Pil1 co-tethering assay to detect protein-protein interactions in the fission yeast
Schizosaccharomyces pombe
Zhao-Qian Pan, Yu-Sheng Yang, and Li-Lin Du

Abstract
Protein-protein interactions play critical roles in biological processes. We havepreviously developed the Pil1 co-tethering assay, an imaging-based method to detect
protein-protein interactions in living Schizosaccharomyces pombe cells. This assay
leverages the distinct localization pattern of the Pil1 protein by fusing a bait protein to
Pil1 and examining whether a prey protein co-localize with the Pil1-fused bait. Here,
we present an improved protocol of the Pil1 co-tethering assay. In this protocol,
modified stable integration vectors (SIVs) with a NotI site as the linearization site areused to express bait and prey proteins. We expect that this protocol will enhance theapplication of the Pil1 co-tethering assay for studying protein-protein interactions.

Keywords: Protein-protein interactions, Live cell imaging, Pil1 co-tethering assay,
Fission yeast, Schizosaccharomyces pombe

1. Introduction
Protein-protein interactions are the basis of numerous cellular processes [1, 2]. A
variety of in vitro and in vivo methods have been developed to detect protein-protein
interactions [3–5]. We have recently developed an imaging-based method called thePil1 co-tethering assay to detect protein-protein interactions in the fission yeast
Schizosaccharomyces pombe [6]. In this method, the S. pombe Pil1 protein, whichforms filamentary structures associated with the plasma membrane [7], is fused withmCherry and a bait protein, and co-expressed with a prey protein fused with mECitrine.
If the bait and prey proteins interact, co-localization of mCherry and mECitrine signals
will be observed in living fission yeast cells (Fig. 1).
In our published method [6], bait and prey proteins are expressed using pDUAL
vector-based integrating plasmids, which integrate into the genome through ends-in

recombination [8, 9]. We have observed that transformants of such plasmids frequently
exhibit failed or incorrect integration. To address this, we have revised the method byadopting stable integration vectors (SIVs) developed by Sophie Martin’s laboratory forexpressing bait and prey proteins [10]. We have modified the SIVs by introducing a
NotI site as the linearization site, making the SIVs more universally applicable (seeNote 1).

2. Materials

2.1 Media
1. Rich medium: yeast extract medium with supplements (YES). For 1 L liquid YES
medium, mix 5 g yeast extract, 30 g glucose, 40 mL 25 × supplements (5 g leucine,
5 g histidine, 5 g adenine, and 5 g uracil in 1 L water), add water to 1 L and autoclave.
For solid medium, add 2% (w/v) agar prior to autoclaving.
2. Minimal medium: Edinburgh minimal medium (EMM). For 1 L liquid EMM
medium, mix 3 g potassium hydrogen phthalate, 5 g ammonium chloride, 2.2 g
sodium phosphate dibasic, 20 g glucose, 20 mL 50 × salt stock (52.5 g magnesium
chloride hexahydrate, 0.735 g calcium chloride dihydrate, 50 g potassium chloride,and 2 g sodium sulfate in 1 L water), 1 mL 1000 × vitamin stock (1 g pantothenic
acid, 10 g nicotinic acid, 10 g inositol, and 10 mg biotin in 1 L water, stored at 4 °C),
0.1 mL 10000 × mineral stock (5 g boric acid, 4.5 g manganese sulfate monohydrate,
4 g zinc sulfate heptahydrate, 2 g iron(III) chloride hexahydrate, 0.4 g molybdic
acid, 1 g potassium iodide, 0.4 g copper sulfate pentahydrate, and 10 g citric acidmonohydrate in 1 L water, stored at 4 °C), add water to 1 L and autoclave. For solid
medium, add 2% (w/v) agar prior to autoclaving.

2.2 Plasmid digestion
1. The restriction enzyme NotI.
2. A 10 × restriction enzyme buffer appropriate for NotI.

2.3 Transformation
1. 1.0 M Lithium acetate: Dissolve 51 g of lithium acetate dihydrate in 450 mL ofwater. Adjust volume to 500 mL with water. Autoclave and store at room
temperature.
2. 50 % w/v PEG MW 3350: Dissolve 100 g of PEG 3350 in 120 mL of water.
Adjust volume to 200 mL with water. Autoclave and store at room temperature (seeNote 2).
3. 2.0 mg/mL carrier DNA: Dissolve 200 mg of salmon sperm DNA in 100 mL of
TE buffer (10 mM Tris-HCl, 1 mM Na2-EDTA, pH 8.0) using a stir plate at 4 °C.
Aliquot and store at -20 °C (see Note 3).

