Large- Scale Protein Localization in Yeast
Proteomics has contributed significantly to our
knowledge of eukaryotic cell biology through largescale
studies describing protein-protein interactions,
protein complex composition, and relative protein
abundance. Recent work in the budding yeast Saccharomyces
(Kumar et al.
, 2002) has extended the
scope of proteomics to encompass the genome-wide
analysis of protein localizationmthe large-scale and
systematic identification of the subcellular compartment
or organelle to which a protein localizes. The
subcellular localization of a protein can be of fundamental
importance in characterizing the function and
regulation of that protein. For example, an uncharacterized
protein that localizes to the mitochondria may
be inferred to function directly or indirectly in cellular
respiration. Many transcription factors are regulated
through subcellular compartmentalization: nuclear
export of the transcription factor Pho4p is tightly regulated
such that it is only localized within the nucleus
(and therefore capable of activating transcription) in
response to phosphate starvation (O'Neill et al.
Protein function can also be addressed on a global
scale by integrating protein localization data with a
variety of other proteomic data sets. In particular,
protein localization data are a strong complement to
large-scale protein-protein interaction studies, corroborating
many putative interactions while also highlighting
potential false-positive results (Gerstein et al.
2002). Ultimately, genome-wide protein localization
data will be helpful in identifying the constituent
proteins of each cellular organelle and, potentially,
mechanisms by which some of these proteins are
Yeast proteins may be localized through any of
several approaches. By one common strategy, antibodies
against a target protein can be used to immunolocalize
that protein within a fixed cell. Antibodies
against native proteins may be prepared by standard
immunology; however, the feasibility of applying this
approach on a proteome-wide scale has yet to be
demonstrated. Alternatively, yeast proteins may be
epitope tagged for subsequent immunolocalization.
Tagged proteins can be localized by indirect immunofluorescence
using monoclonal antibodies directed
against the epitope tag. Epitope tags may be introduced
into a protein-coding sequence by several
methods. Many yeast vectors are available by which
epitope tags may be fused to the amino (N) terminus
or carboxy (C) terminus of a cloned gene (Longtine et
al., 1998). In addition, epitope tags may be introduced
at random within a protein-coding sequence by insertional
mutagenesis using a modified epitope-bearing
transposon (Hoekstra et al.
, 1991; Ross-Macdonald et
al., 1997, 1999). A combination of random and directed
tagging methods has been used successfully in Saccharomyces
to epitope tag approximately 3600 yeast genes;
subsequent immunolocalization of this protein set has
defined the subcellular localization of nearly 2800
yeast proteins (Kumar et al.
As an alternative to immunolocalization, proteins
may be localized by fluorescence microscopy using
yeast open reading frame (ORF)-fluorescent protein
chimeras. Coding sequence for a fluorescent protein
[e.g., green fluorescent protein (GFP)] can be fused to
a protein of interest using any number of appropriate
tagging vectors (e.g., GFP-tagging vectors of the pFA6a
series) (Longtine et al.
, 1998). Yeast ORF-GFP fusions
may be constructed as integrated chromosomal alleles
using these vectors in conjunction with standard polymerase chain reaction (PCR)-based methods. GFP
tagging has been used extensively in S. cerevisiae
the localization of individual target proteins; ongoing
efforts to tag all annotated yeast ORFs with GFP
should enable large-scale analysis of protein localization
by fluorescence microscopy in living yeast cells.
This article presents detailed protocols by which
yeast ORFs may be epitope tagged and yeast strains
containing tagged proteins prepared for immunofluorescence
analysis in a 96-well format. The protocols
presented here utilize the PCR-based tagging method
of Longtine et al.
(1997) to generate C-terminal chromosomal
gene fusions with a sequence encoding three
copies of the hemagglutinin (HA) epitope. The resulting
HA-tagged yeast proteins can be immunolocalized
by indirect immunofluorescence as described; the
immunofluorescence protocol is adapted for performance
in a 96-well format, facilitating proteome-scale
analysis of protein localization.
II. MATERIALS AND
G418 sulfate (geneticin) may be purchased from
Invitrogen Corp. (Cat. No. 11811023). Glusulase is a
trademarked preparation of β-glucuronidase / sulfatase,
each available from Sigma (Cat. Nos. G0258 and S8504). Zymolyase-100T is from Seikagaku America;
Lyticase from Sigma may be substituted (Cat. No.
L4025). The mouse anti-HA monoclonal antibody
16B12 is from Covance (Cat. No. MMS-101R-1000).
Cy3-conjugated affinity-purified goat antimouse IgG is
from the Jackson Laboratories (Cat. No. 115-165-146).
