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Cell Reports, Volume 15
Supplemental Information
APEX Fingerprinting Reveals the Subcellular
Localization of Proteins of Interest
Song-Yi Lee, Myeong-Gyun Kang, Jong-Seok Park, Geunsik Lee, Alice Y.Ting, and Hyun-Woo Rhee
-S1-
Supplemental Information
APEX fingerprinting reveals the subcellular localization of proteins of
interest
Song-Yi Lee, Myeong-Gyun Kang, Jong-Seok Park, Geunsik Lee, Alice Y. Ting* and Hyun-
Woo Rhee*
*Corresponding authors: [email protected], [email protected]
Contents Page
1. Supplemental figures
Figure S1. SA-HRP detection of biotinylated proteins of SEC61B and HMOX1 conjugated
APEX2s S2
Figure S2. EM imaging study of SEC61B- and HMOX1-APEX2 S3 Figure S3. Confocal microscope imaging of biotin-phenol labeling patterns of standard
mitochondrion-localized POI-APEX2s in U2OS cells. S4
Figure S4. Line scan analysis graph of SA-HRP western blot of Fig. 4b S5 Figure S5. Anti-V5 western blots of SA-HRP western blot images in the manuscript S6 Figure S6. Proposed membrane protein topology of various mitochondrial POIs
characterized in this study. S7
Figure S7. Immunofluorescence image of endogenous MGST3
Figure S8. Immunoperoxidase EM image of endogenous MGST3 in Hek293T cells S8
S9
2. Supplemental Experimental procedures
2-1. Materials
2-2. Mammalian cell culturing and Transfection.
2-3. Western blot analysis of biotin-phenol labeling.
2-4. Fluorescence microscope imaging.
2-5. Comprehensive description for correlation value equation (1)
2-6. Plasmids and cloning.
2-7. Sample Preparation of Immunoperoxidase Imaging of endogenous MGST3
Supplemental Table 1. Plasmids Information
S10-S13
-S2-
1. Supplemental figures
Figure S1
Figure S1. SA-HRP detection of biotinylated proteins of SEC61B and HMOX1 conjugated APEX2s (left),
anti-V5 western blot (right), and ponceau staining(bottom). APEX2 was conjugated to C terminus and N
terminus of SEC61B and HMOX1.
-S3-
Figure S2
Figure S2. EM imaging study of SEC61B- and HMOX1-APEX2 (a) Scheme of ER cytosolic face and ER
luminal face DAB labeling patterns of SEC61B-APEX2 and APEX2-Sec61B. (b) Electron microscopic (EM)
imaging of SEC61B-APEX2, APEX2-SEC61B and HMOX1-APEX2. Scale bar: 200 nm
-S4-
Figure S3
Figure S3. Confocal microscope imaging of biotin-phenol labeling patterns of standard mitochondrion-
localized POI-APEX2s in U2OS cells. Labeling was conducted in living cells and imaging was conducted after
fixation/permeabilization. Scale bar: 10 μm.
-S5-
Figure S4
Figure S4. Line scan analysis graph of SA-HRP western blot of Fig. 4b (a) Line scan analysis graphs of IMS
localized proteins in Fig 4. (b) Expanded graph of (a) at low molecular weight range (less than 55kDa) (c) Line
scan analysis graphs of OMM localized proteins in Fig 4. (d) Expanded graph of (c) at low molecular weight
range (less than 55kDa) (e) ball-point (30 point) background subtracted image of Fig. 4b. This image was used
for line scan analysis.
-S6-
Figure S5
Figure S5. Anti-V5 western blots of SA-HRP western blot images in the manuscript (a) anti-V5 western blot
image of Fig. 2b. LACTB-Flag-APEX2 was not shown in this blot. (b) anti-V5 western blot image of Fig. 4b.
-S7-
Figure S6
Figure S6. Proposed membrane protein topology of various mitochondrial POIs characterized in this study.
