Genome-Wide Screening of
Intracellular Protein Localization
in Fission Yeast
Intracellular localization is an important part of the
characterization of a gene product. In order to search
for genes based on the intracellular localization of their
products, we constructed a green fluorescent protein
(GFP)-fusion genomic DNA library of the fission yeast Schizosaccharomyces pombe
(Ding et al.
, 2000). This was
accomplished by fusing random fragments of genomic
DNA to the 5' end of the GFP gene in such a way that
the expression of potential GFP-fusion proteins would
be under the control of the endogenous promoters contained
in the genomic DNA fragments. The intracellular
localization of the fusion proteins was determined
by microscopic observation of individual transformants.
As genes fused to GFP are expressed under
their native promoters, the intracellular localization of
gene products can be examined under physiological
conditions in both mitotic and meiotic cells. This
library provided the foundation for a survey of the
intracellular localization of S. pombe
proteins. Now that
the S. pombe
genome project is completed, genes can
be searched from the intracellular locations of their
protein products using our image database. Our
library of GFP-fusion constructs also provides useful
fluorescent markers, which can be used readily for
microscopic observation in living cells and for various
intracellular structures and cellular activities.
II. MATERIALS AND
Polypeptone is from Nihon Pharmaceutical (Cat.
No. 394-00115), yeast extract is from Difco (Cat. No.
212750), agar is from Wako (Cat. No. 010-15815), ampicillin
is from Wako (Cat. No. 016-10373/TWK6416),
glucose is from Wako (Cat. No. 041-00595), Zymolyase-
100T is from Seikagaku (Cat. No. 120493/109406),
novozym 234 is from Novo Nordisk (Cat. No.
2880/PPM4356), proteinase K is from Merck (Cat. No.
24658-0100/V354968 92), and T4 DNA ligase is from
TaKaRa (Cat. No. 2011A/1511). Water used for the
preparation of the culture medium and all of the solutions
is first treated using the Elix 5 water purification
system (Millipore) and then the MilliQ ultrapure water
purification system (Millipore).
Ten-well 25 x 75-mm glass slides are from Polyscience
(Cat. No. 18357), and glass-bottom culture
dishes are from MatTek (Cat. No. P35G-1.5-10-C). The
DNA sequencer used is the ABI377 from Perkin-Elmer.
A computer-controlled, fluorescence microscope
system (DeltaVision, Applied Precision; Haraguchi et al.
, 1999) is used for visual screening of the GFP
library. This microscope system is based on an inverted
fluorescence microscope (IX70, Olympus Optical)
equipped with a Peltier-cooled, charge-coupled device
(CCD) (CH250 or CH350, Photometrics Ltd., Tucson, AZ). The objective lens used is an Olympus oil immersion
lens (SPlan Apo 60/NA 1.4).
A. Construction of the GFP-Fusion Genomic
|FIGURE 1 Strategy for construction and screening of the
GFP-fusion genomic DNA library. Modified from Ding
et al. (2000)
Figure 1 summarizes our strategy for the construction
and screening of a gene library in which S. pombe
genomic DNA fragments from a wild-type strain were
fused to the 5' end of the GFP-S65T gene.
1. Construction of Plasmid Vectors for the Library
The construction of one such library is described
in detail in Ding et al.
(2000). The multicopy plasmid
used as a vector for our library was created by modification of a popular vector for fission yeast, pREP1
(Maundrell, 1993). In order to express GFP-fusion
genes under the control of endogenous yeast promoters,
the nmtl promoter region in pREP1 was deleted;
therefore, only genes in DNA fragments that also contained
the genes' endogenous promoters would be
expressed. Next, the coding sequence of GFP-S65T
(Heim and Tsien, 1996) was amplified by polymerase
chain reaction (PCR), and inserted upstream of the
nmtl terminator. Linker oligonucleotides were then
designed such that all three reading frames of the
restriction DNA fragments were fused to the GFPcoding
sequence. Each of the resultant plasmids contained
the S. pombe
arsl as a replication origin and the S. cerevisiae
LEU2 gene as a selection marker, both of
which were derived from the pREP1 plasmid (Fig. 1).
