(M) of SARS-Cov-2

Home , BET1L, CCDC8, Moroctocog alfa, Necitumumab, SERP1, UBIAD1

Endomembrane systems are reorganized by ORF3a and Membrane (M) of SARS-CoV-2

Yun-Bin Lee1, Minkyo Jung2, Jeesoo Kim3, Myeong-Gyun Kang1, Chulhwan Kwak1,5, Jong-Seo Kim3,4,*, Ji- Young Mun2,*, Hyun-Woo Rhee1,4,*

1Department of Chemistry, Seoul National University, Seoul 08826, Republic of Korea 2Neural Circuit Research Group, Korea Brain Research Institute, 41062 Daegu, Republic of Korea 3Center for RNA research, Institute for Basic Science, Seoul 08826, Republic of Korea 4School of Biological Sciences, Seoul National University, Seoul 08826, Republic of Korea 5Department of Chemistry, Ulsan National Institute of Science and Technology, 44919 Ulsan, Korea

*Correspondence: [email protected], [email protected], [email protected].

Table of Contents Supplementary Figure 1-15------S2-24

Supplementary Table 1-3------S25-29

-S1-

A GFP V5 BF Merged

+ Pearson R V5

- = 0.64

GFP GFP

SEC61B ORF3a

Pearson R = 0.61

Pearson R

+ V5

- = 0.57

GFP GFP

M SEC61B

Pearson R = 0.64

B GFP Flag BF Merged

+ + Pearson R

= 0.87

Flag

GFP GFP

ORF3a ORF3a

Pearson R = 0.80

+ Pearson R

= 0.67

Flag

GFP GFP

ORF6 ORF6

Pearson R = 0.64

+ Pearson R

Flag = 0.91

GFP GFP

ORF7b ORF7b

Pearson R = 0.90

+ Pearson R

= 0.91

Flag

GFP GFP

M M

Pearson R = 0.89 Supplementary Figure 1. Additional confocal microscopy images of GFP-tagged vPOIs of SARS-CoV-2 (a) Confocal microscopy images of ORF3a-GFP (left) and M-GFP (right) with SEC61B-V5-TurboID. SEC61B-V5- TurboID was visualized by Anti-V5 antibody (AF647-conjugated secondary antibody, Cy5 fluorescence channel). Pearson correlation values were calculated between different fluorescent channel images. BF: bright field, Scale bars: 10 μm. (b) Confocal microscopy images of co-expressed vPOI-GFP and vPOI-Flag. Flag-conjugated vPOIs (ORF3a, ORF6, ORF7b, M of SARS-CoV-2) were visualized by using Anti-Flag antibody (AF647-conjugated secondary antibody, Cy5 fluorescence channel) and GFP-conjugated vPOIs fluorescence was detected in GFP fluorescence channel. Pearson correlation values were calculated between different fluorescent channel images. BF: bright field, Scale bars: 10 μm. -S2-

V5 SA BF

V5 SA GFP BF

TurboID-V5-GBP

TurboID-V5-GBP + ORF3a-GFP

V5 SA GFP BF

TurboID-V5-GBP TurboID-V5-GBP + ORF6-GFP + M-GFP

TurboID-V5-GBP TurboID-V5-GBP + N-GFP + ORF7b-GFP

Supplementary Figure 2. Additional Confocal images of Figure 2B. Confocal microscopy images of GFP-conjugated vPOIs (M, N, ORF3a, ORF6 and ORF7b of SARS-CoV-2) and co-expressed TurboID-V5-GBP in HEK293-AD cells. TurboID-V5-GBP (TurboID-GBP) was visualized by anti-V5 antibody (AF568-conjugated, RFP fluorescence channel). and GFP fluorescence was detected in GFP fluorescence channel. Biotinylated proteins were visualized by AF647- conjugated streptavidin (Cy5 fluorescence channel). BF: bright field, Scale bars: 10 μm.

-S3-

S - S - S - S

S - S - S

- -

- S - - S

- - - S

- - S Supplementary Figure 3. Raw images of western blotting results from Figure 2. (A) Raw images of western blotting and Ponceau membrane stain results with size ladder from Figure 2c (WB: SA-HRP). (B) Raw images of western blotting and Ponceau membrane staining results with size ladder from Figure 2c (WB: anti-V5). Selected areas for Figure 2c are marked with red box. (C) Raw images of western blotting and Ponceau membrane staining results with size ladder from Figure 2d (WB: anti-GFP). -S4-

A ORF3a protein topology B M protein topology

Protter - visualize proteoforms Omasits et al., Bioinformatics. 2013 Nov 21.

Supplementary Figure 4. Predicted transmembrane domain regions of ORF3a and M by Protter. (A) Membrane topology of ORF3a. (B) Membrane topology of M These data was obtained by using Protter (https://wlab.ethz.ch/protter/start/). N-glyco motif was shown with green box.

-S5-

Supplementary Figure 5. Additional EM images of ORF3a or M expressed HeLa cells. (A) Additional EM images of ORF3a-linker-V5-APEX2 transfected HeLa cells. Bright field image of DAB-stained ORF3a-APEX2 transfected HeLa cells were shown in left. An EM image of the cell shown in Figure 3c is marked with black box in a bright field image. Scale bars : 500 m - 1 μm. (B) Additional EM images of M-linker-V5-APEX2 transfected HeLa cells. An EM image of the cell shown in Figure 3d is marked with black box in the bright field image. Untransfected cell was also shown with red box. Mitochondria is indicated as “m”, respectively (b–d). Scale bars: 1 μm. (C) CLEM images of ORF3a-GFP transfected HeLa cells. Scale bars: 1 μm. Distinct membrane structures were observed with higher magnification. Magnified area are marked with various color boxes.) Cubic membranes are indicated as “CM”. (D) CLEM images of M-GFP transfected HeLa cells. Electron-dense autophagic vesicles are marked with green arrows. Scale bars: 1 μm. -S6-

A Untransfected A549 cell

ER G

m m

ER : ER, M : mitochondria, L : ly sosome, G : Golgi

B A549 : SEC61B-APEX2

C A549 : APEX2

Supplementary Figure 6. EM images of control samples of A549 cells. (A) EM images of non-transfected A549 cells. Scale bars: 2 μm. (B) EM images of SEC61B-APEX2 transfected A549 cells. Scale bars: 1 - 2 μm. (C) EM images of APEX2 transfected cells. Scale bars: 1 - 2 μm.

