundblad and Szostak, 1989; Singer and
Gottschling, 1994; Lendvay et al., 1996; Seto et al., 1999;
Hughes et al., 2000). It has remained unclear which cellular
compartments are involved in the assembly and if it occurs in an
ordered manner. In budding yeast, TLC1 is transcribed by RNA
polymerase II and polyadenylated (Chapon et al., 1997).
Whether the RNA moiety of telomerase is exported from the
nucleus to the cytoplasm as other polyadenylated RNAs
remained unclear (Huang and Carmichael, 1996). The TLC1
transcript has a trimethyl G cap and is associated with Sm
proteins, which also bind to snRNAs essential for intron splicing
(Seto et al., 1999). These properties of the TLC1 transcript
suggest that it shares the biogenesis pathway with other snRNAs.
Most vertebrate snRNAs undergo several steps of maturation in
the cytoplasmic compartment (including association with Sm
proteins and hypermethylation to a trimethylguanosine cap)
before being re-imported into the nucleus as mature RNPs
(Mattaj, 1986; Izaurralde et al., 1995; Huber et al., 1998; Ohno
et al., 2000; Will and Luhrmann, 2001). In yeast, hypermethyl-
ation of the cap structure may occur in the nucleolus (Mouaikel
et al., 2002) and it is uncertain whether maturation of snRNPs
involves the cytoplasm.
Here, we report on the subcellular localization of overex-
pressed telomerase components. We found that Est2p and Est1p
localize independently from each other to the nucleolus but that
the assembled Est2p–TLC1 complex is mainly nucleoplasmic.
We also analyzed the TLC1 distribution in heterokaryons and
found that TLC1 is able to migrate from one nucleus to another.
In addition, an open reading frame (ORF) was translated when
provided as a fusion transcript with TLC1. These results support
the notion that TLC1 is exported from the nucleus to the cyto-
plasm as part of the telomerase biogenesis pathway. We
conclude that yeast telomerase biogenesis involves several
cellular compartments and nuclear sub-compartments.
RESULTS AND DISCUSSION
Localization of telomerase components
In order to determine the localization of telomerase, we
replaced the endogenous copy of EST2 with a GFP–EST2 fusion
gene whose expression was controlled with the GAL1 promoter
to obtain the strain YKF15. Cells carrying the fusion construct
+Corresponding author. Tel: +41 21 692 5912; Fax: +41 21 652 6933; E-mail: email@example.com
EMBO reports vol. 3 | no. 7 | 2002 653
Localization of yeast telomerase
did not senesce or show considerable telomere shortening when
grown both on glucose (data not shown) or galactose (see
below). This shows that the chimeric protein is functional and
that low expression due to leakage of GAL1 promoter in glucose
is sufficient to prevent the cells from senescing. Nonetheless,
detection of GFP–Est2p by fluorescence microscopy was only
possible upon induction in galactose. It revealed an enrichment
of the protein in a crescent-shaped zone at the nuclear periphery
(Figure 1). This zone was identified as the nucleolus by co-
staining with the nucleolar protein Nop1p (Schimmang et al.,
1989). A small amount of GFP–Est2p was detected in the
nucleoplasm but not enriched at telomeres that cluster at the
nuclear periphery (Figure 1A, telomeres labeled by immuno-
staining against Rap1p; Gotta et al., 1996). Deletion of the
telomerase RNA gene TLC1 or the telomerase protein subunit
encoding genes EST1 and EST3 did not affect nuclear import or
nucleolar enrichment of Est2p (Figure 2A and B), indicating that
this localization is inherent to Est2p itself.
Nuclear localization of an HA-tagged Est1 was described
previously (Zhou et al., 2000), but nuclear versus nucleolar
localization was not distinguished. To determine the subcellular
localization of Est1p we used the same strategy as for Est2p and
found that a functional fusion protein between GFP and Est1p
expressed from the GAL1 promoter was also present in the
nucleoplasm and enriched in the nucleolus (Figure 1C). To
conclude, both Est2p and Est1p were concentrated in the
nucleolus when expressed from the GAL1 promoter. Several
hypotheses could account for this observation. First, Est2p and
Est1p may have accumulated in the nucleolus because their
overexpression resulted in an accumulation at an intermediate
step of telomerase biogenesis. Secondly, the nucleolus may
serve to stockpile mature telomerase and thus participate in the
control of the access of telomerase to the telomere. Finally, our
finding could simply reflect a general affinity of Est1p and Est2p
for a subcellular site containing a high concentration of RNA.
