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ARTICLES https://doi.org/10.1038/s41592-018-0100-5 A proximity-tagging system to identify membrane protein–protein interactions Qiang Liu 1,2,3,4 , Jun Zheng 1,3,4 , Weiping Sun 1,3 , Yinbo Huo 1,2,3 , Liye Zhang  1 , Piliang Hao 1 , Haopeng Wang  1 * and Min Zhuang  1 * 1 School of Life Science and Technology, ShanghaiTech University, Shanghai, China. 2 Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China. 3 University of Chinese Academy of Sciences, Beijing, China. 4 These authors contributed equally: Qiang Liu and Jun Zheng. *e-mail: [email protected]; [email protected] SUPPLEMENTARY INFORMATION In the format provided by the authors and unedited. NATURE METHODS | www.nature.com/naturemethods

A proximity-tagging system to identify membrane protein ...10.1038...CD28 deficient cell line (a gift from Dr. Oreste Acuto) was transfected with either CD28 or CD28-PafA, cells were

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Page 1: A proximity-tagging system to identify membrane protein ...10.1038...CD28 deficient cell line (a gift from Dr. Oreste Acuto) was transfected with either CD28 or CD28-PafA, cells were

Articleshttps://doi.org/10.1038/s41592-018-0100-5

A proximity-tagging system to identify membrane protein–protein interactionsQiang Liu1,2,3,4, Jun Zheng1,3,4, Weiping Sun1,3, Yinbo Huo1,2,3, Liye Zhang   1, Piliang Hao1, Haopeng Wang   1* and Min Zhuang   1*

1School of Life Science and Technology, ShanghaiTech University, Shanghai, China. 2Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China. 3University of Chinese Academy of Sciences, Beijing, China. 4These authors contributed equally: Qiang Liu and Jun Zheng. *e-mail: [email protected]; [email protected]

SUPPLEMENTARY INFORMATION

In the format provided by the authors and unedited.

NAture MetHodS | www.nature.com/naturemethods

Page 2: A proximity-tagging system to identify membrane protein ...10.1038...CD28 deficient cell line (a gift from Dr. Oreste Acuto) was transfected with either CD28 or CD28-PafA, cells were

Supplementary Figure 1

Identification of modified lysine residues on GST and XIAP.

(a) Protein sequences of GST-PafA, PafA-XIAP and PupE. The modified lysine residues are highlighted in red. (b) Identified peptides from mass spectrometry experiments and the peptide scores (Supplementary Table 1). (c) One peptide spectral example showing the modification site.

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Supplementary Figure 2

Sequence and structure of bio-Pup(E) and bio-DE28.

(a) The sequence of bio-Pup(E). BCCP domain (blue) derived from bacteria carboxylase (Propionobacterium freednreichii, NCBI Reference Sequence: WP_013161729.1) is fused to the N terminus of full length Pup (red). BCCP has been codon optimized for mammalian cell expression (DNA sequence: GCCGGGAAGGCAGGCGAGGGAGAGATCCCCGCACCCTTGGCCGGCACGGTCAGCAAAATCCTGGTCAAGGAAGGCGACACCGTGAAGGCTGGACAGACGGTGTTGGTACTGGAGGCGATGAAGATGGAGACAGAGATCAATGCCCCGACCGATGGGAAGGTGGAGAAGGTGCTGGTTAAGGAGAGGGACGCCGTGCAGGGCGGTCAGGGACTGATCAAGATCGGCGACTACGACATCCCGACAACCGCCAGC). The lysine residue labeled in green is biotinylated in mammalian cells. This version of bio-Pup(E) was used for all intracellular studies in the paper. (b) Recombinant bio-Pup(E) purified from E. Coli. GST-bio-Pup(E) was cloned into pGEX vector. The protein was pull down by GST tag, then GST was cleaved off and the protein was further purified with gel filtration chromatography. * Residual GST left in the sample. (c) The structure and design of chemically synthesized bio-DE28. (d) Mass spectrometry analysis of confirmed peptide mass. (e) Bio-Pup(E) and bio-DE28 have similar activity in vitro. The Puplylation assay was carried out in a 20 μl reaction mix containing 200 nM GST-PafA, 10x reaction buffer (200 mM Tris, 50 mM ATP and 75 mM Mg