-S8-
Figure S7
Figure S7. Immunofluorescence image of endogenous MGST3 (A) Confocal imaging with BFP-ER (ER marker)
and Mito-BFP (mitochondrial marker) in Hela cell (B) Confocal imaging with BFP-ER (ER marker) and Mito-
BFP (mitochondrial marker) in Hek293T cells
-S9-
Figure S8
Figure S8. Immunoperoxidase EM image of endogenous MGST3 in Hek293T cells. The magnified images (red
box) in upper images were shown below.
-S10-
2. Supplemental Experimental procedures
2-1. Materials
All chemicals and reagents were purchased from Sigma-Aldrich and Alfa Aesar otherwise stated. Biotin-phenol
was synthesized and characterized by following reported procedure (Rhee and Zou et al, Science, 2013, 339, 1328-
1331). Anti-MGST3 was purchased from Abcam (catalog number: ab110309).
DNA template of Tom20 (plasmid #54955, NM_014765.2), H2B (plasmid #20972, NM_021062, EBFP2-
CytERM-N-17 (Plasmid #55238), and mito-BFP (Plasmid #49151) were purchased from Addgene (MA, USA)
and DNA template of HMOX1 (clone ID: hMU009959, NM_002133),TRMT61B (clone ID: hMU010636,
NM_017910), MGST3 (clone ID: hMU005608, NM_004528), Sco1 (clone ID: hMU001709, NM_004589),
Sec61B (clone ID: hMU006627, NM_006808) were provided from Korean Human Gene Bank. DNA templates
of AURKAIP1 (NM_001127229.1), BIT1/PTRH2 (NM_001015509.2), COX14 (NM_001257133.1), MRPL12
(NM_002949.3), MRM1 (NM_024864.4), MRPS15 (NM_031280.3), PLGRKT (NM_018465.3), QIL1/MIC13
(NM_205767.2), SFXN1 (NM_022754.5), SVH/ARMC10 (NM_001161009.2), TRMT2B (NM_001167970.1)
were amplified by human cDNA library. Pdk1 (NM_002610) is a kind gift from Prof. In-Kyu Lee (Kyoungbook
National University Medical School).
APEX2 sequence was generated followed by quick-change mutagenesis protocol with A134P primers (fwd:
GGGTCGCTTGCCCGATCCCACTAAGGGTTCTGACC/ rev: GGTCAGAACCCTTAGT
GGGATCGGGCAAGCGACCC). DNA primers for mutagenesis and cloning were ordered from Macrogen,
Korea.
2-2. Mammalian cell culturing and Transfection. HEK-293T cells from ATCC (passages <30) were
cultured in a MEM (Gibco) supplemented with 10% FBS, 50 units/mL penicillin, and 50 μg/mL streptomycin at
37 °C under 5% CO2. U2OS cells from Korean Cell Line Bank were cultured in a McCoy’s 5A medium Gibco)
supplemented with 10% FBS, 50 units/mL penicillin, and 50 μg/mL streptomycin at 37 °C under 5% CO2. For
imaging experiments, cells were grown on 1 cm × 1cm glass coverslips with fibronectin coating in 48-well plates.
For western blotting experiments and EM experiments, cells were grown on 6 well plates without fibronectin
coating. Cells were transfected at 60–80% confluence using Lipofectamine 2000 (Life Technologies), typically
with 1.25 μL Lipofectamine 2000 and 250 ng plasmid per well of 48 well plate for imaging experiment. For 6
well plate transfection, 10 μL Lipofectamine 2000 and 2000 ng APEX plasmid were used per well. Cells were
biotin-labeled and fixed (or lysed for western blotting) 18–24 h after transfection.