Insertion of the GFP PCR product into the nmtl promoter
deleted pREP1, resulting in pEG3-1. This plasmid
has a Bam
HI site immediately 5' to GFP and was used
as the first frame library plasmid. The second frame
plasmid, pEG3-2, was constructed by inserting a 12
base (CCCAGATCTGGG) Bgl
II linker into pEG3-1
that had been digested with Bam
HI and blunt-ended.
The third frame plasmid, pEG3-3, was constructed by
inserting a 10 base (CCAGATCTGG) Bgl
II linker into Bam
HI digested, blunt-ended pEG3-1. Importantly,
restriction enzyme sites that can be used for recloning
of the yeast gene into other GFP-fusion vectors must
be present. In our library, DNA restriction fragments
were cloned into the Bam
HI site of pEG3-1. In pEG3-2
and pEG3-3, however, the Bam
HI site was absent, as
II linkers were blunt ended into this site. Therefore,
HI site present in pEG3-1 was used to
reclone genes from this plasmid, while Bgl
sites were used to reclone genes present in pEG3-2 and
Insertion of the library of DNA restriction fragments
into each of the three plasmids, pEG3-1, pEG3-2, and
pEG3-3, resulted in a library of random fragments of
genomic DNA fused to the GFP-coding sequence.
Microscopic observation was used to eliminate transformants
that did not express GFP-fusion proteins
(Fig. 1). These included transformants that did not
express a GFP-fusion protein due to the absence of an
endogenous promoter to drive the expression of the
fusion gene or because the GFP-coding sequence was
not in-frame with the fission yeast protein-coding
sequence. Thus, after visual screening, a significant
proportion of the library consisted of fission yeast
genes fused in-frame with GFP and driven by their
natural biological promoters.
An important point that needs to be taken into
consideration in this type of library is that the linker
oligonucleotides used to generate alternative GFP reading frames introduce a spacer between GFP and
the yeast gene, and this spacer may affect the localization
of the GFP-fusion protein (see Section IV).
2. Preparation of Genomic DNA
The S. pombe
strain 968 h90
was used to prepare
genomic DNA. Genomic DNA was isolated according
to Matsumoto et al.
(1987), which involves two centrifugation
steps for the isolation of nuclei to reduce
mitochondrial DNA contamination.
- YEade culture medium: 0.5% yeast extract, 3%
glucose, 75µg/ml adenine sulfate dihydrate
- CPS buffer: 50mM citrate/phosphate, pH 5.6, 40mM EDTA, 1M sorbitol
- 10mM TE buffer: 10mM Tris-HCl, pH 7.5, 1 mM EDTA
- 50mM TE buffer: 50mM Tris-HCl, pH 7.5, 50mM EDTA
3. Partial Digestion of Genomic DNA
- Prepare a 200-ml liquid culture of S. pombe cells in
a rich medium (YEade) and grow to midlog phase
(1 × 107 cells/ml) with shaking at 33°C.
- Harvest by centrifugation at 2500rpm for 5 min.
- Wash cells in 10 ml of 10 mM TE buffer. Centrifuge
at 2500rpm for 5 min.
- Resuspend cells in CPS buffer at 3.4 x 108 cells/ml,
add Zymolase-100T and Novozym to a final concentration
of 0.2mg/ml, and incubate at 36°C with
gentle shaking for 30min to digest the yeast cell
- Check digestion of cell walls using a phasecontrast
microscope. Continue the digestion until
80% of the cells become round-shaped protoplasts.
- Spin down at 2000rpm for 5 min.
- Wash with 5 ml CPS buffer and resuspend in 5 ml
ice-cold 10 mM TE buffer containing 1% Triton
- Spin down at 2000rpm for 5 min at 0°C. Transfer
the supernatant to a new tube and centrifuge at
10,000rpm for 30min at 4°C. Discard the supernatant
and dry the pellet.
- Resuspend the pellet in 5 ml of 50mM TE buffer.
Add 0.5 ml 10% SDS, 0.375 ml 4M sodium chloride,
and 0.5 mg protease K and incubate overnight at
- Add 5ml of phenol/chloroform (1:1) and mix
well. Spin down at 10,000 rpm for 10 min.
- Transfer the upper aqueous phase to a new tube
and repeat step 10.
- Transfer the upper aqueous phase to a new tube
and precipitate DNA with 2 volumes of ethanol. Wash the pellet with ice-cold 70% ethanol and dry
- Finally, resuspend DNA in 0.1ml of 10mM TE
buffer containing 100µg/ml RNase A. The DNA
can be quantified by running an aliquot on an
agarose gel alongside a calibrated DNA sample.
