bioRxiv preprint doi: https://doi.org/10.1101/264788; this version posted February 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Liu et al.

An integrated approach to comprehensively map the molecular context of

Xiaonan Liu1,2, Kari Salokas1,2, Fitsum Tamene1,2,3 and Markku Varjosalo1,2,3*

1Institute of Biotechnology, University of Helsinki, Helsinki 00014, Finland 2Helsinki Institute of Life Science, University of Helsinki, Helsinki 00014, Finland 3Proteomics Unit, University of Helsinki, Helsinki 00014, Finland *Corresponding author

Abstract: -protein interactions underlie almost all cellular functions. The comprehensive mapping of these complex cellular networks of stable and transient associations has been made available by affi nity purifi cation mass spectrometry (AP-MS) and more recently by proximity based labelling methods such as BioID. Due the advancements in both methods and MS instrumentation, an in-depth analysis of the whole human proteome is at grasps. In order to facilitate this, we designed and optimized an integrated approach utilizing MAC-tag combining both AP-MS and BioID in a single construct. We systematically applied this approach to 18 subcellular localization markers and generated a molecular context database, which can be used to defi ne molecular locations for any protein of interest. In addition, we show that by combining the AP-MS and BioID results we can also obtain interaction distances within a complex. Taken together, our combined strategy off ers comprehensive approach for mapping physical and functional protein interactions.

Introduction: Majority of proteins do not function in isolation geting the endogenous bait protein, allowing and their interactions with other proteins defi ne purifi cation of the bait protein together with the their cellular functions. Therefore, detailed under- associating proteins (preys). This approach has standing of protein-protein interactions (PPIs) is been proven well suited for even large-scale high- the key for deciphering regulation of cellular net- throughput studies, and to yield highly repro- works and pathways. During the last decade, the ducible data in both intra- and inter-laboratory versatile combination of affi nity purifi cation and usage2. The most commonly used epitope tags in mass spectrometry (AP-MS) revolutionized the medium to large-scale studies include FLAG3, His4, detailed characterization of protein complexes MYC5, HA6, GFP7and Strep8, of which the Strep-tag and protein-interaction networks1. The AP-MS has become the gold-standard in affi nity purifi - approach relies on expression of a bait protein cation proteomics due to unparalleled protein coupled with an epitope tag or antibodies tar- purity in physiological purifi cation conditions as

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An integrated approach to comprehensively map the molecular context of proteins

well as the possibility for native competitive elu- we included as well a nine hemagglu- tion using biotin. tinin (HA)-epitope. The HA-epitope also facilitates AP-MS can also be combined with quantitative additional follow-up approaches such as ChIP- proteomics approaches to better understand the Seq17 and purifi cation of the crosslinked proteins protein complex stoichiometry9 and the dynamics for cross-linking coupled with mass spectrometry of protein–complex (dis)assembly1,10. The combi- (XL-MS)18, making the MAC-tag almost as versatile nation of AP-MS with other techniques, such as as the Swiss Army knife. biochemical fractionation, intact mass measure- To benchmark the usability and performance of ment and chemical crosslinking11,12, has been used the MAC-tag we applied it to 18 bona fi de subcel- to characterize supramolecular organization of lular localization marker proteins. This allowed us protein complexes. to validate the correct localization of the MAC- Although AP-MS remains the most used tagged marker proteins as well as to monitor the method for mapping protein-protein inter- localization of the in vivo biotinylated interactors. actions, the recently developed proximity Additionally the interactions provide new infor- labeling approaches, such as BioID13 and APEX14, mation about these 18 marker proteins and their have become complementary and somewhat cellular functions. Furthermore the 18 localization competing methods. BioID involves expression markers and their 1911 interactions, form a basis of the protein of interest fused with a prokary- of the reference molecular context repository, otic biotin ligase (BirA) and the subsequent which we show can be used for “mass spectrom- biotinylation of the amine groups of the neigh- etry (MS) microscopy” analysis of any protein of boring proteins when excess of biotin is added interest. The combinatory analysis with AP-MS and to the cells. Whereas the wild-type BirA from E. BioID also provided information, which effi ciently Coli is capable of transferring the biotin only to a could be used to derive relative spatial distances substrate bearing a specifi c recognition sequence, for proteins in a complex. Taken together, our the generation of a promiscuous BirA* (Arg118Gly devised combinatory MAC-tag and analysis mutant) allows the biotinylation of any protein approaches around it provide a plethora of infor- found within a 10 nm labeling radius13,15. While mation of the cellular functions and the molecular BioID has the abilities to capture weak and/or context of any studied protein. transient protein-protein interactions, the identi- fi ed interactions are not limited to direct binders RESULTS but can include proximate proteins as well. MAC- tag AP-MS and BioID pipeline for In order to avoid artefactual interactions detection of physical and functional caused by overexpression of the bait proteins, interactions majority of the large-scale interaction proteomic studies employ the Flp-In™ T-REx 293 line To generate a versatile approach for identifi- allowing moderate and inducible bait protein cation of both stable physical and transient expression from isogenic cell clones16. Although functional protein-protein interactions we inte- the system allows rapid generation of transgene grated and optimized the BioID approach with 10,23 stably expressing cell lines, comprehensive our single-strep Strep AP-MS pipeline . Both analyses utilizing complementarily both AP-MS of these approaches have become the method and BioID is resource-intense in the respect of cell of choice for interactomics analyses. We have line generation. To address this caveat and allow recently shown the eff ectiveness of using these 10,23 high-throughput comprehensive interactome approached complimentarily . However, the analyses, we generated a Gateway®-compatible complementary use of the two techniques has MAC (Multiple Approaches Combined) -tag been labor-intense, involving tagging of the bait enabling both the single-step Strep AP-MS as well proteins with BirA* and Strep-tag individually, as the BioID analysis with a single construct, which as well as generation of two set of cell lines per decreases the number of required individual cell bait. To overcome the major limitations, we have lines by 50%. In addition to allow visualization of developed an integrated experimental workfl ow tagged bait protein by immunohistochemistry, utilizing a MAC-tag containing both StrepIII-tag