2-3. Western blot analysis of biotin-phenol labeling. For streptavidin blotting, HEK-293T cells in wells
of a six-well plate were transfected with APEX2 variants and labeled under the same conditions described for
biotin-phenol labeling above. After 1 min of labeling, the cells were washed three times with quencher solution
(10 mM sodium azide, 10 mM sodium ascorbate, and 5 mM Trolox) and then lysed with RIPA lysis buffer (50
mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100), 1× protease cocktail
(Sigma Aldrich, catalog no. P8849), 1 mM PMSF (phenylmethylsulfonyl fluoride), 10 mM sodium azide, 10
mM sodium ascorbate, and 5 mM Trolox) for 10 min at 4 °C. Lysates were transferred to e-tube and was
clarified by centrifugation at 15,000g for 10 min at 4 °C before separation on a 10% SDS-PAGE gel. For
blotting analysis, gels were transferred to nitrocellulose membrane, stained by Ponceau S (10 min in 0.1% (w/v)
Ponceau S in 5% acetic acid/water), and blocked with 2% (w/v) dialyzed BSA and 0.1% Tween-20 in Tris-
buffered saline (TBST) at 4 °C overnight or at room temperature for 1hr. The blots were immersed in
streptavidin-HRP in blot blocking buffer (1:10,000 dilution, Thermo Scientific, cat. no. 21126) at room
temperature for 60 min and then rinsed with blot blocking buffer four times for 5 min (4 × 5 min) before
development with Clarity reagent (Bio-Rad) and imaging on an ImageQuant LAS 4000 (GE healthcare). For
assessing comparative enzyme expression level, membrane was stripped with stripping buffer (100 mM 2-
Mercapto-ethanol, 2% SDS, 62.5 mM Tris-HCl pH 6.8). Blots were washed 4 X 5 min in TBST buffer, and
incubated into 5 % blot blocking buffer for 1-2hr. Blots blocked again using blot blocking buffer, and then
incubated in 10mL of primary antibodies(mouse-anti-V5 or mouse-anti-Flag) for 1-2hr at room temperature.
Blots were washed 4 × 5 min in TBST buffer before incubation in 10 mL of secondary antibody (anti-mouse-
-S11-
HRP) in blot blocking buffer for 30min-1 h at room temperature. The membranes were then washed again 4 × 5
min in TBST buffer before imaging with Clarity reagent (Bio-Rad) as described above.
2-4. Fluorescence microscope imaging. For imaging experiments, mito-BFP or EBFP2-CytERM-N-17 was
co-transfected as mitochondria or ER markers. Biotin-labeled cells were fixed with 4% paraformaldehyde solution
in DPBS at room temperature for 15 min. Cells were then washed with DPBS three times and permeabilized with
cold methanol at –20 °C for 5 min. Cells were washed again three times with DPBS and blocked for 1 h with 2%
BSA in DPBS (“blocking buffer”) at room temperature.
To detect APEX2-fusion expression, cells were incubated with either mouse anti-V5 antibody (Invitrogen, cat.
no. R960-25, 1:5000 dilution) or mouse anti-Flag antibody (Sigma Aldrich, cat. no. F1804, 1:3000 dilution) for 1
h at room temperature. After washing four times with TBST each 5min, cells were simultaneously incubated with
secondary Alexa Fluor 488-goat anti-mouse IgG (Invitrogen, cat. no. A-11001, 1:1000 dilution) and streptavidin–
Alexa Fluor 568 IgG (Invitrogen, cat. no. S11226, 1:1000 dilution) for 30min room temperature. Cells were then
washed four times with TBST each 5min and maintained in DPBS on ice for imaging by FV1000SPD (Olympus)
of UOBC in UNIST, Korea.
2-5. Comprehensive description for correlation value equation (1). Our formulation for the correlation
values is based on a correlation function appearing in the signal process, where the autocorrelation function for a
discrete signal 𝑦(𝑛) is defined as 𝐶(𝑚) = ∑ 𝑦(𝑛)𝑦(𝑛 − 𝑚)𝑛 .
We apply this to quantify the similarity of two barcodes by associating two digitized intensities, 𝐼𝑖(𝑛) and 𝐼𝑗(𝑛),
to two discrete signals. Also 𝑚 = 0, because all the intensities are given for the same sequence of mass density.
Thus the correlation value will be like ∑ 𝐼𝑖(𝑘)𝐼𝑗(𝑘)/𝑁𝑁𝑘=1 . A normalization factor needs to be considered, because
the overall intensity varies from one barcode to another. For this reason we make the correlation value equal to
one, when two barcodes are the same kind. Thereby we formulate the correlation value as following (or Equation
1).