-S7-

A Rep #1 Rep #2 Rep #3

31 31 31 29 29 29 27 27 27 25 25 25 23 23 23 21 y = x 21 y = 1.0298x 21 y = 0.9987x 19 R² = 1 19 R² = 0.7773 19 R² = 0.7378 17 17 17 Rep #1 Rep 15 15 15

13 13 13

ctrl rep #2 log 2 intensity ctrl rep #3 log 2 intensity ctrlrep #1 log 2 intensity 13 18 23 28 33 13 18 23 28 33 13 18 23 28 33 ctrl rep #1 log 2 intensity ctrl rep #1 log 2 intensity ctrl rep #1 log 2 intensity

31 31 33 29 29 27 27 28 25 25 23 23

GBP + + GFP GBP y = x 23 21 21 - y = 0.9699x R² = 1 y = 0.9694x 19 R² = 0.8035 19

Rep #2 Rep 17 17 18 R² = 0.7882 15 15

13 13 ctrl #3 rep log 2intensity 13

ctrl rep #2 log 2 intensity ctrl rep #1 log 2 intensity 13 18 23 28 33 13 18 23 28 33 13 18 23 28 33

ctrl rep #2 log 2 intensity ctrl rep #2 log 2 intensity ctrl rep #2 log 2 intensity TurboID 31 31 31 29 29 29 27 27 27 25 25 25 23 23 23 21 21 21 y = 0.9996x y = x 19 19 y = 1.0302x 19 R² = 0.7242 R² = 1 Rep #3 Rep R² = 0.7413 17 17 17 15 15 15

13 13 13

ctrl rep #2 log 2 intensity ctrl rep #3 log 2 intensity ctrl rep #1 log 2 intensity 13 18 23 28 33 13 18 23 28 33 13 18 23 28 33 ctrl rep #3 log 2 intensity ctrl rep #3 log 2 intensity ctrl rep #3 log 2 intensity

B Rep #1 Rep #2 Rep #3 33 33 33

28 28 28

23 23 23 y = 1.0538x y = x y = 0.9565x R² = 0.9186 R² = 1 18 R² = 0.677 18

18 Rep #1 Rep

13 13

ORF3a rep #3 log 2 intensity ORF3a rep #2 log 2 intensity ORF3a rep #1 log 2 intensity 13 18 23 28 13 13 18 23 28 13 18 23 28

GFP ORF3a rep #1 log 2 intensity ORF3a rep #1 log 2 intensity ORF3a rep #1 log 2 intensity -

33 33 33

28 28 28

23 23 23 y = x y = 1.0099x y = 0.9568x R² = 0.9856 Rep #2 Rep R² = 1 18 R² = 0.6789 18 18

13 13

ORF3a rep #3 log 2 intensity ORF3a rep #2 log 2 intensity ORF3a rep #1 log 2 intensity 13 18 23 28 13 18 23 28

GBP + + ORF3a GBP 13 18 23 28 - ORF3a rep #2 log 2 intensity ORF3a rep #2 log 2 intensity ORF3a rep #2 log 2 intensity

33 33 33

28 28 28

TurboID 23 23 23 y = x

Rep #3 Rep y = 0.9891x y = 0.9465x R² = 0.9855 R² = 1 18 R² = 0.9495 18 18

13 13

ORF3a rep #3 log 2 intensity ORF3a rep #1 log 2 intensity 13 18 23 28 ORF3a rep #2 log 2 intensity 13 18 23 28 13 18 23 28 ORF3a rep #3 log 2 intensity ORF3a rep #3 log 2 intensity ORF3a rep #3 log 2 intensity

-S8-

C Rep #1 Rep #2 Rep #3 33 31 31 29 29 28 27 27 25 25 23 23 23 y = x 21 21 R² = 1 19 19 18 17 y = 1.0018x 17 y = 0.9883x

Rep #1 Rep 15 R² = 0.6414 15 R² = 0.7808 M rep M #2 log 2 intensity 13 13 rep M #3 log 2 intensity 13

M M rep #1 log 2 intensity 13 18 23 28 13 18 23 28 33 13 18 23 28 33

M rep #1 log 2 intensity M rep #1 log 2 intensity M rep #1 log 2 intensity

GFP -

31 31 31 29 29 29 27 27 27 25 25 25 23 23 23 21 21 21

19 19 19 Rep #2 Rep

GBP + M GBP 17 y = 0.9951x 17 y = x 17 y = 0.9866x

- 15 R² = 0.6292 15 R² = 1 15 R² = 0.6083

M rep M #1 log 2 intensity M rep M #2 log 2 intensity 13 13 rep M #3 log 2 intensity 13 13 18 23 28 33 13 18 23 28 33 13 18 23 28 33 M rep #2 log 2 intensity M rep #2 log 2 intensity M rep #2 log 2 intensity

31 31 31 TurboID 29 29 29 27 27 27 25 25 25 23 23 23

21 21 21 Rep #3 Rep 19 19 19 17 y = 1.0101x 17 y = 1.0106x 17 y = x

15 R² = 0.7838 15 R² = 0.6378 15 R² = 1

M rep M #1 log 2 intensity M rep M #2 log 2 intensity 13 13 rep M #3 log 2 intensity 13 13 18 23 28 33 13 18 23 28 33 13 18 23 28 33 M rep #3 log 2 intensity M rep #3 log 2 intensity M rep #3 log 2 intensity

D ctrl : GFP + TurboID-GBP ORF3a-GFP + TurboID-GBP M-GFP + TurboID-GBP rep #1 rep #2 rep #3 rep #1 rep #2 rep #3 rep #1 rep #2 rep #3 rep #1 1 0.924343 0.9441 0.484594 0.428742 0.291283 0.266151 0.210742 0.19369 ctrl : GFP + TurboID-GBP rep #2 0.924343 1 0.976369 0.581784 0.489374 0.396407 0.325799 0.262611 0.226385 rep #3 0.9441 0.976369 1 0.559304 0.454481 0.333395 0.294569 0.216243 0.180331 rep #1 0.484594 0.581784 0.559304 1 0.845807 0.845415 0.869613 0.768219 0.751899 ORF3a-GFP + TurboID-GBP rep #2 0.428742 0.489374 0.454481 0.845807 1 0.93272 0.780329 0.833266 0.765891 rep #3 0.291283 0.396407 0.333395 0.845415 0.93272 1 0.853849 0.887966 0.872773 rep #1 0.266151 0.325799 0.294569 0.869613 0.780329 0.853849 1 0.881657 0.878903 M-GFP + TurboID-GBP rep #2 0.210742 0.262611 0.216243 0.768219 0.833266 0.887966 0.881657 1 0.907887 rep #3 0.19369 0.226385 0.180331 0.751899 0.765891 0.872773 0.878903 0.907887 1