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Liu et al.

and BirA* (Supplementary Fig. 1a). In addition to included: mitochondria (-inducing fac- optimizing the experimental steps, we focused tor 1, AIFM1); (Calnexin, on the compatibility of the two methods and to CALX); peroxisome (Catalase, CATA); early endo- the simplicity of the analysis pipelines to gener- some (Early endosome antigen 1, EEA1); cytoplas- ate a process with improved performance and mic peripheral plasma membrane marker (Ezrin, reproducibility on detecting protein-protein EZRI); nucleolus marker (rRNA 2’-O-methyltrans- interactions. The two pipelines diff er only in the ferase fi brillarin, FBRL); cis-Golgi marker (Golgin activation of the BirA* by addition of biotin to the subfamily A member 2, GOGA2); chromatin (His- cell culture media and harsher lysis condition in tone H3.1, H31); exosome (Heat shock cognate 71 the BioID pipeline (Fig. 1, Supplementary Fig. 1). kDa protein, HSP7C); lysosome (Lysosome-associ- Without biotin addition the BirA* in the MAC-tag ated membrane glycoprotein 1, LAMP1); nuclear remains inactive (Supplementary Fig. 1b, c), result- envelope marker (Prelamin-A/C, LMNA); protea- ing in identical (cor=0.88-0.99) single-step affi nity some (Proteasome subunit alpha type-1, PSA1); purifi cation results as vector with only StrepIII-tag recycling endosome (Ras-related protein -11A, (Supplementary Fig. 1d, e). Similarly, when biotin RAB11A); late endosome (Ras-related protein Rab- was added the results compare (cor=0.95-0.97) to 9A, RAB9A); ( alpha-1A chain, that of a vector with BirA* alone (Supplementary TBA1A) centrosome (Tubulin gamma-1 chain, Fig. 1d, e). TBG1); trans-Golgi (Trans-Golgi network integral The developed integrated approach signifi- 2, TGON2); and ribosome (40S cantly enhances (by two-fold) the throughput ribosomal protein S6, RS6) (Supplementary Table of generating bait‐expressing cell lines, facili- S1). All of the 18 MAC-tagged marker proteins tates a comprehensive analysis of protein-protein localized to their corresponding cellular compart- interactions utilizing both the BioID and AP-MS, ments, illustrating that the MAC-tag or the acti- and allows analysis of protein complexes and vation of the BirA* does not change the correct even transient functional interaction networks localization of these proteins. Furthermore, the with high sensitivity and reproducibility. Addi- localization of the in vivo biotinylated interactors tionally the MAC-tag allows visualization of the correlates well with that of the corresponding bait protein with anti-HA antibody detecting localization marker. In addition to verifying the the HA-epitope. This versatility of our approach correct localization of marker proteins, the results was expected to give detailed view on the bait highlight the usability of our MAC-tag constructs protein formed complexes, interactions, and for fluorescence microscopy on detecting both actual molecular context via the detected stable, the tagged-protein of interest as well as the inter- transient and/or proximal-interactions. acting proteins.

Validation of the correct localization of the Identifying the physical and functional cellular localization markers and their in interactions of the cellular localization vivo biotinylated interactors. markers We then went on and evaluated the MAC-tag sys- Although many proteins and proteins families tem with 18 bona fi de cellular localization mark- have been extensively studied with wide-range ers (Supplementary Table 1) that cover most of of cell biological or biochemical methods, oth- cellular organelles to have more comprehensive ers and we have shown the AP-MS and BioID can view of the application of our integrated multiple reveal wealth of new molecular and functional approach system. Initially 18 localization markers information10,24. However, for many proteins not were cloned to the MAC-tag vector, as a fi rst step much is known and there has not been systematic we explored their localization using fl uorescence methods to effi ciently and comprehensively char- microscopy. The tagged-localization markers acterize them. As shown in Fig. 2, the resolution of were visualized with anti-HA antibody and the in standard fl uorescence microscopy does not allow vivo biotinylated interactors with Alexa Fluor 594 capturing information of the protein dynamic -Streptavidin (Fig. 2). These subcellular markers localization and molecular context. Therefore, we

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An integrated approach to comprehensively map the molecular context of proteins