The results below indicate that the nucleolus does not stockpile
In order to detect the RNA subunit of telomerase, we
performed in situ hybridization using a DNA-based probe
containing Alexa 546-derivatized dUTP. We found that over-
expressed telomerase RNA was distributed over the entire
nucleus with no preferential accumulation in the nucleolus
(Figure 1D). The localization of TLC1 did not change upon
deletion of EST1, EST2 or EST3 (data not shown).
Active telomerase is localized in the nucleoplasm
Telomerase activity requires Est2p and TLC1, which together
make up the catalytic core (Lingner et al., 1997b). We reasoned
that overexpression of both moieties would be necessary to see
the assembled complex. We therefore transformed the PGAL1-GFP–
EST2 fusion gene harboring strain YKF15 with a plasmid
encoding TLC1 under the control of the GAL1 promoter and, as
a control, with the empty plasmid pRS314. We observed no
difference in growth when both strains were grown in galactose,
showing that induction of both GFP–Est2p and TLC1 was not
toxic. Direct observation of GFP–Est2p showed that the catalytic
subunit was redistributed over the entire nucleus upon TLC1
overexpression (Figure 2A, compare panels 1 and 2). Mutants of
TLC1 impaired for binding to Est2p (Livengood et al., 2002) did
not mediate the relocalization of Est2p (Figure 2B, panels 1 and
2). Therefore, the redistribution was dependent on the specific
interaction between Est2p and TLC1. TLC1 RNA localization
remained unchanged under these conditions (data not shown).
This suggested that both subunits co-localize in the nucleoplasm
when assembled. To test this hypothesis, we determined in vitro
telomerase activity as a measure for assembled telomerase, from
strains expressing endogenous levels of telomerase and from
strains overexpressing GFP–Est2p, TLC1 or both. Telomerase
activity was strongly increased in extracts from the strain over-
expressing both Est2p and TLC1 (Figure 2C, lane 4), whereas it
was similar to wild type when only one of the components was
overexpressed (Figure 2C, lanes 2 and 3). When extract derived
from the TLC1 overexpressing strain was mixed with extract
from the Est2p overexpressing strain, no increase in telomerase
activity was detected, ruling out a productive association of the
two subunits in vitro (Figure 2C, compare lanes 2 and 3 with 6).
Together, our results indicate that assembled and active
Est2p–TLC1 complexes colocalize with each other in the
nucleoplasm. However, the increased in vitro telomerase
activity was not sufficient to increase telomere length
(Figure 2D, compare lane 4 with 1–3). Telomere lengthening
may have been limited for example by telomerase recruitment
factors such as Est1p or Cdc13p (Evans and Lundblad, 1999), or
by components of the telomeric chromatin.
The results above indicated that the Est2p–TLC1 telomerase
core complex is mainly nucleoplasmic, whereas unassembled
Est2p accumulates in the nucleolus. To examine the role of Est1p
or Est3p in Est2p–TLC1 assembly or its relocalization, EST1 and
EST3 were deleted separately in the strain overexpressing both
Est2p and TLC1 and in the strain overexpressing only Est2p. All
resultant strains showed a senescence phenotype, yet direct
observation under the microscope revealed that redistribution of
GFP–Est2p from nucleolus to nucleoplasm upon TLC1 over-
expression did not depend on the presence of Est1p (Figure 2A,
panels 3 and 4) or Est3p (Figure 2A, panels 5 and 6). Further-
more, deletion of the Est1p binding site in TLC1 did not affect the
TLC1-mediated relocalization of Est2p to the nucleoplasm
(Figure 2B, panels 3 and 4). Thus, Est1p and Est3p do not influ-
ence the steady-state localization of Est2p and TLC1 or their
association, which is consistent with previous studies showing
that Est1p and Est3p are not required for telomerase activity
in vitro (Cohn and Blackburn, 1995; Lingner et al., 1997a).