Low High

-S9-

E ORF3a interactome : 117 proteins

5.5

GGCX

TMEM165 C1orf21 TMEM110 4.5 NSDHL

EPHA2 SLC22A5 PHACTR4 ITGA6 CLCC1

HMGCR PDE8A SCD5 4 TMEM161A SLC9A1 EPB41L5 STT3B VAMP3 TMEM237 TMX1 SAR1A TMEM38B TMC7 SLC7A3 TMEM57 LSR ROBO1 SLC5A3 EMC6 WLS KIAA0040 HMOX2 3.5 TP53I11 PRAF2 EPB41L2 ZC3HAV1 LNP TMEM187 DNAJC16 DHCR7 UBXN8 UBE2J1 MCAM RNF5 BET1L C2orf44 AAAS ITM2C BET1 EGFR NLGN2 ADCY6 USE1 SPECC1 PTPN1 ZDHHC20 SLC16A1 PI4K2A CCDC8 DNAJC1 3 SLC6A6 ERC2 MAP3K7 ITGB1 SC5D KRTCAP2 VMA21 MMGT1 C16orf58 ACBD5 EHD4 NEO1 TMEM51 KAT2A FYN GLIPR2 SLITRK5 UBXN4 RAB7A PAG1 GTPBP2 PPFIBP1 SHB FKBP8 CDKAL1 SCD NEDD1 2.5 FAM177A1 ASIC1 YIF1B CYP51A1 LRCH4 JAM3 ADAM17 VPS16 ADAM9 DGKE ATP1A1 KIRREL LRP1 SVIP GPR50 NCK1 SEC63 PPP1R16B C1orf43 PTDSS1 CNKSR3 STBD1 ZFAND5 PDLIM7 SEC61B PKP2 PDXDC1 TULP3 AGPAT1 SAR1B 2 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7

M interactome : 191 proteins

TMEM187 PRAF2 6.2 BLZF1 SPECC1 DLGAP4 SCD5 CASKIN2 DNAJC16 TRPM4 PLEKHA8P1

ADAM17 STX10 EI24 STX7 SNAP47 PPP1R16B NAV3 SFT2D2 5.7 UNC93B1 ELMOD3 EPB41L1 JPH3 VMA21 SRC TULP3 SCD PDXDC1 TMEM110 PVRL3 DLG3 ITGB1 MMGT1 5.2 C1orf43 INSR ZC3HAV1 POR GTPBP2 HMGCR ADCY6 SLC7A2 FYN KIAA0040 FAM171A2 CDCA3 SLC12A4 ANKS6 4.7 TBC1D12 HMOX2 RNF5 TRAM1 TMEM51 ARL13B ITM2C VAMP3 ARF1 CC2D2A SNX2 EPB41L2 EPB41L5 TMEM165 SLC3A2 DEPDC7 ROBO1 4.2 KIAA1549 SAR1B MCAM TMEM9B LSR TC2N MAPKAP1 SLC12A7 SHB C2CD5 RAB13 SLC30A1 PLEKHO2 FAT1 LRP8 JAM3 SLC7A3 FAM177A1 ANTXR2 DHCR7 CCDC8 3.7 ESYT2 BET1L UBR3 WLS ZDHHC2 SLC16A1 DSG2 CNNM4 SAR1A FAM84B TMEM237 LMNB2 GAB1 CLCC1 ADD1 RAI14 STX4 CYP51A1 SH3BP4 SLC22A5 PAG1 EPHA2 GLIPR2 MAP4K4 USE1 WDR6 CTNND1 GPR50 PPFIBP1 CNNM3 3.2 LNP ZDHHC20 STX18 UBIAD1 VANGL1 FAM171A1 LBR SLC20A2 SLCO4A1 STEAP3 KLRG2 TMEM161A AP1AR TMX1 IRS4 NDFIP1 NEO1 SNAP23 SLC2A1 ARHGAP32 CISD2 PHACTR4 CNKSR3 PLEKHA1 ZDHHC14 PKP2 FZD5 CXADR KIRREL SCAMP3 RSPRY1 UBE2J1 PHLDB3 RAB7A ARL5B SC5D ASIC1 PDE8A 2.7 PPFIA1 NLGN2 SPTBN1 ATP1A1 SLC9A1 STXBP3 SLC4A7 F11R ERC2 LRP1 MYO18A ITGA6 CDC42EP4 RALA TGOLN2 SLC35B3 SERP1 RAB11A CDKAL1 CAMLG CCNY YKT6 SLC7A11 TMEM2 TMEM201 TP53I11 SLC29A1 SLC7A5 SVIP DNAJC1 EHD4 FARP1 PPAP2B SLC5A3 2.2 ANKRD27 KIAA0195 ZDHHC5 GPRC5B YIF1B 2.3 3.3 4.3 5.3 6.3 7.3

-S10-

F ORF3a interactome : 117 proteins

C1orf21 C2orf44 LRCH4 GTPBP2 ZDHHC20 DNAJC16 CNKSR3 FAM177A1 ERC2 AAAS VAMP3 MAP3K7 PPFIBP1 PDXDC1 NEDD1 PKP2 EPB41L2 TP53I11 JAM3 GPR50 PDE8A ZC3HAV1 KAT2A SLC22A5 ITGA6 PHACTR4 CCDC8 SLC16A1 NLGN2 ADAM9 WLS PDLIM7 NCK1 ACBD5 EPHA2 FYN EPB41L5 SPECC1 LSR KIRREL ATP1A1 ZFAND5 TULP3 47 ITM2C 9 C16orf58 FKBP8 MCAM 1 YIF1B TMEM161A RNF5 2 DGKE 14 NEO1 TMEM51 SLC7A3 LRP1 TMEM237 SVIP Plasma membrane ADAM17 TMEM187 44 ADCY6 PAG1 CLCC1 DHCR7 UBE2J1 MMGT1 SLC5A3 C1orf43 Endomembrane ROBO1 SEC63 TMX1 GLIPR2 VPS16 TMC7 SLC9A1 PTPN1 SCD5 PRAF2 HMGCR ASIC1 Mitochondria SHB EHD4 TMEM110 UBXN8 CYP51A1 PI4K2A SLITRK5 ITGB1 SEC61B SC5D AGPAT1 SLC6A6 Peroxisome SAR1A SCD TMEM38B KRTCAP2 KIAA0040 BET1 LNP STBD1 RAB7A PPP1R16B Nucleus/cy tosol VMA21 HMOX2 NSDHL STT3B BET1L USE1 TMEM57 EMC6