4,6-Diamidino-2-phenylindole (DAPI) is available
from Sigma (Catalog No. D-9542). Poly-L
-lysine is from
Sigma (Cat. No. P1524). Microscope slides are from
Carlson Scientific (Cat. No. 101805).
A. Epitope-Tagging Yeast Genes
Yeast genes may be epitope tagged through a
variety of methods: the protocol described here is
essentially that of Longtine et al.
(1997) in which PCR
is used to amplify a cassette from the vector pFA6a-
3HA-kanMX6 suitable for generating an integrated Cterminal
HA-tagged allele of any nonrepeated yeast
gene. The HA-tagging cassette (Fig. 1A) consists of a
sequence encoding three copies of the HA epitope, the S. cerevisiae ADH1
terminator, and the kanMX6
encoding resistance to G418. Tagging cassettes are
amplified using primers with 5' ends corresponding
to the target gene and 3' ends corresponding to the cassette; primer design is discussed later. Amplified
cassettes are subsequently introduced into yeast by
standard methods of DNA transformation (Gietz et al.
1992). By homologous recombination, the tagging
cassette integrates at its intended genomic locus;
transformants containing this cassette are selected on
medium supplemented with G418. By this PCR-based
approach, yeast genes may be systematically epitope
tagged for subsequent large-scale immunolocalization.
|Figure 1 (A) HA-tagging cassette amplified from the yeast vector pFA6a-3HA-kanMX6. The tagging cassette
contains a sequence encoding three copies of the influenza virus hemagglutinin epitope (3HA), the
S. cerevisiae ADH1 terminator, and the sequence encoding the kanMX6 cassette (encoding resistance to G418
sulfate). PCR primers for amplification of this cassette are designed such that the 5' end of each primer consists
of approximately 40 nucleotides of a gene-specific sequence, whereas the 3' end consists of approximately
20 nucleotides of sequence from the polylinker immediately flanking the tagging cassette.
of PCR primers suitable for C-terminal HA tagging of target genes.
- YPD medium: 1% Bacto-yeast extract, 2% Bactopeptone,
2% glucose, 2% Bacto-agar, distilled water.
Autoclave at 121°C and 15lb/inz. in of pressure for
15 min; cool to ~55°C and pour plates. Omit agar for
- YPD-G418 plates: Prepare YPD medium. After
cooling, supplement YPD with 150mg/liter G418. To
prepare a stock solution of G418 (geneticin), dissolve
in water at a concentration of 80mg dry weight per
milliliter and filter sterilize.
- Design primers enabling targeted integration of
the amplified tagging cassette at its intended genomic
locus. Example primers are shown in Fig. 1B. Design
the 5' end of each primer such that it consists of 40-50
nucleotides of sequence from the target gene. Choose
the gene-specific sequence of the forward primer (A)
such that it ends just upstream of the stop codon;
choose the gene-specific sequence of the reverse
primer (B) such that it ends just downstream of the
- Amplify the HA-tagging cassette from plasmid
pFA6a-3HA-kanMX6 using PCR primers A and B.
Use standard cycling conditions; vary conditions and
primer/template concentrations as needed. Repeat
PCR reaction four or more times. Pool reactions,
extract once with phenol: chloroform:isoamyl alcohol
(25:24:1), precipitate, and resuspend in a small
volume of water (~10µl).
- Introduce concentrated PCR product into S. cerevisiae
by any standard DNA transformation protocol
(e.g., the lithium acetate protocol of Gietz et al., 1992).
This process may be performed in 96-well format as
described previously (Kumar et al., 2000).
- Select transformants on YPD medium supplemented
with G418. Wash cells in water (~1ml), and
resuspend in 200 µl of water. Spread onto YPD plates;
incubate overnight at 30°C and replicate onto YPDG418
plates. Pick individual G418-resistant colonies.
Alternatively, G418 selection may be performed in
large scale. Wash, precipitate, and resuspend transformants
in 400 µl YPD in 96-well plates. Incubate at 30°C for 4-6h with shaking. Streak transformant mixtures
onto rectangular 86 x 128-mm YPD plates supplemented
with G418. Use an eight-pronged replicator,
such that 24 streaks (correponding to 24 transformant
cultures) may be produced per plate. Pick G418-
resistant colonies as described previously.
- Using PCR, confirm that the HA-tagging cassette
has integrated by homologous recombination with the
target gene. Choose one primer such that it anneals
within the HA-tagging cassette and another primer
that anneals to the target gene locus outside of the
insertion region. Identify a PCR product of the
expected size as evidence of homologous integration.