2+, pH 8.0), and 2 μM bio-Pup(E) or bio-

DE28 at 25 °C. The reactions were stopped at different time points for western blot analysis with streptavidin-HRP. The intensity of self-modified GST-PafA were quantitated and plotted (mean ± SEM on the bottom left and individual dots plot on the bottom right, n=3 independent experiments). One representative gel from triplicate experiments was shown on the top. Original full scans of blots are shown in Supplementary Fig. 15.

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Supplementary Figure 3

Mass spectrometry identification of proteins that interact with CD28 cytosolic tail.

(a) The spectral counts are combined from duplicate runs and plotted with each dot represents a protein identified. PUP-ITCD28

and PUP-IT

tailless datasets are compared. The red dots are examples of known CD28 interacting proteins (Supplementary Table 2a). (b)

Identification of CD28 interacting proteins by comparing the spectral counts from PUP-ITCD28

and PUP-IT5AA

datasets (Supplementary Table 2b).

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Supplementary Figure 4

PUP-ITCD28

identified a large number of proteins involved in protein transport.

(a) Venn diagram shows the overlapping proteins identified by different PUP-IT. Proteins identified with more than 2 unique peptides are considered in this plot. (b) 51 unique CD28 tail-binding proteins and c) 202 potential CD28 interacting candidates (Supplementary Table 2) are analyzed with gene ontology, using STRING. Top ten functional enrichment groups are presented.

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Supplementary Figure 5

PUP-ITCD28

maintains correct localization and function.

(a) Immunofluorescence staining shows CD28-PafA is expressed on cell surface in the Pup inducible Jurkat used for mass spectrometry study. Top panel, CD28 in wild type Jurkat was stained. Bottom panel, CD28-PafA-myc was stained with anti-myc antibody. GFP was also expressed in iPUP Jurkat as a control for cytosolic protein. This experiment was repeated independently twice with similar results. (b) CD28-PafA can be detected on cell surface. CD28 deficient cell line (a gift from Dr. Oreste Acuto) was transfected with either CD28 or CD28-PafA, cells were then mixed with anti-CD28-PE antibody and analyzed with flow cytometer. (c) CD28-PafA mediates up regulation of CD69 upon stimulation. CD28 knockout cells were transfected with either CD28 or CD28-PafA. With the stimulation by CD28 ligand mimic antibody, the cell surface activation marker CD69 is up regulated in both CD28 and CD28-PafA transfected cells. (d) Different expression level of CD28 and CD28-PafA in transfected cells. CD28-myc has a much higher expression level than CD28-PafA-myc. Original full scans of blots are shown in Supplementary Fig. 15. (e) The experiments in (c) were repeated three times and CD28 activation index was compared between CD28 and CD28-PafA (mean ± SEM, n=3 biological

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independent samples). CD28-PafA largely maintains the function of wild type CD28. CD28 activation index was defined as percentage of CD69 positive cells/CD28 expression intensity.

Page 9: A proximity-tagging system to identify membrane protein ...10.1038...CD28 deficient cell line (a gift from Dr. Oreste Acuto) was transfected with either CD28 or CD28-PafA, cells were

Supplementary Figure 6

PUP-ITMUL1

and PUP-ITRNF13

identify unique interacting proteins.