Others UBXN4 CDKAL1 DNAJC1 EGFR TMEM165 SAR1B GGCX PTDSS1

M interactome : 191 proteins

CTNND1 ERC2 PPFIBP1 C2CD5 PPFIA1 DLG3 TULP3 YKT6 95 CCDC8 16 UBR3 CNKSR3 ADD1 WDR6 VAMP3 SCAMP3 EPB41L1 JAM3 ZDHHC2 FAM177A1 RAB7A MAP4K4 SLC29A1 RAB13 GPR50 EI24 RSPRY1 ADAM17 STXBP3 PHACTR4 EPB41L2 FZD5 CASKIN2 IRS4 SPTBN1 TGOLN2 FAM84B LMNB2 RAI14 SLC16A1 SLC7A11 PAG1 TC2N SLCO4A1 DEPDC7 UBIAD1 SLC22A5 SLC12A7 INSR NAV3 ZC3HAV1 DSG2 GPRC5B SFT2D2 CC2D2A FAM171A2 GTPBP2 CISD2 VANGL1 PKP2 EPB41L5 LSR RALA CNNM4 TMEM201 TMEM51 ESYT2 NLGN2 KIAA0040 ARL5B RNF5 SH3BP4 SPECC1 CDCA3 SLC7A3 ELMOD3 TMEM187 PLEKHA1 SLC7A5 PHLDB3 SRC CXADR PVRL3 SERP1 28 SLC20A2 TMEM2 TP53I11 JPH3 4 LRP8 TBC1D12 MCAM FYN SLC4A7 MAPKAP1 ANKRD27 SLC35B3 SNAP47 ITGB1 SLC3A2 YIF1B ARHGAP32 PDXDC1 Plasma membrane ATP1A1 TMEM161A ASIC1 CYP51A1 MYO18A TMEM9B LBR C1orf43 ZDHHC5 SLC12A4 CCNY GAB1 EHD4 ZDHHC20 TMEM110 KIAA1549 PRAF2 EPHA2 SNAP23 FAM171A1 Endomembrane ANTXR2 SC5D DHCR7 ITM2C BET1L SLC7A2 PDE8A SLC5A3 RAB11A DLGAP4 UBE2J1 SAR1B STEAP3 LRP1 CDC42EP4 SHB F11R FARP1 PLEKHO2 Mitochondria UNC93B1 MMGT1 HMOX2 SAR1A ADCY6 CAMLG SLC30A1 AP1AR TRAM1 SVIP VMA21 SLC9A1 ARL13B ZDHHC14 PLEKHA8P1 KLRG2 NDFIP1 POR USE1 GLIPR2 KIRREL CNNM3 Peroxisome TMX1 ARF1 WLS SNX2 TMEM237 PPP1R16B 48 ITGA6 KIAA0195 DNAJC16 DNAJC1 LNP SCD NEO1 FAT1 TRPM4 Nucleus/cy tosol STX18 SCD5 STX10 ANKS6 STX4 STX7 CDKAL1 TMEM165 ROBO1 HMGCR CLCC1 SLC2A1 Others PPAP2B BLZF1

-S11-

P-value Function of proteins ORF3a : GO enrichment

P-value Function of proteins M M : GO enrichment

-S12-

Supplementary Figure 7. Additional information of mass analysis results (A) Scatter plot of MS1 intensity of biotinylated peptides from biological triplicate samples ofGFP (control, i.e. TurboID-GBP and GFP). (B) Scatter plot of MS1 intensity of biotinylated peptides from biological triplicate samples of ORF3a-GFP (i.e. TurboID-GBP and ORF3a- GFP). (C) Scatter plot of MS1 intensity of biotinylated peptides from biological triplicate samples of M-GFP (i.e. TurboID-GBP and M-GFP). (D) Correlation value table of MS1 intensities of biotinylated peptides in biological tripicates samples of GFP, ORF3a-GFP and M-GFP (E) Expended view of ORF3a and M interactomes with gene names in the volcano plot of Figure 4b. 117 proteins at ORF3a interactome and 191 proteins at M interactome were all shown. Detail information is shown in Supplementary Dataset 1. (F) Subcellular distribution of ORF3a and M interactome shown in Figure 4c. (G) Functional enrichment of ORF3a interactome (117 proteins) and M interactome (191 proteins). Gene ontology analysis was performed using ShinyGO v0.61: Gene Ontology Enrichment Analysis (http://bioinformatics.sdstate.edu/go/).

-S13-

A Extracellular (or Lumen) C N N N Plasma membrane B B B B B B Cytosol N B N B C C B C C N C ADAM9 EPHA2 JAM3 SLC16A1 SLC22A5 VAMP3 698-718 aa TM 538-558 aa TM 242-264 aa TM 16-36 aa TM, 60-80 aa TM, 21-41 aa TM, 143-163 aa TM, 78-98 aa TM (Uniprot) (Uniprot) (Uniprot) 87-107 aa TM, 112-132 aa TM, 173-193 aa TM, 198-218 aa TM, (Uniprot) K781 K578 K287, K305 144-164 aa TM, 167-187 aa TM, 233-253 aa TM, 258-278 aa TM, K35 263-283 aa TM, 299-319 aa TM, 342-362 aa TM, 374-394 aa TM, 329-349 aa TM, 354-374 aa TM, 407-427 aa TM, 431-451 aa TM, 390-410 aa TM, 423-443 aa TM 463-483 aa TM, 489-509 aa TM (Uniprot) (Uniprot) K216, K219, K224 K553

N Lumen C C C N Endo membrane B B B B B B B B B B Cytosol B B B B C B B B N N B N N C N C C

BET1L CLCC1 : DHCR7 : proposed model SCD SCD5 SEC61B SEC63 87-107 aa TM proposed model 40-60 aa TM, 154-174 aa TM, 73-93 aa TM, 50-70 aa TM, 71-91 aa TM 15-35 aa TM, (Uniprot) 185-205 aa TM, 177-197 aa TM, 266-286 aa TM, 98-118 aa TM, 73-93 aa TM, (Uniprot) 70-90 aa TM, K31, K36 217-237 aa TM, 306-326 aa TM, 331-351aa TM, 218-237 aa TM, 194-214 aa TM, K20 189-209 aa TM 330-350 aa TM 420-440 aa TM 242-263 aa TM 216-238 aa TM (Uniprot) (Uniprot) (Uniprot) (Uniprot) (Uniprot) K534, K535 K414, K437, K11, K13 K196, K338, K10, K17 K463, K470, K341 K472, K518

N C Lumen N N N C Endo membrane B B B B B B B Cytosol C B B B B B C N B N B B B C C C N