MAC-tagged 18 known cellular localization mark- chromatin organization, and framework for the ers and subjected them to our integrated method nuclear envelope and telomere dynamics. Not to obtain detailed molecular context proteome surprisingly both the AP-MS and BioID identify map with information from both the physical interactions with -B (LMNB)1, LMNB2, and functional interactions formed by these pro- lamin-B receptor (LBR), lamina-associated poly- teins. The analysis resulted in 26527 interactions peptide (LAP)2A and LAP2B, inner nuclear from BioID and 9390 from AP-MS, of which 2118 membrane protein Man1 (MAN1), and emerin high-confi dence interactions from BioID and 679 (EMD). Another group of interacting proteins interactions from AP-MS were retained after using are nuclear pore complexes (NPCs) components: stringent statistical filtering (Fig. 3a-c, Supple- nuclear envelope pore membrane protein POM mentary Table 1). The identifi ed average connec- (P121A, P121C), Nuclear pore complex protein tivity (38) of the 18 localization markers, identifi ed Nup (NU) 107, NU133, NU153, NU155, NU160, using AP-MS, matches well with the published NUP50, NUP85, NUP98, NUP37, NUP43, nucleo- large-scale studies10,25. As the BioID is also able to porin SEH1, nucleoprotein TPR, ELYS, SEC13, of capture highly transient and close-proximity inter- which only components of the nuclear basket actions, the total number of identifi ed interactors NUP50, TPR, SEC13 are detected of low abundance as well as interactions per bait is higher than that with AP-MS (Fig. 3d, and Supplementary Table 1c). of AP-MS (Fig. 3a, b). This is seen for example with This suggests that for the correct localization to Rab9A and Rab11A, two regulators of endosomal the nucleoplasmic side of the nuclear envelope, transport, for which BioID provides 16 times and LMNA needs to pass through nuclear pore and 11 times more high-confi dence interacting pro- during this process it transiently interacts and in teins (HCIPs) than AP-MS, respectively (Supple- vivo biotinylates the nuclear pore complex (NPCs) mentary Fig. 2). In this case, the proteins detected components. Similarly, the importin transport solely with BioID likely represent cargo proteins in proteins importin subunit alpha (IMA1, IMA3, endosomal transit. Interestingly the ratio of identi- IMA4, IMA5, IMA6, IMA7), importin subunit beta- fi ed novel vs. known interactions in total is almost 1(IMB1) are detected with BioID, but only IMA5 two-fold higher with BioID (11.3) than with AP-MS and IMA7 in AP-MS. In addition several histone (6.8), refl ecting the sensitivity of BioID to identify modifi ers and chromatin remodelers are detected more transient interactions (Fig. 3a). (AN32E, EDF1, MYSM1 and PARP1). Somewhat However, the complementary nature of these surprisingly our analysis also identifies several two methods is illustrated by their overlap as well proteins involved in cell cycle and mitosis (RAD50, as with their individually detected interactions, ARF, KI67, HDGR2, WRIP1, AKP8L, BCCIP and P53). such as the ones formed by proteosomal marker Both of the examples show that the detected PSA126 and nuclear envelope marker LMNA (Fig. 3c, high-confi dence interacting proteins are highly d). With PSA1 the overlap of AP-MS (green edges) specifi c for the studied location, as illustrated by and BioID (yellow edges) identifi ed interactions the retrieval of the HCIPs localization informa- is 17 components of the 20S core proteasome tion from CellWhere database 29 (Fig. 3c, d). The complex involved in the proteolytic degradation proteins with the highest ranking for the particular of most intracellular proteins (Fig. 3c, Supple- location from CellWhere are shown in dark green mentary Table 1c). BioID also captures and for the rest of the ranks the node color is light (MYH10 and MYH14) and unconventional myosins green. Proteins with no CellWhere ranking are (MYO1B-D and -6), which have high turnover rates shown in grey. and after use they are either refolded for reuse or degraded by the proteasome 27. Additionally, Reference molecular context proteome map BioID identifies proteasome activator complex reveals unique profi les for diff erent cellular subunits 1 (PSME1) and 2 (PSME2), which are part organelles of the 11S (PA28) immunoproteasome28. In addition to lacking molecular level resolution, LMNA is a component of the nuclear lamina, standard fl uorescence microscopy is often used playing an important role in nuclear assembly, to produce static images representing the par-

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Liu et al.