Typical yields are about 5-15µg per a starting
culture of 200ml at midlog phase.
Genomic DNA is partially digested with a four-base
cutter restriction enzyme, e.g., Sau
3AI (Ding et al.
2000). A combination of several four- or six-base
cutter restriction enzymes is also applicable (Sawin
and Nurse, 1996). Considering the average size of
fission yeast genes, including their promoters, 3- to
6-kb DNA fragments are appropriate. A suitable
digestion time should be determined empirically in a
small-scale pilot experiment. Centrifuge the partially
digested DNA through a sucrose gradient (10-40%)
at 50,000rpm for 15min using a RP-50T2 rotor in a
Hitachi ultracentrifuge (Himac CP70G). Collect
fractions and run aliquots out on an agarose gel. Pool
the fractions that contain DNA fragments with the
preferred length. Recover the DNA by ethanol
4. Construction of Plasmid Libraries
in Escherichia Coli
Digest each plasmid vector-when constructing our
library, these were pEG3-1, pEG3-2, and pEG3-3mwith
an appropriate restriction enzyme and treat with alkaline
phosphatase to inhibit self-ligation of the plasmid.
Run small-scale pilot ligation experiments to find ligation
conditions that minimize background transformants
derived from self-ligation. In our large-scale
experiments, 30 ng of the alkaline phosphatase-treated
vector and 150ng of DNA fragments were incubated
with 350 units of T4 ligase in a total volume of 30µl
overnight at 16°C. Competent E. coli
transformed with 30µl of each of the three plasmid
DNA libraries and were plated on LB plates containing
50µg/ml ampicillin at a dilution suitable for the
appearance of isolated single colonies. This procedure
yielded approximately 12,000 E. coli
for each library. Ampicillin-resistant colonies were
scraped from plates with LB medium. A portion of this
pool of cells (1/10 volume) was saved in small aliquots
at -80°C as a frozen stock. DNA was prepared from
the remaining cells. Small-scale preparations of DNA
from 30 individual transformants showed that 76-85%
of the plasmids had insert DNAs with an average
length of 5 kbp.
B. Transformation of S. pombe Cells with
Three sets of transformation were carried out using
the libraries based on the three plasmid vectors, pEG3-
1, pEG3-2, and pEG3-3. Homothalic S. pombe
leul-32 ura4 his2) and CRL152 (h90
32 ura4 lysl) were used for transformation with the
GFP-fusion genomic DNA libraries. Homothalic (h90
strains can readily be induced to undergo meiosis and
thus can be conveniently screened for mitotic and
meiotic localization of fluorescently labeled proteins.
A standard lithium chloride method (Moreno et al.
1991) can be used for the transformation of fission
yeast cells with the library DNA. Transformants were
selected on plates of EMM2 (Edinburgh minimal
medium; Moreno et al.
, 1991) lacking leucine. In our
experiments, 50 µg of one library transformed into 7 × 108
cells generated about 40,000 fission yeast transformants
on 45 plates of EMM2 in 90-mm dishes.
C. Microscopic Screening of S. pombe Transformants
A total of about 50,000 transformants from the
three libraries were selected for microscopic screening
(Table I). Single colonies were picked with toothpicks
and transferred onto new plates of EMM2. Cells were
grown for 24h at 33°C and then for 12h at 26°C. Culture of the cells at 26°C enhances GFP fluorescence. Cells were then suspended in liquid EMM2 lacking a
nitrogen source on a 10-well glass slide (Polysciences)
and were observed with our CCD microscope system
using an Olympus oil immersion objective lens (SPlan
Apo 60/NA 1.4) and high-selectivity excitation and
barrier filters for fluorescence (Chroma Technology,
Brattleboro, VT). GFP signals are usually bright and
easily detected by eye, but when the signal is weak it
is recommended that images be taken on the CCD with
relatively long exposure times (a few seconds on our
system). Note that dead cells often produce strong autofluorescence
at the emission wavelength of GFP and
should therefore be avoided when ascertaining the
intracellular localization of GFP-fusion proteins.