Nuclear export and re-import of TLC1
In order to test whether yeast telomerase RNA is exported from
the nucleus to the cytoplasm, we analyzed TLC1 RNA redistribu-
tion in yeast heterokaryons containing nuclei that overexpress
TLC1 and nuclei that express TLC1 at endogenous levels. In
yeast, heterokaryons can be obtained by mating two strains of
appropriate genotype and by preventing nuclear fusion in the
resulting zygote with the dominant kar1-∆15 allele (Vallen et al.,
1992; Flach et al., 1994). We therefore mated one strain carrying
a plasmid encoding TLC1 under the control of the GAL1
promoter with another strain carrying the kar1-∆15 allele and
the GFP–NOP1 marker (Figure 3A). The population obtained
contained up to 25% of zygotes (determined cytologically).
It was subsequently incubated for 12 h in selective medium
containing galactose. This prevented growth of the parental
654 EMBO reports vol. 3 | no. 7 | 2002
M.T. Teixeira et al.
strains and allowed expression of TLC1 from the GAL1
promoter. In accordance with previous results (Conde and Fink,
1976; Vallen et al., 1992), we obtained a final population with
∼15% of multinucleated cells. Only 2–3% of diploids arose due
to leakage of the kar1-∆15 allele. The population was fixed and
prepared to allow TLC1 detection by in situ hybridization. Multi-
nucleated cells derived from zygotes were unambiguously iden-
tified by containing at least one nucleus that stained positive for
TLC1 and GFP–Nop1p. In Figure 3B, two representative
heterokaryons are shown. The top row shows a heterokaryon
(representing 10–60% of identified heterokaryons, depending on
the experiment) in which TLC1 RNA was present in all the nuclei
Fig. 1. Localization of telomerase components. Confocal images of strains YKF5 (A), YKF15 (B) and YT39 (C) grown in galactose-containing medium at 25°C
and prepared for indirect immunofluorescence using an antibody against Rap1p (A) or Nop1p (B and C). (D) Confocal images of FYC2-6A/BL2 transformed with
both pRS314-PGAL1-TLC1 and pUN100-GFP–NOP1, growningalactose-selectivemediumandprepared for in situ hybridizationusingthe PCR Alexa 546-coupled
probe. (E) Signal obtained with YT1 (tlc1-∆) under the same conditions as (D) is shown as a control.
EMBO reports vol. 3 | no. 7 | 2002 655
Localization of yeast telomerase
Fig. 2. Est2p is redistributed to the nucleoplasm upon TLC1 overexpression. (A) YKF15 (1 and 2), YT21 (3 and 4) and YT23 (5 and 6) were transformed either
with the empty plasmid pRS314 (1, 3 and 5) or with pRS314-PGAL1-TLC1 (2, 4 and 6) and grown in selective medium containing galactose at 25°C. Cells were
visualized with a Zeiss Axiovert 100. Bar: 6 µm. (B) Redistribution of Est2p from the nucleolus to the nucleoplasm upon TLC1 overexpression is dependent on
the Est2p binding site in TLC1. YKF15 was transformed with pRS314-PGAL1-TLC1-derived plasmids expressing deletion alleles of TLC1 as indicated, which
impair the binding to Est2p (2), Est1p (3 and 4) or both (1) (Livengood et al., 2002). Cells were analyzed as in (A). (C) Telomerase assays performed as described
(Forstemann and Lingner, 2001) using extracts from FYBL1-4D (lanes 1 and 2) and YKF15 (lanes 3 and 4) transformed either with pRS314 (lanes 1 and 3) or
pRS314-PGAL1-TLC1 (lanes 2 and 4). Lane 5, extract that was pre-treated with RNase A. Lane 6, mixture of extracts derived from the strain overexpressing Est2p
(lane 3) or TLC1 (lane 2). Cells were grown in galactose-containing selective medium. (D) Telomere length determined by telomere-PCR as described previously
(Forstemann et al., 2000) using an oligonucleotide (o286, see Supplementary data) that specifically amplified the telomere of chromosome IL. Two independent
clones were analyzed for each strain. Lanes 1–4 as in (B).