𝐶𝑖𝑗 =
1𝑁
∑ 𝐼𝑖(𝑘)𝐼𝑗(𝑘)𝑁𝑘=1
√1𝑁
∑ 𝐼𝑖(𝑘)2𝑁𝑘=1
√1𝑁
∑ 𝐼𝑗(𝑘)2𝑁𝑘=1
2-6. Plasmids and cloning. Genes were cloned into the specified vectors using standard enzymatic
restriction digest and ligation with T4 DNA ligase. To generate constructs where short tags (e.g., V5 or Flag
epitope tag) or signal sequences were appended to the protein, the tag was included in the primers used to PCR-
amplify the gene. PCR products were digested with restriction enzymes and ligated into cut vectors (e.g., pcDNA3,
and pDisplay). In all cases, the CMV promoter was used for expression in mammalian cells. Table S1 below
summarizes the genetic constructs cloned and used for this study.
The nuclear export sequence, NES (LQLPPLERLTLD), was derived from residues 6–17 of the HIV-1 Rev
protein30. The ER-targeted C1 sequence was derived from the N-terminal 27 amino acids of rabbit P450 C1
(MDPVVVLGLCLSCLLLLSLWKQSYGGG). Protein processed size during translocation was obtained by
programs: Mitoprot (http://ihg.gsf.de/ihg/mitoprot.html) and SignalP 4.1 Server
(http://www.cbs.dtu.dk/services/SignalP/) .
2-7. Sample Preparation of Immunoperoxidase Imaging of endogenous MGST3 HEK293T cells were
grown on 6-well cell culture plates. Cells were fixed on the ice using cold 4% formaldehyde (Electron Microscopy
Sciences, Hatfield, PA, USA) in PBS buffer for 15 min. Cells were washed in PBS three times. After washing,
permeabilization of the cells was performed by 0.5% Tween-20 (sigma Aldrich) for 5 min and then washed again
two times. Cells were blocked by 2% BSA in PBS for 0.5~1 hour and anti-MGST3 (Abcam) in 2% blocking
-S12-
solution (1:500 of dilution) was replaced without washing. After overnight incubation in refrigerator, antibodies
were washed with PBS 6 x 5 min each. Anti-mouse-HRP in 2% blocking solution (1:3000 of dilution) was added
into cells after washing and incubated for 30 min. Secondary antibodies were washed with PBS 6 x 5 min each.
Cells were again fixed using 2% glutaraldehyde (Electron Microscopy Sciences) in PBS. After 30–60 min, the
cells were rinsed 5 × 2 min each in chilled buffer and then treated for 5 min in buffer containing 20 mM glycine
to quench the unreacted glutaraldehyde, followed by another 3 5 min rinses in chilled buffer. DAB staining was
initiated by adding freshly diluted 1 mg/mL (2.8 mM) DAB (Sigma; from a stock of the free base dissolved in 0.1
M HCl) and 10 mM H2O2 in PBS. After 20 min, the reaction was stopped by removing the DAB solution, and the
cells were again washed with PBS 3 × 5 min each. Post-fixation staining was performed using 2% (w/v) osmium
tetroxide (Electron Microscopy Sciences) for 1 h in chilled buffer. Cells were rinsed 5 × 2 min each in chilled
distilled water. Cells were brought to room temperature, washed in distilled water, and then carefully scraped off
the plastic, resuspended, and centrifuged at 1600 g for 1 min to generate a cell pellet. The supernatant was
removed and the pellet was dehydrated in a graded ethanol series (50%, 60%, 70%, 80%, 90%, 100%) for 15 min
each time; then, it was infiltrated into EMBED-812 (Electron Microscopy Sciences) using 1:1 (v/v) resin and
anhydrous ethanol for 1 h. The second change of 2:1 (v/v) resin and anhydrous ethanol was incubated overnight.