(a) Cellular localization of MUL1-PafA and RNF13-PafA. HeLa cells were transfected with either MUL1-PafA-myc or RNF13-PafA-myc, then cells were fixed and stained with anti-myc antibody. This experiment was repeated independently twice with similar results. (b) Volcano plot of PUP-IT

MUL1 and PUP-IT

CD28. The logarithmic ratios of protein LFQ-intensity (CD28/MUL1) was plotted against negative

logarithmic P value of a two-sided two samples t-test in Perseus. Mitochondria located proteins, including RHOT1, TOM70 and HK2 are uniquely identified in PUP-IT

MUL1 datasets (Supplementary Table 3b). (c) Volcano plot of PUP-IT

MUL1 and PUP-IT

RNF13. The

logarithmic ratios of protein LFQ-intensity (RNF13/MUL1) was plotted against negative logarithmic P value of a two-sided two samples t-test in Perseus. Ubiquitin ligase RNF13 uniquely binds the ubiquitin conjugating enzyme UBE2H (Supplementary Table 3c). In (b) and (c), the green and blue dots represent significantly enriched proteins from common hits (FDR≤0.05, n=3 independent experiments).

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Supplementary Figure 7

Validation of potential MUL1-interacting proteins.

(a) Pathway analysis of MUL1 interaction proteins. Each dot represents a protein identified in Supplementary Table 4 as a MUL1 specific interacting protein. These proteins are either connected in primary metabolic process or related to ubiquitin-proteasome system. The gene ontology software STRING (functional protein association networks) was used to analyze protein interactions (https://string-db.org/). The thickness of the lines indicates the strength of data support. (b) Co-immunoprecipitation of MUL1 and interacting candidates. Myc tagged MUL1 was co-transfected with different V5 tagged candidate in HeLa cells. LCK-V5 was used as the negative control. MUL1 associates with TOMM22, CYB5R1 and PEX19, but not with OCIAD1 and RHOT1. (c) TOMM22 and PEX19 interact with MUL1. The co-immunoprecipitation experiments were repeated with TOMM22 and PEX19 to further confirm the specific interaction. Experiments in (b) and (c) were repeated independently twice with similar results. Original full scans of blots are shown in Supplementary Fig. 15.

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Supplementary Figure 8

PUP-IT expression does not introduce more background for mass spectrometry experiments.

Gene ontology analysis using protein lists identified in (a) iPUP cells without PafA or Pup expression (no PUP-IT transfection and no doxycycline) (n=801 proteins) (b) iPUP cells induced with doxycycline but no PafA expression (n=795 proteins), (c) common background proteins identified with PUP-IT

CD28, PUP-IT

MUL1 and PUP-IT

RNF13 (n=543 proteins). The data (Supplementary Table 3) was

analyzed with DAVID (https://david.ncifcrf.gov) and plotted with Graphpad Prism 6.

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Supplementary Figure 9

Raji cell-surface labeling by PUP-ITCD28-ex

.

Page 14: A proximity-tagging system to identify membrane protein ...10.1038...CD28 deficient cell line (a gift from Dr. Oreste Acuto) was transfected with either CD28 or CD28-PafA, cells were

(a) PUP-ITCD28-extracellular

mediated Raji cells labeling. Similar experiments were set up as in Fig. 5d but the fluorescence was examined with flow cytometer. The percentage of biotin positive cells is shown in the gated window. This experiment was repeated independently twice with similar results. (b) mCherry positive Jurkat cells were transfected with FKBP-CD28 and mixed with GFP positive Jurkat cells. Cell surface Pup modification assay was performed in the same way as in (a). This experiment was repeated independently twice with similar results.

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Supplementary Figure 10

Recombinant protein expression and purification.

(a) Construct design and protein sequence used to express and purify IL2-FKBP. Human IL2-FKBP fusion with GS linker and His tag was subcloned into pCDNA3.1 and transfected 293T cells. Medium containing IL2-FKBP was collected and the protein was purified with Ni beads. FRB-PafA was subcloned into pGEX6p-1 and fused with N terminal GST (see Methods for details). (b) Purified IL2-FKBP and (c) Purified FRB-PafA are analyzed with SDS-PAGE and coomassie staining. Lane 1 in (c) is purified GST-FRB-PafA cleaved by PreScission protease and lane 2 is further purified FRB-PafA.