656 EMBO reports vol. 3 | no. 7 | 2002
M.T. Teixeira et al.
of the heterokaryon. Thus, overexpressed TLC1 RNA was able to
migrate from one nucleus to another. Furthermore, our results
show that both export and import pathways for TLC1 RNA exist.
The re-import of TLC1 into the nucleus argues that trafficking
Fig. 3. Heterokaryon analysis. (A) Scheme of the analysis. Heterokaryons were created by mating a Mata strain overexpressing TLC1 (FYBL1-4D transformed
with pRS314-PGAL1-TLC1) with a Matα strain carrying the kar1-∆15 allele and the GFP–NOP1 marker [MS113 (Vallen et al., 1992) transformed with pUN100-
GFP–NOP1]. Upon mating and incubation, we tested whether the TLC1 signal was detected in all the nuclei of heterokaryons indicating that there was export
(heterokaryonin the left) or whetherTLC1 was restricted to the nucleus whereit was overexpressed, indicatingnuclear retention(heterokaryononthe right). (B) The first
two rows correspond to confocal images of representative heterokaryons obtained. Percentages report to the total of heterokaryons as defined in the text. The two
lower rows show parental cells treated under the same conditions. Bar: 6 µm.
EMBO reports vol. 3 | no. 7 | 2002 657
Localization of yeast telomerase
may also occur at physiological levels and that the export of
overexpressed TLC1 was not solely due to the saturation of a
putative nuclear retention signal.
Our inability to detect TLC1 RNA at endogenous levels
precludes performing this experiment without overexpression of
TLC1. Therefore, we undertook an independent approach to
determine whether TLC1 RNA is exported to the cytoplasm. We
reasoned that a transcript present transiently in the cytoplasmic
compartment would be translatable by ribosomes. In this case,
an ORF provided as a fusion transcript with the TLC1 RNA
would allow production of a functional protein. Our results
presented as Supplementary data available at EMBO reports
Online show that a chimeric transcript containing an ORF
embedded in TLC1 is translatable. This suggests that endo-
genous TLC1 may also be exported from the nucleus to the cyto-
plasm as part of its life cycle. However, we cannot rule out the
possibility that the ORF itself recruited the mRNA export
machinery (Ohno et al., 2002) or that translation occurred in the
nucleus, although to date translation in the nucleus has only
been demonstrated for very small peptides (Iborra et al., 2001).
Taken together with the heterokaryon analysis, our results
support the notion that TLC1 RNA maturation involves a step in
the cytoplasm. However, more sensitive detection methods to
localize endogenous TLC1 are required to clarify the issue.
In this report, we provide evidence that telomerase biogenesis in
S. cerevisiae involves several distinct subcellular compartments:
i.e. the nucleolus, the nucleoplasm and the cytoplasm. Intra-
cellular trafficking supports a multistep character of telomerase
enzyme assembly. The RNA subunit is subject to several maturation
and assembly steps involving polyadenylation (Chapon et al.,
1997), hypermethylation at the 5′ end, association with the Sm
proteins (Seto et al., 1999) and association with Est proteins. Our
results suggest that some of these maturation steps may occur
in the cytoplasm. In addition, assembly steps that occur in the
nucleolus are more clearly emerging. The association of the
telomerase RNA and the catalytic moiety may take place in
the nucleolus, as supported by the nucleolar accumulation of
overexpressed Est2p and Est1p. The assembly of telomerases
from other species is a chaperone-assisted process (Holt et al.,
1999; Licht and Collins, 1999). Since the nucleolus is consid-
ered to be a site with a high concentration of trans-acting factors
required for the assembly of ribosomes and other RNPs
(Pederson, 1998; Sleeman and Lamond, 1999; Venema and
Tollervey, 1999), it may provide factors that assist in the
assembly of telomerase. One of these factors could be the
recently identified methyltransferase responsible for the cap
hypermethylation of snRNAs and snoRNAs (Mouaikel et al.,
2002). In support of this, it was shown that vertebrate telomerase
RNA contains an H/ACA motif that targets the RNA to nucleoli
where it was hypothesized to associate with the catalytic subunit
of telomerase (Mitchell et al., 1999; Lukowiak et al., 2001). We
found that the assembled Est2p–TLC1 telomerase core is local-
ized in the nucleoplasm. Thus, association of Est2p with TLC1
may trigger its relocation to or its retention in the nucleoplasm.