VMA21 ITGB1 TMX1 UBXN8 BET1 TMEM110 : TMEM165 26-46 aa TM, 729-751 aa TM 181-203 aa TM 2-22 aa TM, 95-115 aa TM proposed model 90-110 aa TM, 66-86 aa TM (Uniprot) (Uniprot) 34-54 aa TM (Uniprot) 29-49 aa TM, 127-147 aa TM, (Uniprot) K784, K794 K222, K223, (Uniprot) K42, K47, K58, 69-89 aa TM, 152-172 aa TM, K6 K233, K259 K104, K154, K62 102-122 aa TM, 229-249 aa TM, K160 156-176 aa TM, 268-288 aa TM, 194-214 aa TM 300-320 aa TM (Uniprot) (Uniprot) K281 K208 C Lumenal IMS Endo OMM membrane

Cytosolic B Cytosolic B C B N B B N

RNF5 : FKBP8 : proposed model proposed model 118-138 aa TM, 390-410 aa TM 160-180 aa TM (Uniprot) (Uniprot) K272, K314, K75 K334, K340

-S14-

C N N C B Extracellular N N C Plasma membrane

B B B B B B B B B Cytosolic B B B B B N C N C N N C C C CXADR VAMP3 JAM3 LSR MCAM TMEM2 UBR3 : ESYT2 238-258 aa TM 78-98 aa TM 242-264 aa TM 260-280 aa TM 560-583 aa TM 83-103 aa TM proposed model 104-124 aa TM, (Uniprot) (Uniprot) (Uniprot) (Uniprot) (Uniprot) (Uniprot) 761-781 aa TM, 128-148 aa TM K339 K35, K42 K287, K305 K410, K638 K616, K640 K31, K48 919-939 aa TM, (Uniprot) 1806-1826aa TM K405, K750 (Uniprot) K36

C C N Extracellular Plasma membrane B B N B B B B B B B N B N B Cytosolic B B N B B B C B C B B C B N B B SLC22A5 SLC29A1 SLC3A2 SLC7A3 SLC16A1 21-41 aa TM, 143-163 aa TM, 13-29 aa TM, 83-107 aa TM, 185-205 aa TM 37-57 aa TM, 62-82 aa TM, 16-36 aa TM, 60-80 aa TM, B B 173-193 aa TM, 198-218 aa TM, 112-130 aa TM, 139-157 aa TM, (Uniprot) 108-128 aa TM, 163-183 aa TM, 87-107 aa TM, 112-132 aa TM, B 233-253 aa TM, 258-278 aa TM, 175-199 aa TM, 207-227 aa TM, K140, K145, 192-212 aa TM, 234-254 aa TM, 144-164 aa TM, 167-187 aa TM, B 342-362 aa TM, 374-394 aa TM, 292-311 aa TM, 324-342 aa TM, K147, K160, 286-306 aa TM, 336-356 aa TM, 263-283 aa TM, 299-319 aa TM, 407-427 aa TM, 431-451 aa TM, 360-378 aa TM, 393-413 aa TM, K166 383-403 aa TM, 407-427 aa TM, 329-349 aa TM, 354-374 aa TM, C 463-483 aa TM, 489-509 aa TM 432-452 aa TM 476-496 aa TM, 507-527aa TM, 390-410 aa TM, 423-443 aa TM (Uniprot) (Uniprot) 541-561aa TM, 570-590aa TM (Uniprot) K553 K249, K255, K263 (Uniprot) K211, K216, K219, K224, K473, K605 K479

DSG2 610-634 aa TM (Uniprot) K697, K706, K746, K779, K832, K834, K844, K901, K919, K1109 Lumenal N N C B Endo membrane

B B B Cytosolic B B C B B B B N B N C C B C B C B N N STEAP3 ITGB1 TMEM110 : proposed model VMA21 LBR BET1L 208-228 aa TM, 259-279 aa TM, 729-751 aa TM 29-49 aa TM, 69-89 aa TM, 26-46 aa TM, 212-232 aa TM, 258-278 aa TM, 87-107 aa TM 305-325 aa TM, 359-379 aa TM, (Uniprot) 102-122 aa TM, 156-176 aa TM, 66-86 aa TM 299-319 aa TM, 326-346 aa TM, (Uniprot) 391-411 aa TM, 434-454 aa TM K784, K794 194-214 aa TM (Uniprot) 386-404 aa TM, 447-467 aa TM, K31, K36 (Uniprot) (Uniprot) K6 481-501 aa TM, 561-581 aa TM K7, K484 K281 (Uniprot) K123, K147, K178, K186, K190, K524

IMS N

OMM

B B B Cytosolic B C B N C

UBIAD1 : proposed model CISD2 83-103 aa TM, 134-154 aa TM, 38-60 aa TM 160-180 aa TM, 188-208 aa TM, (Uniprot) 209-229 aa TM, 245-267 aa TM, K95, K105, K131, 277-297 aa TM, 315-335 aa TM K132 (Uniprot) K10 Supplementary Figure 8. Proposed membrane topology of host membrane proteins of ORF3a and M of SARS- CoV-2 from Figure 5. (A) Results of biotin-labeled sites on membrane protein by ORF3a-GFP and TurboID-GBP. Among 30 proteins at ORF3a interactome of Figure 4e, 22 proteins are integral membrane proteins. Biotin-labeled sites in ORF3a-GFP sample, not in M-GFP sample are shown as red-colored biotin (“B”). Biotin-labeled sites in both ORF3a- and M-GFP samples are shown as purple-colored biotin. Proposed models of topology (e.g. CLCC1, DHCR7, TMEM110, FKBP8, RNF5) based on our mass data were colored in yellow. Among depicted 22 proteins, proteins overlapped with MAM proteome were shown with light blue colored boxes. IMS meant mitochondrial intermembrane space. OMM meant outer mitochondrial membrane. (B) Results of biotin-labeled sites on membrane protein by M-GFP

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and TurboID-GBP. Among 30 proteins at M interactome of Figure 4f, 22 proteins are integral membrane proteins. Biotin-labeled sites by M not ORF3a are shown as blue-colored biotin. Biotin-labeled sites by both ORF3a and M are shown as purple-colored biotin. Proposed models of topology (e.g. UBR3, TMEM110, UBIAD1) based on our mass data were colored in yellow. Among depicted 22 proteins, proteins overlapped with MAM proteome were shown with light blue colored boxes. IMS meant mitochondrial intermembrane space. OMM meant outer mitochondrial membrane.