ticular time point when the image is taken. How- fact that protein translation requires ribosomes, ever, cellular proteins are highly diverse in their which are after the synthesis of the MAC-tagged spatiotemporal properties, thus making their protein immediately in vivo biotinylated in the characterization with microscopy alone extremely BioID approach. Therefore the ribosome (RS6) challenging. The BioID, in principle, overcomes was excluded from the further analyses. However, these limitations as monitoring of the biotinylated the other localization markers, such as mito- close-proximity proteins and their quantities chondria (AIFM1), cytoplasmic peripheral plasma should allow defi ning the BirA* -tagged bait pro- membrane (EZRI), exosome (HSP7C), peroxi- teins detailed molecular context within certain some (CATA), microtubule (TBA1A), proteasome time period (Supplementary Fig. 2). Using the 2118 (PSA1) and centrosome (TBG1) had highly unique high-confi dence interactions from BioID, we gen- molecular context signature, which suggest the erated a cellular compartment-specific protein usability of this reference set in tracking of protein interaction map to the 18 bona fi de localization of interests dynamic localization in intracellular markers (Fig. 3e and Supplementary Table1). The environment. Additionally comparison of the HCIPs domain as well as the ontology (GO) HCIPs cellular locations from CellWhere database term profi les for each marker were unique (Sup- showed them to be assigned to the correct cellular plementary Table 2 a-f and Supplementary Fig. localization according to their bait protein, further 3, 4). However, we identified also shared HCIs reinforcing the idea that proteins that share their between the endomembrane system consist- interaction profi les are proximal. Finally, total of ing of ER (CALX), the Golgi (GOGA2 and TGON2), 1911 HCIs (excluding RS6 interactions), collapsed endosomes (EEA1, RAB9A and RAB11A) and lyso- to 14 subcellular localizations were integrated to some (LAMP1). The four organelles shared 17 build up the reference molecular context map interactors, and the combination of any three (Fig. 3e). Overlaying of any protein of interests locations shared in total 87 interactions (Sup- BioID PPIs with our molecular map should allow plementary Fig. 5a). These four organelles are defi ning the dynamic localization of the protein. In involving in two major intracellular trafficking principle the developed MS-microscopy approach pathways: The exocytic pathway (ER via Golgi (53 could have high impact on cellular quantitative shared interactions) to the plasma membrane); biology (Fig. 4a). and the endocytic pathway (plasma membrane via endosomes to 1) Golgi (101 interactions) and 2) Mass spectrometry microscopy using the lysosome (61 interaction) to ER (86 interactions)). reference molecular context proteome map This organization is also well visible with, within a Despite the biological significance of dynamic cell, the physically farthest from each other locat- subcellular localizations of proteins, simple tools ing endosome and ER, sharing the least interac- for detecting the relative subcellular distribution tions of the all possible binary combinations of the have not been extensively developed. To test four locations. the applicability of the MS-microscopy on this, Similarly, chromatin, nucleolus and nuclear we selected dynamic cytoplasmic signaling mol- envelope are all sub-structures in the nucleus and ecules aurora kinase B (AURKB), cyclin-dependent shared interactors with each other (Supplemen- kinase (CDK) 7, CDK8, and glycogen synthase tary Fig. 5b); nuclear envelope (LMNA) 22 with kinase-3 beta (GSK3B), as well as additional mark- chromatin (H31) and chromatin 31 with nucleolus ers for cellular locations ras-related protein Rab-5A (FBRL). We previously already discussed the role (RAB5A) and Golgi vesicular membrane-traffi ck- of nuclear envelope on chromatin organization, ing protein p18 (BET1) and applied our approach and chromatin control the structure of nucleolus to them (Fig. 4, Supplementary Table 1a-c, and via ribosome DNA. Nucleolus is the place where Supplementary Fig. 6). Aurora kinase plays an ribosomal RNA transcription and the ribosome important role in cellular division by controlling assembly occur. Ribosome (RS6) was detected as chromatic segregation, which matches well to its an outlier as it shared many of the interactors with interactions overlaying with “chromatin” marker other localization markers. This is explained by the H31 (Fig. 4b, c, d). Similarly SNARE protein BET1,

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An integrated approach to comprehensively map the molecular context of proteins

involved in the docking process of ER-derived trons directly from ubiquinol to oxygen in a non- vesicles with the cis-Golgi membrane is assigned proton-motive manner36. Transgenic expression of to Golgi (Fig. 4b, c, d). Essential component of the AOX in mammalian systems has been suggested transcription factor II H (TFIIH), CDK7, and media- as a therapeutic option for treating mitochondrial tor complex associating CDK8 are predominantly disease induced by OXPHOS dysfunction37, and associating with “chromatin” (Fig. 4b, c, d). This additionally it has been shown that even broad fi nding is in line with their important role in tran- expression of AOX does not disturb normal physi- scription regulation. Importantly, these examples ology in mice38. The exact molecular context of show the high resolution of the MS-microscopy AOX in mitochondria membrane is not currently to distinct exact molecular locations, which could known, but it is thought to locate close to com- not be resolved by fl uorescence microscopy (Sup- plex II based on its alleviating eff ects after toxic or plementary Fig. 6). Glycogen synthase kinase β pathological inhibition of the mitochondrial res- (GSK3B) phosphorylates many substrates in mam- piratory chain39. Therefore, we decided to apply malian cells, and functions in many physiological our MS-microscopy method to defi ne the molecu- processes, and acts as an important regulator in lar context of Ciona intestinalis’ AOX in human cells Wnt and Hedgehog signaling pathways30. Some- and possibly shed some light on its interactions what, to our surprise our MS-microscopy showed with respiratory chain. Our approach identifies GSK3B localization to Golgi and exosomes. Recent AOX predominantly to localize to mitochondria research have demonstrated that a portion (Fig. 5a, Supplementary Fig. 6). More specifi cally, of GSK3B is localized to the trans-Golgi network >90% of AOX interactors (Fig. 5b) belong to mito- through peripheral protein p23031 and that cyto- chondria according to CellWhere database and plasmic GSK3B relocalizes to the same endosome 48% among them have the GO term annotation as the internalized Wnt ligand32. It is plausible of mitochondria inner membrane. Furthermore, that this colocalization of GSK3B continues with 38 of the interactors are components of the mito- active Wnt through endosomal organelles onto chondrial respiratory complexes I-V. From these exosomes33. For validation of our endosomal loca- AOX prefers interactions with complex II (2/4 com- tion, we choose RAB5A which is known to localize ponents detected), complex I (19/44) and com- to early endosomes and is involved in the recruit- plex V (9/19) (Fig. 5c), which is also visible from ment of RAB7A and the maturation of these orga- the quantitative interactor abundance (Fig. 5d). nelles to late endosomes34. In our analysis we can Therefore, our MS-microscopy findings confirm confi rm the (early)-endosomal location as well as the functionally suggested location of transgenic detect a fraction of Golgi localization, which could AOX in human cells. be related to the fusion of trans-Golgi network- derived vesicles with the early endosome35. These Defi ning interaction distances within a examples clearly establish the sensitivity and protein complex applicability of the MS-microscopy in defi ning the Others and we have shown that AP-MS offers molecular context of a(ny) protein. In addition to accurate quantifi cation of complex composition analysis of wild type proteins our system should allowing calculations on complex stoichiom- be useful for defining possible altered molecu- etry9,10,40. With BirA* the labeling radius is limited lar context in human diseases caused by either (circa 10 nm), and it has been used to obtain rough somatic or germline genetic alteration, as well maps of spatial distribution of proteins within as for example analyzing functions of transgenic structures by reciprocally analyzing BirA*-tagged proteins not expressed in human cells. proteins throughout the structure15. As the in vivo biotinylation is enzymatic reaction deriving rela- Transgenic Alternative Oxidase localizes tional interaction abundances of participants can- in the mitochondria intermembrane and not be done. However, it can be reasoned that the functionally associates with Complex I-V more proximal proteins will be more efficiently Alternative oxidase (AOX) present in many lower biotinylated and purified in larger abundances eukaryotes, but not in vertebrates, transfer elec- than proteins further away15,41. On the AP-MS side