B. Large-Scale Immunolocalization of
|Figure 2 Examples of immunofluorescence patterns
protein localization studies in
S. cerevisiae. (Top) Immunofluorescence
vegetative yeast cells stained with monoclonal
antibodies directed against HA. (Bottom) The same
with the DNA-binding dye 4,
Staining of the
nucleus is evident in cells carrying a tagged allele of
the homocitrate synthase Lys21p, while nucleolar
staining can be
seen in cells HA tagged for the
chaperone Srp40p. HA-tagged
Nup159p localizes to
the nuclear rim, while staining of the vacuole
observed in cells containing a tagged form of the
Strains containing HA-tagged proteins may be
immunolocalized by indirect immunofluorescence
using monoclonal antibodies directed against the HA
epitope (primary antibody) and the Cy3-conjugated
secondary antibody. Immunolocalization in yeast
necessitates permeabilization of the cell wall; here,
yeast cells are spheroplasted using the trademarked
enzyme preparations glusulase and Zymolyase-100T.
The provided immunolocalization protocol is tailored
for implementation in a 96-well format. Using this
procedure, a single researcher can easily prepare 192
samples (two 96-well microtiter plates) for immunofluorescence
in a period of 3 days (Kumar et al.
Sample immunofluorescence patterns in cells containing
HA-tagged proteins are presented in Fig. 2.
- Solution A: 1.2M sorbitol, 50mM KPO4, pH 7.
Prepare sorbitol as a 2M stock solution in water. Store
solution A up to 1 week at 4°C.
- Solution A / glusulase / Zymolyase: Supplement
solution A with 1 µl/ml 2-mercaptoethanol, 1 µl/ml
glusulase, 1µl/ml Zymolyase (at a concentration of
10mg/ml). Prepare a stock solution of 10mg/ml
Zymolyase in 5% glucose in 20mM phosphate buffer,
pH 7.4. Store stock solution of Zymolyase at -20°C. Add 2-mercaptoethanol under a chemical fume hood.
Prepare this mixture immediately before use.
- Phosphote-buffened Saline (PBS) buffer: 20x PBS
buffer per liter, 160g NaCl, 4g KCl, 28.8g Na2HPO4,
4.8g KH2PO4, 800ml water. Adjust pH to 7.4; adjust
volume to 1 liter. Autoclave and store at room temperature.
Dilute 20-fold prior to use. PBS buffers supplemented
with BSA may be stored for approximately
1 week at 4°C.
- Antibody solutions: Primary and secondary antibodies
should be added to PBS buffer immediately prior to use. Centrifuge antibodies at 4°C for 10min at
maximum speed in a variable-speed microcentrifuge
(e.g., Eppendorf Model 5415C centrifuge). Store the
Cy3-conjugated secondary antibody in the dark at
- Poly-L-lysine solution: 0.5mg/ml poly-L-lysine.
Dissolve 50mg poly-L-lysine powder in 10ml H2O;
dilute 10-fold in H2O. Filter sterilize if desired and
aliquot into 1.5-ml tubes. Store at -20°C. Centrifuge
poly-L-lysine solution for 10min prior to use.
- DAPI stock solution: Dissolve DAPI at a concentration
of 1 mg/ml in sterile H2O. Wear gloves and
safety glasses when working with DAPI, as it is a possible
carcinogen. Store at -20°C.
- Mount medium with DAPI: 1mg/ml p-phenylenediamine
in PBS supplemented with DAPI
(final concentration of 1 µg/ml). Dissolve 100mg p-phenylenediamine in 10ml PBS buffer; prepare solution
in a small beaker covered with aluminum foil.
Add 90ml glycerol and continue stirring. Aliquot into
1.5-ml tubes and store at -70°C. Before use, add 1µl
DAPI stock / ml mount medium. Mount medium with
DAPI may be stored for up to 2 weeks.
- Inoculate strains containing HA-tagged proteins
in 96-well microtiter plates containing 75 µl YPD media
per well. Grow cultures overnight (approximately 12 h)
at 30°C with gentle shaking on an orbital platform shaker (240rpm). Alternatively, incubate plates on a
vortex shaker set to its lowest speed. After overnight
growth, add 45µl YPD; grow for an additional 90min
to an OD600 of 0.75-1.
- Fix cells in formaldehyde at a final concentration
of 3.75% (v/v). Agitate cells gently for 30min using a
vortex shaker set to its lowest speed. Collect cells by
centrifugation at 2000rpm for 4min in a Sorvall H-1000B rotor. Wash pelleted cells three times in 100µl
solution A. Resuspend cells in 100 µl of solution A supplemented
with 0.1% (v/v) 2-mercaptoethanol, 0.02%
(w/v) glusulase, and Zymolyase-100T (5 µg/ml).
