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: rhee@unist.ac.kr, ating@mit.edu

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)