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Liu et al.

this would correlate with the likelihood of more limits. The Mediator complex is composed of 4 abundant interactors being more direct than low modules, the head, the middle, the tail and the abundant, in which the interaction could be medi- kinase module. The evolutionarily conserved and ated by other proteins and the interaction with dissociable kinase module is formed by CDK8 the bait would be secondary or tertiary etc. There- together with cyclin C (CCNC), mediator of RNA fore, by blotting both the BioID and AP-MS data, polymerase II transcription subunit mediator in theory, we could obtain relative distance of the complex subunit (MED) 12 and 13, (Fig. 6e)51-53. MAC-tagged bait protein to its interacting pro- To test and validate the reproducibility of our tein in a complex. For testing this hypothesis, we approach, in addition to CDK8, we addition- selected CDK7 and CDK8 for which we have previ- ally choose MED13 for analysis. Additionally this ously identifi ed successfully quantitative complex would allow more accurate prediction of the compositions25. kinase-module docking surface to the Mediator We applied our dual-approach, and with both core complex. To our surprise the overall corre- AP-MS and BioID we could detect the CDK7 lation of CDK8 and MED13 distances from the interactions with TFIIH core components (Fig. Mediator core is extremely high (c=0.95) (Fig. 6a)42-44. The size of the TFIIH is estimated to be 6b, c, g), confi rming that these two proteins are ~10 nm42, which is still within the BirA* biotinyla- highly proximal. Based on the analysis the closest tion range and should allow measurement of the Mediator subunits for both CDK8 and MED13 are CDK7 interaction distances for all of the complex MED12, MED14, MED1, MED24, MED23, MED17, components. Before associating with TFIIH, CDK7 MED15, MED27, MED16 and MED6/453. This associates and forms cyclin-dependent kinase suggests that the kinase module is docking hori- (CDK)-activating kinase (CAK) complex with two zontal to the MED14 ranging from RM1 and RM2, regulatory subunits; cyclin H (CCNH) required the two repeats of a structural domain on MED14. for CDK7 activity and with RING fi nger protein Both the CDK7, CDK8 and MED13 studies CDK-activating kinase assembly factor MAT1 benchmark another utility of our MAC-tag system and shows that by integration of AP-MS and BioID (MAT) which modulates the substrate specifi city it is possible to derive information on complex 45 of the complex . In agreement, both CCNH and structure, interaction distances and possible MAT1 are detected as closest to CDK7, followed distance constraints. by ERCC2, ERCC3 and TFIIH1. The ERCC2, TFIIH basal transcription factor complex helicase XPD Discussion subunit is the bridge linking the CAK module with TFIIH ring-like core and has been shown to In this study, we developed and optimized an directly interact with TFIIH basal transcription integrated workflow based around MAC-tag, factor complex helicase XPB subunit (ERCC3)46 for characterization of the molecular context and TFIIH147. CDK7 also has been reported to of any protein of interest from human cells. This directly interact with TFIIH1 (Fig. 6d)48,49. Our workflow features state-of-the art affinity puri- results are in line with both hypothesis, the fi cation using Strep-tag to identify and quantify evidence suggesting that TFIIH1 and ERCC3 have protein-protein interaction and protein complex short inter-distances as well as that they both are stoichiometry; identifi cation of transient or close- close to the CAK module (Fig. 6d, f). The core ring proximity interactions with BioID; visualization of structure of TFIIH composed of TFIIH2-4, CDK7 are the bait protein and the proximal interactors with located adjacent, having highly similar distance to immunofluorescence microscopy; and defining CDK7 (Fig. 6a, f). Similarly, ERCC5 and TFIIH5 are in the molecular context with MS-microscopy utiliz- longer distances from CDK7, suggesting that they ing the reference dataset obtained by identify- are located on the opposite side of the complex ing proximal interactors for bona fi de subcellular from CAK (Fig. 6a, d, f). localization markers. Additionally our integrated The transcriptional co-activator Mediator workfl ow reduces the generation of the required complex has more than 30 subunits and is ~30 cell lines for APMS and BioID to half. nm in size50, and therefore on the upper detection