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Supplementary Figure 11

PUP-ITIL2

-dependent cell-surface modification of bio-DE28.

(a) Activated T cell surface can be modified by PUP-ITIL2

. T cells are first activated then mixed with PUP-ITIL2

, bio-DE28, ATP and rapamycin. The same reaction was also set up without either ATP or rapamycin. A complete assembly of PUP-IT

IL2 is required for bio-

DE28 labeling. This experiment was repeated independently more than three times with similar results. (b) Cell surface labeling increases with increased PUP-IT

IL2. 1×10

5 Jurkat cells were first activated over night by anti-CD3 and anti-CD28. Cell surface Pup

modification assays were set up the same as in Fig. 6. In 150 μl reaction volume, IL2-FKBP was added to reach the final concentration at 0.5, 1, 2, 4, and 14.8 ng/μl. With increased IL2-FKBP protein, more cell surface labeling of bio-DE28 can be observed. This experiment was repeated independently twice with similar results. (c) Representative flow cytometer data from three experimental repeats as shown in Fig. 6d. (d) The gating strategy used in (a)-(c).

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Supplementary Figure 12

PUP-IT is more active than BioID both in vitro and in cells.

(a) Recombinant purified GST-BirA* (R118G, BioID) and GST-PafA (PUP-IT) were analyzed with coomassie stain. (b) In vitro modification assay of BioID and PUP-IT to compare the activity. For BioID reaction, the 40 μl reaction contains 1μM GST-BirA* and 50 μM biotin. For PUP-IT reaction, the 40 μl reaction contains 1μM GST-PafA and 10 μM bio-DE28. The experiments were carried out at 37 °C in the presence of ATP and Mg

2+. At each time point, 10 μl reaction mixture were added to SDS loading dye to stop the reaction.

Anti-GST blot shows the enzyme level while the streptavidin blot shows the product level. This experiment was repeated independently twice with similar results. (c) BirA* and PafA are fused to the C terminus of CD28 individually to generate BioID

CD28 and PUP-IT

CD28 with

myc tag. Jurkat cells are transiently transfected with either BioID or PUP-IT (with bio-PupE). BioID transfected cells were cultured in medium supplemented with 50 μM biotin. 24 hours after transfection, cells are harvested and lysed for immune-blot analysis. This experiment was repeated independently three times with similar results. Original full scans of blots are shown in Supplementary Fig. 15.

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Supplementary Figure 13

Side-by-side comparison of BioIDCD28

and PUP-ITCD28

to identify CD28-interacting proteins.

(a) Volcano plot for BioIDCD28

and BioIDCD28 tailless

comparison (Supplementary Table 5a). (b) Volcano plot compare PUP-ITCD28

and PUP-IT

CD28 tailless comparison (Supplementary Table 5b). In (a) and (b), the logarithmic ratios of protein LFQ-intensity was plotted

against negative logarithmic P value of a two-sided two samples t-test. The green and blue dots represent significantly enriched proteins from common hits (FDR≤0.05, n=4, 2 biologically independent samples in 2 independent experiments). (c) BioID background protein analysis. The common proteins (n=688 proteins) that are identified in BioID

CD28 WT, BioID

CD28 tailless and BioID

CD28 5AA were

analyzed the same way as in supplementary Fig. 8.

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Supplementary Figure 14

Unprocessed scans of original blots shown in the main text figures, and a plot with individual data points.

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Supplementary Figure 15

Unprocessed scans of original blots shown in the Supplementary Figures.

Page 24: A proximity-tagging system to identify membrane protein ...10.1038...CD28 deficient cell line (a gift from Dr. Oreste Acuto) was transfected with either CD28 or CD28-PafA, cells were

SUPPLEMENTARY NOTES

Identification of MUL1 interacting proteins

An alternative method using spectral counts has been used to analyze the same data.