2.4 Modified SIV plasmids for expressing bait and prey proteins
We have constructed SIV plasmids that contain the P41nmt1 promoter, the ScADH1
terminator, and the coding sequence of Pil1-mCherry, mECitrine, or mTurquoise2(Tables 1, 2, and 3). These plasmids are available from Addgene(https://www.addgene.org/) and the Yeast Genetic Resource Center of Japan(YGRC/NBRP, https://yeast.nig.ac.jp/). Table 1 includes four plasmids (pDB5225pDB5228) that contain the coding sequence of Pil1-mCherry and can be used forexpressing bait proteins. They differ in the selection marker and the locus of
integration. Table 1 also includes a control bait plasmid (pDB5230) expressing Pil1mCherry fused with SpAtg8(1-115). Table 2 lists four plasmids (pDB4875pDB4884) for expressing prey proteins fused with mECitrine and a control prey
plasmid (pDB5253) expressing CtNbr1 fused with mECitrine. Table 3 lists four
plasmids (pDB4887-pDB4896) for expressing prey proteins fused withmTurquoise2.

3. Methods

3.1 Plasmid construction
To generate a plasmid expressing a bait protein, the coding sequence of the bait

protein can be cloned into the BglII site of the SIV plasmids containing the coding
sequence of Pil1-mCherry (Fig. 2). To generate a plasmid expressing a prey protein,
the coding sequence of the prey protein can be cloned into either the NheI or BglII
site of the SIV plasmids containing the coding sequence of mECitrine or
mTurquoise2 (Fig. 2). The availability of plasmids containing these two different
fluorescent proteins facilitates the detection of ternary protein interactions (see Note4).

3.2 Yeast transformation
1. Inoculate a yeast strain in 5 mL of liquid YES medium and incubate overnight at
30 °C with rotary shaking. Determine the cell concentration by measuring theoptical density at 600 nm (OD600). Dilute the culture into a flask containing freshliquid YES medium to a OD600 of 0.1-0.2. Incubate the flask at 30 °C with rotary
shaking (see Note 5).
2. When the OD600 of the culture reaches 0.4-0.8, harvest the cells by centrifuging
at 3000 g for 2 min, and wash the cells with sterilized water once. 5 OD600 units ofcells are used for each transformation.
3. Perform plasmid digestion by mixing 3 μL 10 × restriction enzyme buffer, 1 μL
NotI, 1 μg plasmid, adding water to a total volume of 30 μL, and incubating at 37 °C
for 1 h.
4. Boil carrier DNA for 5 min and then chill in an ice-water bath immediately.
5. Prepare the transformation mix (240 μL 50% PEG 3500, 36 μL 1 M LiAc, and50 μL carrier DNA for one transformation) and add the mix into a 1.5 mL tube
containing the pellet of 5 OD600 units of cells (see Note 6).
6. Add the 30 μL NotI digestion mix into the tube containing the cells and thetransformation mix. Add sterilized water to a final volume of 360 μL. Resuspend
the cells by vortexing. A bait plasmid and a prey plasmid can be co-transformed
together (see Note 7).
7. Incubate the tube in a 42 °C water bath for 40 min.
8. Centrifugation at the top speed of a microcentrifuge for 1 min and remove thesupernatant.
9. Resuspend the cells with 100 μL of sterilized water and spread the cells on a YES
plate. Incubate the plate at 30 °C for 24 h.
10. Replica plate onto an antibiotic-containing YES plate. Incubate the plate at
30 °C for 2-3 days (see Note 8).

3.3 Transformant clone verification
1. Pick colonies grown on the selection plate, and re-streak to single colonies on a
YES plate.
2. When the single colonies are grown up, replica plate onto a selection plate. If atransformant clone contains stably integrated plasmid(s), all single colonies derived
from it should grow on the selection plate.
3. Further verify the clones by examining the fluorescence signals with microscopy
and by performing colony PCR (see Note 9).