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An integrated approach to comprehensively map the molecular context of proteins

In addition to analyzing the physical and func- Methods tional interactions formed by 18 cellular localiza- Generation of MAC-tag Gateway® tion markers, we used our integrated workfl ow destination vectors to map interactions for four kinases (AURKB, CDK7, CDK8 and GSK3B), as well as for two addi- To generate Gateway compatible destination tional localization markers (BET1 and RAB5A). In vectors, plasmids containing the tags (C terminal: addition to identifying 539 interactions for these StrepIII/HA/BirA*, N terminal: BirA*/HA/StrpIII) six proteins, we could validate the accuracy of the were synthesized by GeneArt®, Life Technologies. MS-microscopy method for identifying correct These were digested with restriction enzymes cellular localization for these proteins. Further- and inserted into N terminal: pcDNA5/FRT/TO/ 19 more, we could show with an exogenous protein, StrepIII/HA/GW or C terminal: pcDNA5/FRT/TO/ 2 AOX that our MS-microscopy correctly identi- StrepIII/HA/GW in which entire StrepIII/HA tag fi es AOX to localize to mitochondria. Additional was removed. All the Gateway compatible entry analysis using our integrated workfl ow shows that clones, which contain subcellular marker gene of AOX localizes to inner mitochondrial membrane interested, were from Human ORFeome collec- and is in close-proximity with Complex II. Our tion. fi ndings validate, for the fi rst time, the functionally suggested vicinity of AOX with Complex II. Immunofl uorescence Identifying the complex components in a stoi- HeLa cells (American Type Culture Collection) chiometry fashion has been shown to be possible were transfected with vectors containing MAC- with affi nity purifi cation mass spectrometry9,10. tagged gene of interest and cultured either with However, obtaining any further spatial information or without supplemental biotin. Bait proteins of the complex formation has only been possibly were detected with anti-HA antibody, followed by in combination with XL-MS18. We could now show Alexa Fluor488-conjugated secondary antibody. with the TFIIH and Mediator complex as model Biotinylated proteins were detected with Alexa- complexes, that by utilizing both the AP-MS and Fluor 594 streptavidin. DAPI staining was used to BioID approaches we can obtain relative interac- determine the nuclei. Wide fi eld fl uoresce micro- tion distances for proteins in a complex. Based on scope (Leica DM6000, Leica) with HCXPL APO the interaction distances it is possible to obtain an 63x/1.40-0.60 oil objective was used to image the estimate for the interaction surfaces for proteins samples. The image fi les were processed with LAS or structures, such as with the kinase submodule X (Leica), and ImageJ softwares. of the Mediator complex. In summary, our study showed that the inte- Cell culture grated workflow and the reference molecular For generation of the stable cell lines inducibly context proteome map generated here, allows expressing the MAC-tagged versions of the baits, an easy way to probe the molecular localization Flp-In™ 293 T-REx cell lines (cultured in DMEM (4.5 of protein of interest, and additionally an online g/L , 2 mM L-glutamine) supplemented resource of our BioID based MS-microscopy with 10% FBS, 50 mg/mL penicillin, 50 mg/mL approach is available at http. The “molecular streptomycin) were co-transfected with the image” obtained from the MS-microscopy analysis expression vector and the pOG44 vector (Invit- considers the weights of interactors and provides rogen) using the Fugene6 transfection reagent more dynamic localization information at the (Roche Applied Science). Two days after trans- molecular level. The developed MAC-tag and the fection, cells were selected in 50 mg/mL strepto- integrated approach should empower, not only mycin and hygromycin (100 μg/mL) for 2 weeks, the interaction proteomics community, but also and then the positive clones were pooled and cell biologist, with an experimentally proven inte- amplifi ed. Stable cells expressing MAC-tag fused grated workfl ow for mapping in detail the physical to green fl uorescent protein (GFP) were used as and functional interactions and the molecular negative controls and processed in parallel to the context of any protein in human cells. bait proteins.

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Liu et al.