We compared the spectral counts in all the PUP-IT samples to identify those proteins

that are uniquely associated with different bait (Supplementary table 4). Similar

results were obtained for PUP-ITCD28 with the enrichment of GTPase and GTPase

regulators. More MUL1 interacting candidates are identified. Pathway analysis

reveals they are enriched in primary metabolic process and ubiquitin-proteasome

system (Supplementary Fig. 7a). We examined the interaction between MUL1 and

five interacting candidates, including OCIAD1, RHOT1, TOMM22, CYB5R1 and

PEX19 by co-immunoprecipitation. V5 tagged TOMM22, CYB5R1 and PEX19 pull

down more MUL1, compared to the non-related control LCK (Supplementary Fig.

7b). We further validated the specific interaction between MUL1 and TOMM22,

MUL1 and PEX19 (Supplementary Fig. 7c). These data suggest PUP-ITMUL1 can

also help identify novel interacting proteins.

Identification of GTPases that may mediate cytoskeleton remodeling by CD28

CD28 mediated-costimulation is considered to be one of the key mechanisms to

maintain peripheral tolerance. Abatacept, a CTLA4-Ig fusion protein that competitively

blocks the ability of CD28 to interact with its ligands B7s (extracellular region of

CD86/CD80), has been approved for the therapy of rheumatoid arthritis. Therefore,

the study of CD28 interactome may help us identify more potential therapeutic target.

Our previous study, in which a synthesized CD28 cytoplasmic tail was used to affinity

purify CD28-interacting proteins from T cell lysate, identified 28 proteins that bind with

CD28 in a phosphorylation-dependent manner 1. Here we used PUP-IT approach to

identified >50 proteins. Some of these proteins, such as LCK, p85 and CSK are

revealed in both studies. Proteins that not show in this study may be more

dependent on phosphorylation of CD28 to bind.

Previous study suggested one important CD28-mediated function is to regulate actin

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cytoskeleton remodeling, which intersects with signaling events mediated by TCR 1, 2.

However, it is not clear how CD28 affects the actin dynamics. Interestingly, here we

find a significant fraction of CD28-binding proteins are cytoskeleton-remodeling

regulators. Small GTPases are key components for cytoskeleton remodeling. We

find several small GTPases, such as Cdc42 and RHOA, and other GTPase regulators.

IQGAP2 is GTPase-activating Protein (GAP), while ARHGDIB and GDI2 are

GDP-dissociation inhibitors (GDIs), which reduce the rate of GDP dissociation from

GTPase. In addition, ROCK1 is a downstream effector of small GTPase (RhoA) and

DOCK2 is a guanine nucleotide exchange factor (GEF) that activates GTPase 3,

4. Taken together, these data suggest CD28 might regulate actin dynamics by

transiently interacting with multiple small GTPases and their regulators as well as

downstream effectors. Further functional validations will be required to dissect the

molecular mechanism with or without CD28 signaling.

References

1. Tian, R. et al. Combinatorial proteomic analysis of intercellular signaling

applied to the CD28 T-cell costimulatory receptor. Proc Natl Acad Sci U S A

112, E1594-1603 (2015).

2. Tan, Y.X. et al. Inhibition of the kinase Csk in thymocytes reveals a

requirement for actin remodeling in the initiation of full TCR signaling. Nat

Immunol 15, 186-194 (2014).

3. Kulkarni, K., Yang, J., Zhang, Z. & Barford, D. Multiple factors confer specific

Cdc42 and Rac protein activation by dedicator of cytokinesis (DOCK)

nucleotide exchange factors. J Biol Chem 286, 25341-25351 (2011).

4. Maekawa, M. et al. Signaling from Rho to the actin cytoskeleton through

protein kinases ROCK and LIM-kinase. Science 285, 895-898 (1999).