3.4 Microscopy analysis
1. Inoculate a yeast strain in 2 mL of liquid EMM medium and incubate overnightat 30 °C with rotary shaking (see Note 10).
2. Dilute and grow the culture at 30 °C for 24 h, diluting at least twice during thisperiod to ensure the culture remains in continuous log-phase growth prior to
microscopy.
3. Collect 1 mL of culture in a 1.5-mL tube and centrifuge at 3000 g for 30 s (seeNote 11).
4. Using a micropipette, collect 1-1.5 μL of cell pellet from the bottom of the tube
and place it onto a coverslip. Gently place a glass slide on top to create a sandwichwith the cells between the slide and coverslip (see Note 12). If the cells do not
spread into a single layer, make a new sandwich.
5. Acquire micrographs of the cells using a fluorescence microscope (see Note 13).

3.5 Quantification of the co-localization

To quantify the degree of co-localization between bait and prey proteins in the Pil1
co-tethering assay, we routinely use the Pearson correlation coefficient (PCC)
calculated by the Coloc 2 plugin of the Fiji distribution of the ImageJ software [6].
1. Open a multi-channel image file in Fiji and use the “Image > Color > Split
Channels” command to obtain separate files for different wavelength channels.
2. Use the “Freehand selections” tool (the fourth tool in the Fiji toolbar) to outline
individual cells in one channel (see Note 14). The outlined cells are called regionsof interest (ROIs) in Fiji.
3. Open the Coloc 2 plugin in Fiji (Analyze > Colocalization > Coloc 2). In the“ROI or mask” pull-down menu, choose the channel in which ROIs have been
selected in the last step.
4. After running the Coloc 2 plugin, the plugin-reported “Pearson’s R value (nothreshold)” for a cell is recorded as the PCC value for that cell. We usually analyze
a total of 10 cells and present the PCC values as mean±s.d. (see Note 15).

Here we use the positive control plasmids pDB5230 and pDB5253 (Tables 1 and 2;
available from Addgene and YGRC/NBRP) to demonstrate PCC-based colocalization analysis. pDB5230 expresses the bait protein Pil1-mCherry-fused
SpAtg8(1-115), a C-terminally truncated version of S. pombe Atg8. pDB5253
expresses the prey protein mECitrine-fused Chaetomium thermophilum Nbr1
(CtNbr1). We have previously shown that these two proteins interact in the Pil1 cotethering assay [11]. Fluorescent signals from these two proteins exhibit strong
visual co-localization in both top/bottom and mid-plane Z-sections (Fig. 3). As anegative control, Pil1-mCherry alone does not co-localize with mECitrine-CtNbr1(Fig. 3). PCC values obtained using the Coloc 2 plugin in Fiji align with the visualobservation (Fig. 3).

4. Notes
1. The original SIV plasmids developed by the Martin laboratory use the restriction
site of AfeI, PmeI, BstZ17I, or StuI as the linearization site [10]. AfeI, BstZ17I, andStuI are 6-base cutters and their restriction sites frequently exist in intended inserts,
thus rendering a SIV plasmid incompatible with an intended insert. We modified
the SIVs so that all of them use the restriction site of the 8-base cutter NotI as thelinearization site.
2. The bottle containing the PEG 3350 solution should be tightly capped to prevent
evaporation, as an increased concentration of PEG in the transformation reaction
can negatively affect the yield of transformants.
3. Carrier DNA should be denatured by boiling in a heat block for 5 min and chilled
immediately in an ice-water bath before use. Denatured carrier DNA can be used
three or four times without loss of activity, and it should be boiled again afterwards
or a new aliquot should be used.
4. To detect a ternary interaction, one bait protein fused with Pil1-mCherry and two
prey proteins fused respectively with mECitrine and mTurquoise2 can be coexpressed, as has been done in our published study [6].
5. It is important to allow the cells to complete at least two doublings before
collecting the cells for transformation.
6. When preparing multiple transformation reactions, make a transformation mix
with a volume for n+1 reactions (where n is the number of planned transformations),
then aliquot the mix into the individual tubes. This ensures sufficient volume isprepared to account for any pipetting losses during the aliquoting process.
7. The transformation efficiency when co-transforming more than one plasmid islower compared to transforming a single plasmid. To account for this, at least 1 μg
of DNA should be used for each plasmid when performing co-transformations.
8. For co-transformation of two plasmids, replica plate onto a selection platecontaining two different antibiotics (e.g. YES + hygromycin + G418).
9. In our experience, the rate of correct SIV plasmid integration is quite high. The
pUra4NotI, pAde6NotI, and pHis5NotI plasmids show rates of correct integration of 90%
or above, while the pLys3NotI plasmids exhibit a lower rate that is still above 50%.
PCR primers for assessing whether correct integration has happened are thosereported by the Martin laboratory [10].