This localization does not depend on the telomerase associated
proteins Est1p or Est3p. Thus it is possible that both of these
proteins modulate telomerase activity downstream of the relocation
of the assembled complex. It is already known that Est1p is
required for telomerase recruitment to telomeres (Evans and
Lundblad, 1999). In the case of Est3p, it is at present unclear
how it affects the action of telomerase at telomeres in vivo.
Yeast strains and plasmids. See Table I and Supplementary data.
Immunostaining and TLC1 detection. Rap1p and Nop1p immuno-
staining was performed as described previously (Gotta et al.,
Table I. Yeast strains
aGasser strain collection.
bObtained from B. Dujon.
cFairhead et al. (1996).
eVallen et al. (1992).
Strain Relevant genotype
Mata ura3-∆851 trp1-∆63 leu2-∆1 his3-∆200 DIA5-1
Mata ura3-∆851 trp1-∆63 leu2-∆1 his3-∆200 lys2-∆202
Matα ura3-∆851 trp1-∆63 leu2-∆1 his3-∆200
Mata/Matα ura3-∆851/+ trp1-∆63/+ leu2-∆1/+ his3-∆200/+ lys2-∆202/+
Mata ura3-∆851 trp1-∆63 leu2-∆1 his3-∆200 DIA5-1 His3MX6:PGAL1-GFP–EST2
Mata ura3-∆851 trp1-∆63 leu2-∆1 his3-∆200 lys2-∆202 His3MX6:PGAL1-GFP–EST2
Mata/Matα ura3-∆851/+ trp1-∆63/+ leu2-∆1/+ his3-∆200/+ lys2-∆202/+ kanMX6:PGAL1-GFP–EST1/+
Matα ura3-∆851 trp1-∆63 leu2-∆1 his3-∆200 tlc1-∆::kanMX6
ura3-∆851 trp1-∆63 leu2-∆1 his3-∆200 est1-∆::KANMX6 His3MX6:PGAL1-GFP–EST2
ura3-∆851 trp1-∆63 leu2-∆1 his3-∆200 est3-∆::HIS3MX6 His3MX6:PGAL1-GFP–EST2
Matα ura3-52 trp1-∆1 leu2-3,112 kar1-∆15
Matα ura3-∆851 trp1-∆63 leu2-∆1 his3-∆200 cox17-∆::His3MX6
658 EMBO reports vol. 3 | no. 7 | 2002
M.T. Teixeira et al.
1996; Teixeira et al., 1997). The probe for in situ hybridization
was created by incorporating Alexa Fluor 546-dUTP (Molecular
Probes) in a PCR using pRS314-TLC1 as a template and oligo-
nucleotides o1 and o2 as primers. The product was purified from
unincorporated nucleotides and digested with RsaI and MboI.
Fixation and spheroplasting of cells was performed as described
previously (Teixeira et al., 1997). Slides were treated for in situ
hybridization as described in Gotta et al. (1999) without the
RNase treatment. Images were acquired on Zeiss LSM410 or
LSM510 confocal microscopes.
Heterokaryon assays. Mating experiments were performed
essentially as described previously (Flach et al., 1994). Mata
parental cells (5 × 106), grown in raffinose tryptophan-dropout
medium, were mixed in equal amounts with the Matα cells and
concentrated on 0.8 µm pore size filters. The filters were placed
on galactose-containing media and incubated at 30°C for 5.5 h.
The cells were recovered by vortexing the filter in 10 ml of
synthetic medium containing galactose and lacking leucine and
tryptophan. The suspension was then incubated for 12 h at 30°C
with agitation before in situ hybridization.
Supplementary data. Supplementary data are available at
EMBO reports Online.
We thank T. Laroche and P. Heun for advice, and M. Rose,
A. Thierry, B. Dujon, D. Gottschling, V. Doye, A. Livengood and
T. Cech for sharing strains and plasmids. M.T.T. is a recipient of
a postdoctoral fellowship from the Human Frontiers Science
Program and K.F. was supported by a PhD fellowship from the
Boehringer Ingelheim Fonds. This work was supported by grants
from the Swiss National Science Foundation and the Human
Frontiers Science Program.
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