Each stable cell line was expanded to 80% and reconstituted to a fi nal volume of 30 μL in

confl uence in 20 × 150 mm cell culture plates. 0.1% TFA and 1% CH3CN. Ten plates were used for AP-MS approach, in which 1 μg/ml tetracycline was added for 24 h Liquid chromatography–mass spectrometry induction, and ten plates for BioID approach, in (LC-MS) which in addition to tetracycline, 50 μM biotin Analysis was performed on a Q-Exactive mass was added for 24 h before harvesting. Cells from spectrometer using Xcalibur version 3.0.63 cou- 5 × 150 mm fully confl uent dishes (~ 5 × 107 cells) pled with an EASY-nLC 1000 system via an electro- were pelleted as one biological sample. Thus, each spray ionization sprayer (Thermo Fisher Scientifi c). bait protein has two biological replicates in two In detail, peptides were eluted and separated with diff erent approaches. Samples were snap frozen a C18 precolumn (Acclaim PepMap 100, 75μm x and stored at − 80 °C. 2cm, 3 μm, 100 Å, Thermo Scientifi c) and analyti- cal column (Acclaim PepMap RSLC, 50 μm × 15 cm, Affi nity purifi cation of the interacting 2 μm, 100 Å; Thermo Scientifi c), using a 60 min- proteins ute buff er gradient ranging from 5 to 35% buff er For AP-MS approach, the sample was lysed in B, followed by a 5 min gradient from 35 to 80% 3 ml of lysis buff er 1 (0.5% IGEPAL, 50 mM Hepes, buffer B and 10 min gradient from 80 to 100%

pH 8.0, 150 mM NaCl, 50 mM NaF, 1.5 mM NaVO3, buffer B at a flow rate of 300 nl/min (buffer A: 5 mM EDTA, supplemented with 0.5 mM PMSF 0.1% formic acid in 98% HPLC grade water and 2% and protease inhibitors; Sigma). acetonitrile; buff er B: 0.1% formic acid in 98% ace- For BioID approach, Cell pellet was thawed in tonitrile and 2% water). For direct LC-MS analysis, 3 mL ice cold lysis buff er 2 (0.5% IGEPAL, 50 mM 4 μl peptide samples were automatically loaded Hepes, pH 8.0, 150 mM NaCl, 50 mM NaF, 1.5 mM from an enclosed cooled autosampler. Data-

NaVO3, 5 mM EDTA, 0.1% SDS, supplemented with dependent FTMS acquisition was in positive ion 0.5 mM PMSF and protease inhibitors; Sigma). mode for 80 min. A full scan (200-2000 m/z) was Lysates were sonicated, treated with benzonase. performed with a resolution of 70,000 followed Cleared lysate was obtained by centrifuga- by top10 CID-MS2 ion trap scans with resolution tion and loaded consecutively on spin columns of 17,500. Dynamic exclusion was set for 30 sec- (Bio-Rad) containing lysis buffer 1 prewashed onds. Acquired MS2 spectral data files (Thermo. 200 μl Strep-Tactin beads (IBA, GmbH). The beads RAW) were searched with Proteome Discoverer were then washed 3 x 1 ml with lysis buff er1 and 1.4 (Thermo Scientific) using SEQUEST search 4 × 1 mL with wash buff er (50 mM Tris-HCl, pH 8.0, engine of the selected human component of Uni- 150 mM NaCl, 50 mM NaF, 5 mM EDTA). Following ProtKB/SwissProt database (http://www.uniprot. the fi nal wash, beads were then resuspended in org/, version 2015-09). The following parameters 2 x 300 μL elution buff er (50 mM Tris-HCl, pH 8.0, were applied: Trypsin was selected as the enzyme 150 mM NaCl, 50 mM NaF, 5 mM EDTA, 0.5mM and a maximum of 2 missed cleavages were per- Biotin) for 5 mins and eluates collected into an mitted, precursor mass tolerance at ±15 ppm and Eppendorf tubes, followed by a reduction of the fragment mass tolerance at 0.05 Da. Carbamido- cysteine bonds with 5 mM Tris(2-carboxyethyl) methylation of cysteine, was defined as static phosphine (TCEP) for 30 mins at 37 °C and alkyla- modifi cations. Oxidation of methionine and bioti- tion with 10 mM iodoacetamide. The proteins nylation of and N-termini were set as vari- were then digested to peptides with sequencing able modifi cations. grade modifi ed trypsin (Promega V5113) at 37 °C overnight. After quenching with 10% TFA, the Identifi cation of the HCIs samples were desalted by C18 reversed-phase Significance Analysis of INTeractome (SAINT) spin columns according to the manufacturer’s express version 3.6.020 and Contaminant Reposi- instructions (Harvard Apparatus). The eluted tory for Affinity Purification (CRAPome, http:// peptide sample was dried in vacuum centrifuge www.crapome.org/)21 were used as statistical tools for identifi cation of specifi c high-confi dence

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An integrated approach to comprehensively map the molecular context of proteins