10. The P41nmt1 promoter is a thiamine-repressible promoter. Since the commonlyused rich medium for fission yeast (the YES medium) contains thiamine, minimal
media lacking thiamine, such as the EMM medium, should be used for expressionof proteins under the control of the P41nmt1 promoter.
11. Gently pouring off some, but not all, of the supernatant can make the following
pipetting step easier.
12. Cleanness of the coverslip and slide is critical, as dust particles can impede
proper cell spreading. Gently placing the heavier glass slide on top aids in spreading
cells into a single layer without applying additional pressure that may cause cell
rupture.
13. We typically use images focused on the top or bottom plasma membrane forvisualizing co-localization. At least 5 optical Z-sections with a 0.2 μm Z-interval
should be acquired to ensure that at least one Z-section has the plasma membraneassociated Pil1 filaments in focus. Performing deconvolution can enhance image
quality.
14. Choose a channel in which the cell boundary is more visually discernable foroutlining.
15. PCC values range from −1 to 1, with higher values indicating stronger colocalization between the two channels.

References
1. Alberts B (1998) The Cell as a Collection of Protein Machines: Preparing the
Next Generation of Molecular Biologists. Cell 92:291–294
2. Gavin A-C, Superti-Furga G (2003) Protein complexes and proteomeorganization from yeast to man. Current Opinion in Chemical Biology 7:21–27
3. Wiens MD, Campbell RE (2018) Surveying the landscape of optogenetic
methods for detection of protein-protein interactions. Wiley Interdiscip Rev Syst Biol
Med 10:e1415
4. Zhou M, Li Q, Wang R (2016) Current Experimental Methods for Characterizing
Protein-Protein Interactions. ChemMedChem 11:738–756
5. Xing S, Wallmeroth N, Berendzen KW et al (2016) Techniques for the Analysis
of Protein-Protein Interactions in Vivo. Plant Physiol 171:727–758
6. Yu Z-Q, Liu X-M, Zhao D et al (2021) Visual detection of binary, ternary andquaternary protein interactions in fission yeast using a Pil1 co-tethering assay. J Cell
Sci 134:jcs258774
7. Kabeche R, Baldissard S, Hammond J et al (2011) The filament-forming protein
Pil1 assembles linear eisosomes in fission yeast. Mol Biol Cell 22:4059–4067
8. Matsuyama A, Shirai A, Yashiroda Y et al (2004) pDUAL, a multipurpose,
multicopy vector capable of chromosomal integration in fission yeast. Yeast 21:1289–
1305
9. Wei Y, Wang H-T, Zhai Y et al (2014) Mdb1, a Fission Yeast Homolog of
Human MDC1, Modulates DNA Damage Response and Mitotic Spindle Function.

PLoS ONE 9:e97028
10. Vještica A, Marek M, Nkosi PJ et al (2020) A toolbox of stable integration
vectors in the fission yeast Schizosaccharomyces pombe. J Cell Sci.
https://doi.org/10.1242/jcs.240754
11. Zhang J, Wang Y-Y, Pan Z-Q et al (2022) Structural mechanism of protein
recognition by the FW domain of autophagy receptor Nbr1. Nat Commun 13:1–11

TablesTable 1. SIV plasmids for expressing a bait protein fused with Pil1 and mCherry
Plasmid