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Supplementary Figure 9. RNF5 ubiquitinylates ORF3a of SARS-CoV-2. (a) Confocal microscopy images of Flag- RNF5 (left) and SEC61-V5-TurboID with mCherry-RNF5. Flag-RNF5 was visualized by anti-Flag antibody (AF488- conjugated, GFP fluorescence channel). SEC61B-V5-TurboID was visualized by anti-V5 antibody (AF488- conjugated, GFP fluorescence channel). mCherry-RNF5 was visualized by RFP fluorescence channel. BF: bright field, Scale bars: 10 μm. (b) Anti-GFP Western blot results of ORF3a-GFP or M-GFP expressed HEK293T cells with or without MG132 treatment (2 μM, 16 h). (c) Expression of ORF3a and M shown in (b). After MG132 treatment, expression of ORF3a and M increased. (d) Additional western blotting results are shown in Figure 6d. Anti-HA Anti- V5, and Anti-ub western blotting results of ORF3a-Flag, HA-RNF5 and V5-UBB co-expressed HEK293T cells. Band with red arrow is the expected size of ORF3a-Flag. Band with blue arrows is the ubiquitinylated ORF3a-Flag. Band with green arrow is HA-RNF5. (e) Raw images of western blotting and Ponceau membrane staining with size ladder from (d) (WB: Anti-Flag, Anti-HA, Anti-V5 and Anti-ub). -S18-

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Supplementary Figure 10. RNF5C42S mutant do not induce ubiquitination of SARS-CoV-2 ORF3a Anti- Flag, Anti-HA, Anti-V5 and Anti-ubiquitin (ub) western blot results of ORF3a-Flag, HA-RNF5(C42S) and V5-UBB co-expressed HEK293T cells. Red arrow marked unmodified molecular weight of ORF3a-Flag. Band with green arrow is HA-RNF5.

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HEK293T (transfected)

IP FT Elu

TOM20-BioID(C-term) + + + + + + + + +

BioID(N-term)-SEC61B + + + + + + + + +

Empty vector + + +

GFP + + +

M-GFP + + +

Biotinylation (O/N) + + + + + + + + + +

Lane number 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10

kDa

245 180 130

100

WB : SA-HRP 25

WB : SA-HRP WB : SA-HRP (shorter exposure time) (longer exposure time)

Lane number 1 2 3 4 5 6 7 8 9 10 1 2 3 4 1 2 3 4

kDa kDa kDa 245 245 245 180 180 180 130 130 130 100 100 100 75 75 75

63 63 63

48 48 48

35 35 35

25 25 25 20 20 20

17 17 17

WB : Anti-GFP WB : Anti-HA WB : Anti-FLAG for TOM20-BioID(C-term) for BioID(N-term)-SEC61B Supplementary Figure 12. Western blot results of M expressed cells using Contact-ID. SA-HRP, Anti- GFP, Anti-HA and Anti-Flag western blot results of whole cell lysate of GFP and M-GFP expressed HEK293T cells with co-expression of Contact-ID constructs. These samples were enriched by SA-bead. M was biotinylated by Contact-ID constructs, indicating M localized to MAM. IP, FT and Elu meant input, flow through and elution of samples. Bands with red arrow showed M protein biotinylated by Contact-ID.

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Supplementary Figure 13. ORF3a affects secretome profiles of host cells. (A) Raw images of western blotting and Ponceau membrane staining with size ladder from Figure 7g (WB: SA-HRP). (B) Line scan analysis results of Figure 7g. Expression of proteins from Figure 7g is shown in detail. Total expression of secreted proteins from Figure 7g. From densitometric analysis of the western blotting lanes, we observed that an increase in biotinylated proteins of over 6.23 folds was detected in ORF3a-expressing cells. (C) Raw images of western blotting and Ponceau membrane staining with size ladder from Figure 7h (WB: anti-strep).

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Supplementary Figure 15. M affects secretome profiles of host cells. (a) SA-HRP western blotting result of secreted proteins from SEC61B-TurboID expressed HEK293T cells with or without co-expression of M. (b) Anti-Flag western blotting results of the same samples as (a). (c) Whole cell lysate sample from (a).

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Supplementary Table 1. Construct Information Templates of nsp7, nsp8, nsp9, nsp16, S, E, M, N, ORF3a, ORF6, ORF7a, ORF7b, and ORF8 were obtained from professor Kim, Ho Min (KAIST). Templates of ORF3b, ORF9b, ORF9c, and ORF10 were obtained from professor Kim, V. Narry (SNU).

Name (expected size) Features Promotor/Vector Details nsp7-linker-eGFP HindIII-KpnI-nsp7- CMV/pCDNA5 linker : (expected MW : 37.7 BsiWI-linker-NheI- GAPGSAGSAAGSG kDa) eGFP-Stop-NotI-XhoI nsp8-linker-eGFP HindIII-KpnI-nsp8- CMV/pCDNA5 linker : (expected MW : 50.3 BsiWI-linker-NheI- GAPGSAGSAAGSG kDa) eGFP-Stop-NotI-XhoI nsp9-linker-eGFP AflII-nsp9-HindIII- CMV/pCDNA5 linker : (expected MW : 40.7 linker-NotI-eGFP-Stop- GAPGSAGSAAGSG kDa) AsCI-XhoI nsp16-linker-eGFP AflII-nsp16-HindIII- CMV/pCDNA5 linker : (expected MW : 61.6 linker-NotI-eGFP-Stop- GAPGSAGSAAGSG kDa) AsCI-XhoI S-linker-eGFP HindIII-KpnI-Spike- CMV/pCDNA5 linker : (expected MW : 169.5 BsiWI-linker-NheI- GAPGSAGSAAGSG kDa) eGFP-Stop-NotI-XhoI E-linker-eGFP AflII-Envelope-HindIII- CMV/pCDNA5 linker : (expected MW : 36.5 linker-NotI-eGFP-Stop- GAPGSAGSAAGSG kDa) AsCI-XhoI M-linker-eGFP AflII-Membrane- CMV/pCDNA5 linker : (expected MW : 53.3 HindIII-linker-NotI- GAPGSAGSAAGSG kDa) eGFP-Stop-AsCI-XhoI M-Flag (expected AflII-Membrane- CMV/pCDNA5 MW : 27.6 kDa) HindIII-Flag-Stop-XhoI M-linker-mCherry AflII-Membrane- CMV/pCDNA5 linker : (expected MW : 53.9 HindIII-linker-NotI- GAPGSAGSAAGSG kDa) mCherry-Stop-AsCI- XhoI N-linker-eGFP HindIII-KpnI- CMV/pCDNA5 linker : (expected MW : 73.9 Nucleocapsid-BsiWI- GAPGSAGSAAGSG kDa) linker-NheI-eGFP- Stop-NotI-XhoI ORF3a-linker-eGFP AflII-ORF3a-HindIII- CMV/pCDNA5 linker : (expected MW : 59.3 linker-NotI-eGFP-Stop- GAPGSAGSAAGSG kDa) AsCI-XhoI ORF3a-Flag (expected AflII-ORF3a-HindIII- CMV/pCDNA5 MW : 33.6 kDa) Flag-Stop-AsCI-XhoI ORF3b-linker-eGFP AflII-ORF3b-BamHI- CMV/pCDNA5 linker : (expected MW : 37.9 HindIII-linker-NotI- GAPGSAGSAAGSG kDa) eGFP-Stop-AsCI-XhoI ORF6-linker-eGFP AflII-ORF6-HindIII- CMV/pCDNA5 linker : (expected MW : 35.4 linker-NotI-eGFP-Stop- GAPGSAGSAAGSG kDa) AsCI-XhoI ORF6-Flag (expected AflII-ORF6-HindIII- CMV/pCDNA5 MW : 9.8 kDa) Flag-Stop-XhoI ORF7a-linker-eGFP AflII-ORF7a-HindIII- CMV/pCDNA5 linker : (expected MW : 41.9 linker-NotI-eGFP-Stop- GAPGSAGSAAGSG kDa) AsCI-XhoI ORF7b-linker-eGFP HindIII-KpnI-ORF7b- CMV/pCDNA5 linker : (expected MW : 33.5 BsiWI-linker-NheI- GAPGSAGSAAGSG kDa) eGFP-Stop-NotI-XhoI ORF7b-Flag (expected HindIII-KpnI-ORF7b- CMV/pCNDA5 MW : 7.7 kDa) BsiWI-Flag-Stop-XhoI ORF8-linker-eGFP AflII-ORF8-HindIII- CMV/pCDNA5 linker : -S25-