- Spheroplast cells in this mixture at 37°C with
gentle shaking for 13-20 min (optimal incubation times
must be determined empirically). Check cells periodically
during this incubation; once translucent, recover
cells and wash in 100 µl solution A. Resuspend pelleted
cells in 100t, tl PBS buffer supplemented with 0.1%
(v/v) Nonidet P-40 and 0.1% (w/v) bovine serum
albumin (BSA). Incubate samples at room temperature
without shaking for 15 min. Pellet cells by centrifugation
as before. Remove excess liquid by aspiration, and
resuspend pellets in 100 µl PBS supplemented with 3%
(w/v) BSA. Gently shake cells at room temperature for
30 min to 1 h.
- Add 40µl of mouse anti-HA monoclonal antibody
16B12 at a final dilution of 1:1000 in PBS supplemented
with 3% (w/v) BSA. Incubate overnight at
4°C with gentle shaking.
- Wash cells as follows: once in 150µl PBS supplemented
with 0.1% (w/v) BSA, once with 100µl PBS
supplemented with 0.1% (w/v) BSA and 0.1% (v/v)
Nonidet P-40, and once in 100µl PBS supplemented
with 0.1% (w/v) BSA. During this final wash, gently
shake cells for 5min at room temperature prior to
- Treat pelleted cells with Cy3-conjugated affinitypurified
goat antimouse IgG (secondary antibody) at a
final dilution of 1:200 in 40µl of PBS supplemented
with 3% (w/v) BSA. Shake cells gently at room temperature
for 2 h in the dark (to minimize Cy3 exposure
- Prepare poly-L-lysine-coated slides for use in
step 9. To prepare two slides, add a total volume of
40µl poly-L-lysine solution (0.5mg/ml in H2O) to one
slide; distribute the poly-L-lysine in five or six wells
spaced evenly over the surface of the slide. Place the
other slide (with no added polylysine) onto the poly-
L-lysine-treated slide such that the two faces of the
slides are sandwiched together. Wait 15 min. Separate
slides and rinse in H2O; allow slides to air dry before
use in step 9.
- Wash cells (from step 6) as follows: once in 150µl
PBS plus 0.1% (w/v) BSA, once in 100 µl PBS plus 0.1% (w/v) BSA with gentle shaking for 5min, twice in
100µl PBS plus 0.1% (w/v) BSA and 0.1% (v/v) Nonidet
P-40, and once in 100µl PBS plus 0.1% (w/v) BSA.
- Resuspend cells in 75 µl PBS. Using a multichannel
pipetter, transfer 15µl of this cell suspension into
12 individual wells on a poly-L-lysine-treated slide.
Allow cells to sit for a minimum of 15min before
removing excess solution by aspiration. Add mounting
medium supplemented with DAPI such that a total
volume of 40µl is spotted at five or six locations in
between the wells. Place a coverslip on the slide; avoid
air bubbles and ensure that mounting medium is
spread over all wells. Seal slides with nail polish and
store in the dark at -20°C do not store slides longer
than 1 month prior to examination.
In designing protein localization studies, consideration
should be paid to the placement of
reporter/epitope tags relative to each target open
reading frame. Typically, reporters and epitope tags
are fused to either N or C termini of target genes;
however, each tagging strategy possesses associated
advantages and drawbacks. Organelle-specific targeting
signals (e.g., mitochondrial targeting peptides
and nuclear localization signals) are often located at
the N terminus (Silver, 1991). N-terminal reporter
fusions may disrupt these sequences, resulting in aberrant
protein localizations. In other cases, C-terminal
sequences may be important for proper function and
regulation. For example, isoprenylation motifs are typically
C-terminal, and sequences important for plasma
membrane localization may be found throughout
the gene-coding sequence (Nakai and Horton, 1999).
While no single tagging strategy can be all-encompassing,
a greater proportion of the yeast proteome
should remain functional as C-terminal fusions:
several studies have generated genome-wide Cterminal
fusions for large-scale protein localization
(Ding et al.
, 2000; Kumar et al.
Gene copy number may also impact upon the accuracy
with which a tagged protein is localized. Overexpressed
protein products can saturate intracellular
transport mechanisms, potentially resulting in anomalous
protein localization. However, weakly expressed
single copy genes may yield insufficient protein levels
for analysis, although the protocol presented here-in
which tagged proteins are expressed endogenously
under control of their native promoters-has been
used successfully to localize nonabundant proteins
(Kumar et al.
The immunofluorescence protocol presented here is
reliable, but care should be taken to empirically determine
optimal incubation times for spheroplasting
(Zymolyase/glusulase treatment). The efficiency of
spheroplasting may be monitored by phase-contrast
microscopy. Ideally, cells should be a dark, translucent
gray. Bright cells may have been insufficiently
digested, whereas pale gray cells with little internal
structure have likely been overdigested (Adams et al.
1998). In our hands, cells spheroplasted for 13-20min
yield good staining patterns upon subsequent antibody
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