After removing the resin, the sample was exchanged once more with 100% resin for 2 h before transferring the
sample to fresh resin, followed by polymerization at 60°C for 24 h. Embedded cell pellets were cut with a diamond
knife into 50-nm sections and imaged on a FEI-Tecnai G2 Spirit bio transmission electron microscope (operated
at 120 kV) at the Korea Basic Science Institute in Daejeon, Korea.
Supplemental Table 1. Plasmids Information
Name
(expected size)
Features Promotor/
Vector
Details
H2B-V5-APEX2
(42.9 kDa)
KpnI-H2B(126aa)-
BamHI-V5-APEX2-
Stop-XbaI
CMV/
pCDNA3
H2B (NM_021062)
Soybean APEX2 has 4 mutations
relative to wtAPX: K14D, W41F,
E112K, and A134P.
V5: GKPIPNPLLGLDST
V5-APEX2-NLS
(31.9 kDa)
NotI-V5-APEX2-
EcoRI-NLS-Stop-
XhoI
CMV/
pCDNA3
NLS:
PKKKRKVDPKKKRKVDPKKKRK
V (nuclear localized signal*)
Mito-V5-APEX2
(unprocessed: 32kDa,
processed: 29kDa)
NotI-Mito-BamHI-
V5-APEX2-Stop-
XhoI
CMV/
pCDNA3
Mito-:
MLATRVFSLVGKRAISTSVCVRA
H (matrix targeting sequence,
Fornuskova et al., 2010)
ScoI-V5-APEX2
(unprocessed: 62kDa,
processed: 59kDa)
NotI-ScoI-BamHI-
V5-APEX2-Stop-
XhoI
CMV/
pCDNA3
Sco1 (NM_004589)
Tom20-V5-APEX2
(45.7 kDa)
NotI-Tom20-10aa
linker-NheI-V5-
APEX2-Stop-XhoI
CMV/
pCDNA3
10aa linker :
GGSGDPPVAT
Tom20 (NM_014765.2)
TRMT61B-V5-APEX2
(81.5 kDa)
NotI-TRMT61B-
NheI-V5-APEX2-
Stop-XhoI
CMV/
pCDNA3
TRMT61B (NM_017910),
HMOX1-V5-APEX2
(61.4kDa)
NotI-HMOX1-NheI-
V5-APEX2-Stop-
XhoI
CMV/
pCDNA3
HMOX1 (Heme oxygenase 1,
NM_002133)
V5-APEX2-HMOX1
(61.5kDa)
NotI-V5-APEX2-
NheI-HMOX1-Stop-
XhoI
CMV/
pCDNA3
HMOX1 (Heme oxygenase 1,
NM_002133)
MGST3-V5-APEX2
(45.1 kDa)
NotI-MGST3-
BamHI-V5-APEX2-
CMV/
pCDNA3
MGST3 (NM_004528)
-S13-
Stop-XhoI
Pdk1-V5-APEX2
(unprocessed, 81.5 kDa,
processed: 76.0 kDa)
NotI-Pdk1-NheI-V5-
APEX2-Stop-XhoI
CMV/
pCDNA3
Pdk1 (NM_002610)
C1(1-29)-V5-APEX2
(31.4 kDa)
NotI-C1(1-29)-NheI-
V5-APEX2-Stop-
XhoI
CMV/
pCDNA3
C1(1-29):
MDPVVVLGLCLSCLLLLSLW
KQSYGGGKL(endoplasmic
reticulum membrane anchor*)
V5-APEX2-Sec61B
(38.6 kDa)
NotI-V5-APEX2-
NheI-Sec61B-Stop-
XhoI
CMV/
pCDNA3
Sec61B (NM_006808)
Sec61B-V5-APEX2
(38.5 kDa)