(expected MW : 42.0 Flag-NotI-eGFP-Stop- GAPGSAGSAAGSG kDa) AsCI-XhoI ORF9b-linker-eGFP AflII-ORF9b-BamHI- CMV/pCDNA5 linker : (expected MW : 39.6 HindIII-linker-NotI- GAPGSAGSAAGSG kDa) eGFP-Stop-AsCI-XhoI ORF9c-linker-eGFP AflII-ORF9c-BamHI- CMV/pCDNA5 linker : (expected MW : 39.5 HindIII-linker-NotI- GAPGSAGSAAGSG kDa) eGFP-Stop-AsCI-XhoI ORF10-linker-eGFP AflII-ORF10-BamHI- CMV/pCDNA5 linker : (expected MW : 33.2 HindIII-linker-NotI- GAPGSAGSAAGSG kDa) eGFP-Stop-AsCI-XhoI G3BP1-V5-APEX2- HindIII-KpnI-G3BP1- CMV/pCDNA5 for confocal imaging with AP NheI-V5-APEX2-AP- co-expression of (expected MW : 82.6 Stop-NotI-XhoI Nucleocapsid kDa) eGFP HindIII-KpnI-eGFP- CMV/pCDNA5 for mass sampling as control (expected MW : 26.9 Stop-NotI-XhoI kDa) TurboID-V5-GBP HindIII-TurboID-KpnI- CMV/pCDNA5 (expected MW : 49.4 V5-NotI-GBP-Stop- kDa) XhoI ORF3a-linker-V5- AflII-ORF3a-HindIII- CMV/pCDNA5 linker : APEX2 linker-NotI-V5- GAPGSAGSAAGSG (expected MW : 62.6 APEX2-AP-Stop-XhoI for APEX-EM imaging of kDa) ORF3a expression M-linker-V5-APEX2 AflII-Membrane- CMV/pCDNA5 linker : (expected MW : 56.6 HindIII-linker-NotI-V5- GAPGSAGSAAGSG kDa) APEX2-AP-Stop-XhoI for APEX-EM imaging of M expression Flag-RNF5 (expected AflII-Flag-RNF5-Stop- CMV/pCDNA5 For co-imaging with vPOI MW : 22.3 kDa) XhoI mCherry-RNF5 AflII-mCherry- CMV/pCNDA5 For co-imaging with (expected MW : 47.3 EcoRI-BamHI-RNF5- SEC61B kDa) Stop-XhoI HA-RNF5 (expected HindIII-HA-BamHI- CMV/pCNDA5 For ubiquitination of vPOI MW : 21.2 kDa) RNF5-Stop-XhoI HA-RNF5(C42S) HindIII-HA-BamHI- CMV/pCDNA5 Inactivation version of RNF5 (expected MW : 21.2 RNF5(C42S)-Stop- kDa) XhoI V5-UBB (expected AflII-V5-HindIII-UBB- CMV/pCDNA5 For ubiquitination of vPOI MW : 10.3 kDa) Stop-XhoI TOM20-pBirA(79aa-C KpnI-TOM20-BamHI- CMV/pCDNA5 linker : SGGSGGSR term)-HA linker-pBirA(79aa-C TOM20(NM_014765.2) (expected MW : 45.0 term)-HA-Stop-NotI for applicating Contact-ID kDa) under ORF3a expression ref : Kwak et al., 2020 Flag-pBirA(N term- NotI-Flag-pBirA(N CMV/pCDNA3 linker : 78aa)-SEC61B term-78aa)-linker- GGASGGSGSGPVAT (exepcted MW : 20.7 EcoRI-SEC61B-Stop- SEC61B (NM_006808) kDa) XhoI for applicating Contact-ID under ORF3a expression ref : Kwak et al., 2020 ORF3a-twin strep HindIII-ORF3a- CMV/pCDNA3.1 (expected MW : 34.3 BamHI-twin strep-Stop- kDa) XhoI SEC61B-V5-TurboID HindIII-SEC61B- CMV/pCDNA5 for identification of secreted (expected MW : 46.9 BamHI-KpnI-ClaI-V5- proteins under ORF3a kDa) NheI-TurboID-Stop- expression NotI-XhoI

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Supplementary Table 2. ORF3a interactome : approved drug ID