D. Recovery of Plasmids from Selected
Transformants and Determination of
Nucleotide Sequences of a DNA Insert
Out of 50,000 transformants, 728 transformants
exhibited distinct patterns of GFP fluorescence (Table
I). Examples of localizations of GFP-fusion proteins
are shown in Fig. 2. Plasmids from these transformants
were recovered using standard methods (Moreno et al.
1991). Isolated plasmids were retransformed into
fission yeast cells to confirm the intracellular localizations
determined from the first screening. Partial
nucleotide sequencing of the ORF of the inserted yeast
gene was performed using a portion of the GFP gene as a sequencing primer (Fig. 1). Sequencing identified
250 independent genes (Ding et al.
E. Construction of Image Database of
|FIGURE 2 Examples of localizations of GFP-fusion proteins. (A) General nuclear staining, (B) nuclear
dots, (C) nucleolus, (D) nuclear rim, (E) cell periphery, (F) cell pole and septum, (G) membrane, and (H) cytoplasmic
dots. Scale bar: 10 µm.
We constructed an image database of the intracellular
localization of these 250 gene products and categorized
them into 11 groups as shown in Table I (Ding et al.
, 2000). Occasionally, a GFP-fusion protein was
observed in more than one intracellular localization, as
some proteins localize to several structures simultaneously
or localize to various different structures during
passage through the cell cycle. In these cases, we categorized
the gene by the intracellular location that
exhibited the most prominent staining. Secondary
localizations are also listed in the database and
genes can be searched for by any of their detected
locations. Using this database, DNA sequences can be
searched based on the localization of their products
(see our Web site, http://www.karc.crl.go.jp/d332/CellMagic/index.html).
F.Time-Lapse Observation in Living Cells
Time-lapse images of GFP fluorescence in living
cells were obtained on a cooled CCD using our
computer-controlled fluorescence microscope system
(Ding et al.
, 1998; Haraguchi et al.
, 1999). For time-lapse
observation, living fission yeast cells expressing a
GFP-fusion construct are mounted between two coverslips
(Method 1) or in a 35-mm glass-bottom culture dish (MatTek Corp., Ashland, MA) coated with concanavalin
A (Method 2), and observed in EMM2
medium. Sandwiching between coverslips generates
clearer images, but allows observation for only about
2h due to the limited amount of nutrition and oxygen
available to the cells in such an arrangement. For
longer observation, the use of glass-bottom culture
dishes is recommended.
To make 1 liter of EMM2, add 20g glucose, 5g
Cl, 2.2 g Na2
, 3 g potassium hydrogen phthalate,
20ml salts, 1
0.1 ml trace elements, 2
1 ml vitamins, 3
and 20ml amino acids 4
Salts (50x stock): 52.5 g/liter MgCl2·
O, 0.735 g/liter
O, 50 g/liter KCl, and 2 g/liter Na2
Trace elements (10,000x stock): 5g/liter boric acid (HBBO3),
4 g/liter MnSO4, 4 g/liter ZnSO4·
O, 2 g/liter FeCl2·
0.4g/liter molybdic acid (H2
), 1g/liter KI, 0.4g/liter
O, and 10 g/liter citric acid.
Vitamins (1000x stock): 1g/liter pantothenic acid, 10g/liter
nicotinic acid, 10 g/liter myo-inositol, and 10mg/liter biotin.
Amino acids (50x stock): 10mg/ml L-leucine, 3.75mg/ml
adenine sulfate dihydrate, 3.75 mg/ml uracil, 3.75 mg/ml L
-histidine hydrochloride monohydrate, and 3.75mg/ml L
|FIGURE 3 Living cells between coverslips. (A) living
fission yeast cells are mounted between coverslips and
sealed with silicon grease. Silicon grease is packed in a
syringe capped with a micropipette tip and applied to the
edges of the smaller. upper coverslip. (B) A side view of a
specimen on the microscope stage.
Complete EMM2 medium containing NH4
Cl as the
nitrogen source generates optical turbidity after autoclaving,
disturbing microscopic observation. The use
of EMM2 depleted of NH4
Cl (EMM2-N) is recommended
because of its optical clarity. Usually EMM2-
N is used for the induction of meiosis and the
observation of meiotic cells. For mitotic growth, however, a nitrogen source is necessary; in this case,
autoclave EMM2-N and add a filtered 50x stock solution
Cl to the autoclaved EMM2-N.