NotI-Sec61B-NheI-
V5-APEX2-Stop-
XhoI
CMV/
pCDNA3
Sec61B (NM_006808)
APEX2-V5-KDEL
(unprocessed: 31.8 kDa,
processed: 29.5kDa)
EcoRV-ss-HA-ApaI-
APEX2-V5-KDEL-
Stop-NotI
CMV/
pDisplay
ss:
METDTLLLWVLLLWVPGSTGD
(IgK chain leader sequence for ER
lumen)
KDEL: ER retention motif
V5-APEX2-NES
(29.8 kDa)
NotI-V5-APEX2-
NES-Stop-XhoI
CMV/
pCDNA3
NES: LQLPPLERLTLD (nuclear
exclusion signal)
LACTB-Flag-APEX2
(unprocessed: 36
kDa ,processed: 30 kDa)
NotI-LACTB(1-68)-
linker-NheI-V5-
APEX2-Stop-XhoI
CMV/
pCDNA3
LACTB(1-68):
MYRLLSSVTARAAATAGPAWD
GGRRGAHRRPGLPVLGLGWAG
GLGLGLGLALGAKLVVGLRGA
VPIQS (IMS targeting signal,
Polianskyte et al., 2009)
Flag: DYKDDDDK
LACTB-Flag-APEX2
(unprocessed: 35
kDa ,processed: 29 kDa)
NotI-LACTB(1-68)-
BamHI-Flag-APEX2-
Stop-XhoI
CMV/
pCDNA3
LACTB(1-68):
MYRLLSSVTARAAATAGPAWD
GGRRGAHRRPGLPVLGLGWAG
GLGLGLGLALGAKLVVGLRGA
VPIQS (IMS targeting signal,
Polianskyte et al., 2009)
AURKAIP1-V5-APEX2-
AP
(53.0kDa)
HindIII-KpnI-
AURKAIP1-BamHI-
NheI-V5-APEX2-
AP-Stop-NotI-XhoI
CMV/
pCDNA5
AURKAIP1 (NM_001127229.1)
BIT1/PTRH2-V5-APEX2
(47.8kDa)
HindIII-KpnI-
BIT1/PTRH2-
BamHI-V5-APEX2-
Stop-NotI-XhoI
CMV/
pCDNA5
BIT1/PTRH2 (NM_001015509.2),
COX14-V5-APEX2
(35.2kDa)
HindIII-KpnI-
COX14-BamHI-V5-
APEX2-Stop-NotI-
XhoI
CMV/
pCDNA5
COX14 (NM_001257133.1)
MRM1-V5-APEX2-AP
(69.3kDa)
HindIII-KpnI-
MRM1-BamHI-NheI-
V5-APEX2-AP-Stop-
NotI-XhoI
CMV/
pCDNA5
MRM1 (NM_024864.4),
MRPS15-V5-APEX2-AP
(60.5kDa)
HindIII-KpnI-
MRPS15-BamHI-
NheI-V5-APEX2-
AP-Stop-NotI-XhoI
CMV/
pCDNA5
MRPS15 (NM_031280.3)
PLGRKT-V5-APEX2
(45.8kDa)
HindIII-KpnI-
PLGRKT-BamHI-
CMV/
pCDNA5
PLGRKT (NM_018465.3)
-S14-
V5-APEX2-Stop-
NotI-XhoI
QIL1-V5-APEX2
(41.7kDa)
HindIII-KpnI-QIL1-
BamHI-V5-APEX2-
Stop-NotI-XhoI
CMV/
pCDNA5
QIL1/MIC13 (NM_205767.2)
SFXN1-V5-APEX2-AP
(66.3kDa)
HindIII-KpnI-
SFXN1-BamHI-
NheI-V5-APEX2-
AP-Stop-NotI-XhoI
CMV/
pCDNA5
SFXN1 (NM_022754.5)
SVH-V5-APEX2
(62.4kDa)
NotI-SVH-NheI-V5-
APEX2-Stop-XhoI
CMV/
pCDNA3
SVH/ARMC10 (NM_001161009.2)
TRMT2B-V5-APEX2-AP
(87.4kDa)
HindIII-KpnI-
TRMT2B-BamHI-
NheI-V5-APEX2-
AP-Stop-NotI-XhoI
CMV/
pCDNA5
TRMT2B (NM_001167970.1)