UNIPROT ID Gene Name Approved drug ID (drugbank.ca) : Drug name (Accession Number) P05556 ITGB1 Antithymocyte immunoglobulin (DB00098) O76082 SLC22A5 Levocarnitine (DB00583) P53985 SLC16A1 Pyruvic acid (DB00119) Q9UBM7 DHCR7 NADH (DB00157) P29317 EPHA2 Dasatinib (DB01254), Regorafenib (DB08896), Fostamatinib (DB12010) P30519 HMOX2 NADH (DB00157) Q8WY07 SLC7A3 L-Lysine (DB00123), Fluciclovine (DB13146) P19634 SLC9A1 Amiloride (DB00594) Q16850 CYP51A1 Tioconazole (DB01007), Itraconazole (DB01167) Q15738 NSDHL NADH (DB00157) Coagulation factor VIIa (DB00036), Coagulation Factor IX (DB00100), Glutamic acid (DB00142), Menadione (DB00170), Phylloquinone P38435 GGCX (DB01022), Anisindione (DB01125), Kappadione (DB09332), Coagulation factor IX (DB13152) P06241 FYN Dasatinib (DB01254), Fostamatinib (DB12010) Digoxin (DB00390), Acetyldigitoxin (DB00511), Hydroflumethiazide (DB00774), Etacrynic acid (DB00903), Trichlormethiazide (DB01021), Deslanoside (DB01078), Ouabain (DB01092), Diazoxide (DB01119), Bretylium (DB01158), Ciclopirox (DB01188), Bepridil (DB01244), P05023 ATP1A1 Potassium cation (DB01345), Aluminium (DB01370), Magnesium cation (DB01378), Digitoxin (DB01396), Almitrine (DB01430), Magnesium acetate (DB13996), Potassium acetate (DB14498), Potassium sulfate (DB14499), Potassium (DB14500), Aluminium phosphate (DB14517), Aluminum acetate (DB14518) NADH (DB00157), Lovastatin (DB00227), Cerivastatin (DB00439), P04035 HMGCR Simvastatin (DB00641), Atorvastatin (DB01076), Fluvastatin (DB01095), Rosuvastatin (DB01098), Pitavastatin (DB08860), Cannabidiol (DB09061) P52429 DGKE Alpha-Tocopherol succinate (DB14001) Antihemophilic factor (DB00025), Tenecteplase (DB00031), Coagulation Q07954 LRP1 Factor IX (DB00100), Coagulation factor IX (DB13152), Lonoctocog alfa (DB13998), Moroctocog alfa (DB13999) Cetuximab (DB0000), Lidocaine (DB00281), Gefitinib (DB00317), Erlotinib (DB00530), Lapatinib (DB01259), Panitumumab (DB01269), Vandetanib (DB05294), Afatinib (DB08916), Osimertinib (DB09330), P00533 EGFR Necitumumab (DB09559), Foreskin keratinocyte (DB10772), Neratinib (DB11828), Dacomitinib (DB11963), Fostamatinib (DB12010), Brigatinib (DB12267), Zanubrutinib (DB15035) P78348 ASIC1 Amiloride (DB00594) P48651 PTDSS1 Phosphatidyl serine (DB00144) O60658 PDE8A Caffeine (DB00201)

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Supplementary Table 3. M interactome : approved drug ID

UNIPROT ID Gene Name Approved drug ID (drugbank.ca) P05556 ITGB1 Antithymocyte immunoglobulin (DB00098) Q99808 SLC29A1 Troglitazone (DB00197), Ethanol (DB00898), Fostamatinib (DB12010) P53985 SLC16A1 Pyruvic acid (DB00119) O76082 SLC22A5 Levocarnitine (DB00583) Q8WY07 SLC7A3 L-Lysine (DB00123), Fluciclovine (DB13146) O95819 MAP4K4 Fostamatinib (DB12010) Digoxin (DB00390), Acetyldigitoxin (DB00511), Hydroflumethiazide (DB00774), Etacrynic acid (DB00903), Trichlormethiazide (DB01021), Deslanoside (DB01078), Ouabain (DB01092), Diazoxide (DB01119), Bretylium (DB01158), Ciclopirox (DB01188), Bepridil (DB01244), P05023 ATP1A1 Potassium cation (DB01345), Aluminium (DB01370), Magnesium cation (DB01378), Digitoxin (DB01396), Almitrine (DB01430), Magnesium acetate (DB13996), Potassium acetate (DB14498), Potassium sulfate (DB14499), Potassium (DB14500), Aluminium phosphate (DB14517), Aluminum acetate (DB14518) P29317 EPHA2 Dasatinib (DB01254), Regorafenib (DB08896), Fostamatinib (DB12010) P19634 SLC9A1 Amiloride (DB00594) P11166 SLC2A1 Carboxymethylcellulose (DB11059) Cystine (DB00138), Glutamic acid (DB00142), Riluzole (DB00740), Q9UPY5 SLC7A11 Sulfasalazine (DB00795), Acetylcysteine (DB06151), Thimerosal (DB11590) P30519 HMOX2 NADH (DB00157) Q9UP95 SLC12A4 Potassium chloride (DB00761), Bumetanide (DB00887) P52569 SLC7A2 L-Lysine (DB00123), Ornithine (DB00129) Antihemophilic factor (DB00025), Tenecteplase (DB00031), Coagulation Q07954 LRP1 Factor IX (DB00100), Coagulation factor IX (DB13152), Lonoctocog alfa (DB13998), Moroctocog alfa (DB13999) P49069 CAMLG Cyclosporine (DB00091) Q16850 CYP51A1 Tioconazole (DB01007), Itraconazole (DB01167) Q9Y666 SLC12A7 Potassium Chloride (DB00761) P06241 FYN Dasatinib (DB01254), Fostamatinib (DB12010) P78348 ASIC1 Amiloride (DB00594) Q9UBM7 DHCR7 NADH (DB00157) Q8TD43 TRPM4 Glyburide (DB01016) Insulin (DB00030), Insulin lispro (DB00046), Insulin glargine (DB00047), Insulin prok (DB00071), Mecasermin (DB01277), Insulin aspart P06213 INSR (DB01306), Insulin detemir (DB01307), Insulin glulisine (DB01309), Chromic chloride (DB09129), Insulin degludec (DB09564), Fostamatinib (DB12010), Brigatinib (DB12267), Mecasermin rinfabate (DB14751) Flavin adenine dinucleotide (DB03147), Flavin mononucleotide P16435 POR (DB03247) P84077 ARF1 Glycerin (DB09462) O60658 PDE8A Caffeine (DB00201) Dasatinib (DB01254), Citric acid (DB04272), Bosutinib (DB06616), P12931 SRC Ponatinib (DB08901), Nintedanib (DB09079), Fostamatinib (DB12010)

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NADH (DB00157), Lovastatin (DB00227), Cerivastatin (DB00439), P04035 HMGCR Simvastatin (DB00641), Atorvastatin (DB01076), Fluvastatin (DB01095), Rosuvastatin (DB01098), Pitavastatin (DB08860), Cannabidiol (DB09061) Q9HB21 PLEKHA1 Citric acid (DB04272)

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