Addgene

NBRP

ID

ID

ID

Descriptive name

Locus of

Antibiotic

integration in-resistancethe S. pombe

markergenomepDB5225 215492

FYP6282

pUra4NotI-

ura4

natMX

ade6

hphMX

lys3

bsdMX

P41nmt1-Pil1mCherryterminatorScADH1natMX
pDB5226 215493

FYP6283

pAde6NotIP41nmt1-Pil1mCherryterminatorScADH1hphMX

pDB5227 215494

FYP6284

pLys3NotIP41nmt1-Pil1mCherryterminatorScADH1-

bsdMX
pDB5228 215495

FYP6285

pHis5NotI-

his5

kanMX

ade6

hphMX

P41nmt1-Pil1mCherryterminatorScADH1kanMX
pDB5230 215496

FYP6299

pAde6NotIP41nmt1-Pil1mCherrySpAtg8(1-115)
terminatorScADH1hphMX

Table 2. SIV plasmids for expressing a prey protein fused with mECitrine
Plasmid

Addgene

NBRP

ID

ID

ID

Descriptive name

Locus of

Antibiotic

integration in-resistancethe S. pombe

markergenomepDB4875 215497

FYP6300

pUra4NotI-

ura4

natMX

ade6

hphMX

lys3

bsdMX

P41nmt1mECitrineterminatorScADH1
natMX
pDB4878 215498

FYP6301

pAde6NotIP41nmt1mECitrineterminatorScADH1
hphMX

pDB4881 215499

FYP6302

pLys3NotI-

P41nmt1mECitrineterminatorScADH1bsdMX
pDB4884 215500

FYP6303

pHis5NotI-

his5

kanMX

ura4

natMX

P41nmt1mECitrineterminatorScADH1kanMX
pDB5253 215501

FYP6304

pUra4NotIP41nmt1mECitrineCtNbr1
terminatorScADH1natMX

Table 3. SIV plasmids for expressing a prey protein fused with mTurquoise2
Plasmid

Addgene

NBRP

ID

ID

ID

Descriptive name

Locus of

Antibiotic

integration in-resistancethe S. pombe

markergenomepDB4887 215502

FYP6305

pUra4NotI-

ura4

natMX

ade6

hphMX

P41nmt1mTurquoise2terminatorScADH1
natMX
pDB4890 215503

FYP6306

pAde6NotIP41nmt1mTurquoise2-

terminatorScADH1
hphMX
pDB4893 215504

FYP6307

pLys3NotI-

lys3

bsdMX

his5

kanMX

P41nmt1mTurquoise2terminatorScADH1bsdMX
pDB4896 215505

FYP6308

pHis5NotIP41nmt1mTurquoise2terminatorScADH1kanMX

Figure legends
Fig. 1 A schematic of the Pil1 co-tethering assay. The bait protein is fused with Pil1mCherry and is localized on filamentary structures. If the mECitrine-fused prey protein
can interact with the bait protein, it will exhibit co-localization with the bait protein on
the same filamentary structures.

Fig. 2 Schematics of SIV plasmids for expressing bait or prey proteins (pUra4 NotI
backbone as example). SIV plasmid backbone is shown in grey and the NotI
linearization site is indicated. The coding sequence of a bait protein can be cloned into
the BglII site of the plasmid shown on top, resulting in a plasmid expressing a Pil1mCherry-bait fusion protein. The coding sequence of a prey proteins can be cloned intoeither the NheI or BglII site of a plasmid shown at the bottom, resulting in a plasmid
expressing the prey protein either C-terminally or N-terminally fused with mECitrine
or mTurquoise2.

Fig. 3 Detection of the interaction between SpAtg8(1-115) and CtNbr1. SpAtg8(1-115),

lacking the glycine 116 residue required for lipidation, was fused to Pil1-mCherry as
bait and co-expressed with mECitrine-fused CtNbr1 in S. pombe. Pil1-mCherry served
as a negative control bait. Deconvolved images focused on the top/bottom or mid-planesections of the cells are shown. PCC values for the cells indicated by arrows are
displayed. Scale bar, 2 μm.

mECitrine-prey

Pil1-mCherry-bait+

bait and preyinteract

bait and prey
do not interact

Figure 1

NotI

e
Ch
ADH1 mator
terminpUra4NotIP41nmt1-Pil1-mCherry
BglII
Nh
eI

rry

NheI

Pil1

1

nmt

P41

NotI

NotI

pUra4NotIP41nmt1-mECitrine

pUra4NotIP41nmt1-mTurquoise2

1 r
H ato
AD in
rm
te

mEC

NheI

itrine

mt1

n
P41

1 r
H ato
AD in
rm
te

BglII

BglII

mTur

Figure 2

NheI
mt1

n
e2 P41

quoisPil1mCherry- mECitrineSpAtg8(1-115) CtNbr1
top/bottom

Merge

0.93

Pil1mCherry

0.78

mECitrineCtNbr1

Merge
0.36
0.03
0.25

Z-section

0.04

mid-plane
0.88
0.86

Figure 30.13