Method 1: Sandwiching between
- Prepare two coverslips (60 x 24mm and 18 x
- Suspend fission yeast cells in the EMM2 medium.
- Place 2.5µl of cells on the larger coverslip (60 x
- Cover with the smaller coverslip (18 x 18 mm) carefully
so as not to introduce air bubbles.
- Seal with silicon grease to avoid evaporation during
observation (Fig. 3A). Do not use organic solventcontaining
sealing reagents such as nail enamel as
they are toxic to living cells. If the sealing is poor,
cells can move due to streaming of media caused by
- Observe the specimen on an inverted microscope
Method 2: Glass-Bottom Culture Dishes (Fig. 4)
|FIGURE 4 Living cells in a glass-bottom culture dish.
(A) A 35-mm glass-bottom culture dish.
(B) A side view of a specimen on the microscope stage.
- Coat a glass-bottom culture dish (Fig. 4A) with
concanavalin A shortly before use. Spread 50µl of
1 mg / ml concanavalin A solution on the glass bottom
of the dish. Then, remove excess solution and dry.
- Suspend fission yeast cells in EMM2 medium.
- Place 50 µl of cells on the glass bottom in the center
of the dish. The cell wall will adhere to the concanavalin
- Place small drops of water, or small pieces of wet
paper or cotton, at the periphery of the dish to avoid
evaporation during observation. Seal the lid of the
dish with parafilm.
- Place the dish on an inverted microscope (Fig. 4B).
Leave the specimen on the microscope stage for
about 10min before observation to allow the cells to
settle down in the dish.
A spacer between GFP and the gene may affect the
localization of the GFP-fusion protein. We constructed
our library using three plasmid vectors, pEG3-1,
pEG3-2, and pEG3-3. In pEG3-1, there was no spacer
codon between GFP and the yeast coding sequence,
whereas in pEG3-2, three amino acid residues, Trp,
Gly, and Ser, were inserted between GFP and the yeast
coding sequence, and in pEG3-3 three amino acid
residues, Leu, Gly, and Ser, were inserted. The best
results were obtained when Leu, Gly, and Ser were
inserted between GFP and the fission yeast coding
sequence (Ding et al.
, 2000). Fusing the GFP directly
to the protein backbone or with a spacer made of a
heterocyclic aromatic amino acid, such as tryptophan,
may affect the secondary structure of the fusion
protein and, consequently, may disturb its intracellular
An obvious limitation of our library is that those
gene fragments that abate cell viability cannot be
obtained. Another limitation of our library is occasional
mislocalization of the fusion gene products.
Because we fused GFP to random fragments of the
genomic DNA, the C-terminal portion of the gene
product was truncated to various extents and replaced
by GFP. Gene products that have localization signals
at their C termini could be mislocalized or excluded
during screening. Also, cryptic localization signals
can occasionally be contained within the amino acid
sequences fused to GFP. Now that all the proteincoding
genes of S. pombe
have been identified by the
completion of the S. pombe
genome project (Wood et al.
2002), these limitations can be partly eliminated by
constructing an ordered library in which the GFP gene
is fused to full-length open reading frames. In the
budding yeast S. cerevisiae
, a collection of strains have
been constructed, in which the GFP-fusion construct
is integrated into the native chromosomal locus and
expressed under their own promoter (Huh et al.
- For observing living fluorescently stained cells,
use a high-sensitive camera to avoid damage to the
cells caused by the excitation light and also minimize
the excitation by the use of a shutter.
- Occasionally an isolated plasmid will contain
two different GFP-fusion genes, presumably as a
result of heterodimer formation. This is seen during
nucleotide sequence determination as two sequences
overlapping each other. In such cases, the fusion proteins
must be recloned into separate plasmids and the
intracellular localization of the GFP-fusion products
- Because the GFP-fusion constructs are expressed
on a multicopy plasmid, overexpression of the
GFP-fusion protein may occur and affect its intracellular
localization. To minimize this problem, it is recommended
to use fresh preparations of exponentially
growing cells for observation. Cells with physiological
perturbations tend to fluoresce brightly or to accumulate
fluorescent protein aggregates in the cell. Avoid
observing such cells that give atypically bright fluorescence.
In principle, localization patterns representative
of the major population of cells in a microscope
field are reliable.
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