interactions from our AP-MS data. 16 GFP con- PSM values of all bait interactors that match any trol runs (8 N-terminal MAC-GFP and 8 C-terminal localization group. This is then multiplied by the MAC-GFP) were used as control counts for each sum of PSM values of interactors of the localization hit and the fi nal results only considering proteins group that match the bait of interest interactors with SAINT score ≥0.73. This corresponds to an divided by the sum of PSM values of all interactors estimated protein-level Bayesian FDR of <0.05. of the localization group. The score refl ects sub- Furthermore, we used the CRAPome database cellular localization by numerically describing the with a cut-off frequency of ≥20% (≥82) except similarity of the subcellular environment and the the average spectral count fold change ≥ 3 was time spent there between the bait of interest and set for assigning high confidence interactions each localization group. (HCIs). Clustering analysis Prey protein frequency count matrix was generated using DAVID gene Results visualization functional classifi cation tool to provide the (GO) terms (domains, biological process The MS-microscopy analyses are presented as and molecular function). The p-values associated polar plots with in-house python script, where with each annotation terms has p< 0.01. the circle has been equally divided into 14 sectors, Hierarchical cluster was performed by centered each sector representing one specifi c subcellular correlation (both baits and interactors; average location. Diff erent colored sector areas indicates linkage) using Cluster 3.0 and the clusters the possible location score of query bait, with were visualized with Tree View 1.1.6 and the scores between 0 and 0.5 marked in red, between matrix2png web server (http://www.chibi.ubc.ca/ 0.5 and 0.75 in yellow, and 0.75 and 1 in green. matrix2png/). Online interface Networks and maps We have developed web application (R-shiny; Protein interaction networks are constructed from http://www.biocenter.helsinki.fi /bi/protein/ SAINT data that were imported into Cytoscape msmic) for observation of protein localization by 3.2.122. The Known prey-prey interaction data MS-microscopy. A user can upload an input fi le were obtained from PINA2 database (http://omics. (after SAINT and CRAPome fi ltering) and visualize bjcancer.org/pina/) the bait protein dynamic localization. The image as well as a parsed data can also be downloaded. MS-microscopy database construction Determining relative intra molecular The high-confi dence interacting proteins (HCIPs) distances of the protein complexes obtained from previous fi ltering steps were sorted according to the corresponding bait protein The PSMs of preys were normalized with interact- localization information to build the reference ing bait PSM abundance respectively. The scatter database, containing the following localization plots were based on the normalized PSMs from information: peroxisome, microtubule, endosome both BioID and AP-MS approach. Then the Euclid- (combined: early, late and recycling endosome), ean distances were calculated between bait to proteasome, nuclear envelope, Golgi (combined: preys according to the scatter plot. Subsequently, Trans- and Cis-Golgi), lysosome, nucleolus, plasma the correlation was calculated. membrane, endoplasmic reticulum, mitochon- dria, centrosome, chromatin, exosome. Data deposition Mass spectrometry data are available at the Pep- Score calculation tideAtlas (http://www.peptideatlas.org/) raw data Final localization scores for all localization groups repository (PASS01076). Interactome data will be for given bait of interest were calculated by divid- available at the IntAct database. ing the sum of peptide-spectrum match (PSM) values of the interactors that match between bait of interest and a localization group by the sum of

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Liu et al.

References 13. Roux, K.J., Kim, D.I., Raida, M. & Burke, B. A promiscuous biotin ligase fusion protein iden- 1. Dunham, W.H., Mullin, M. & Gingras, A.C. tifies proximal and interacting proteins in Affinity-purification coupled to mass spec- mammalian cells. J Cell Biol 196, 801-10 (2012). trometry: basic principles and strategies. Prot- eomics 12, 1576-90 (2012). 14. Rhee, H.W. et al. Proteomic mapping of mito- chondria in living cells via spatially restricted 2. Varjosalo, M. et al. Interlaboratory reproduc- enzymatic tagging. Science 339, 1328-31 ibility of large-scale human protein-complex (2013). analysis by standardized AP-MS. Nat Methods 10, 307-14 (2013). 15. Kim, D.I. et al. Probing nuclear pore complex architecture with proximity-dependent bioti- 3. Einhauer, A. & Jungbauer, A. The FLAG peptide, nylation. Proc Natl Acad Sci U S A 111, E2453-61 a versatile fusion tag for the purifi cation of (2014). recombinant proteins. J Biochem Biophys Methods 49, 455-65 (2001). 16. Ward, R.J., Alvarez-Curto, E. & Milligan, G. Using the Flp-In T-Rex system to regulate GPCR 4. Bornhorst, J.A. & Falke, J.J. Purification of expression. Methods Mol Biol 746, 21-37 (2011). proteins using polyhistidine affinity tags. Methods Enzymol 326, 245-54 (2000). 17. Kidder, B.L., Hu, G. & Zhao, K. ChIP-Seq: technical considerations for obtaining high- 5. Kolodziej, P.A. & Young, R.A. Epitope tagging quality data. Nat Immunol 12, 918-22 (2011). and protein surveillance. Methods Enzymol 194, 508-19 (1991). 18. Leitner, A., Faini, M., Stengel, F. & Aebersold, R. Crosslinking and Mass Spectrometry: An 6. Tai, T.N., Havelka, W.A. & Kaplan, S. A broad- Integrated Technology to Understand the host-range vector system for cloning and Structure and Function of Molecular Machines. translational lacZ fusion analysis. Plasmid 19, Trends Biochem Sci 41, 20-32 (2016). 175-88 (1988). 19. Glatter, T., Wepf, A., Aebersold, R. & Gstaiger, 7. Gerdes, H.H. & Kaether, C. Green fl uorescent M. An integrated workfl ow for charting the protein: applications in cell biology. FEBS Lett human interaction proteome: insights into the 389, 44-7 (1996). PP2A system. Mol Syst Biol 5, 237 (2009). 8. Skerra, A. & Schmidt, T.G. Use of the Strep-Tag 20. Teo, G. et al. SAINTexpress: improvements and and streptavidin for detection and purifi cation additional features in Signifi cance Analysis of of recombinant proteins. Methods Enzymol INTeractome software. J Proteomics 100, 37-43 326, 271-304 (2000). (2014). 9. Wepf, A., Glatter, T., Schmidt, A., Aebersold, R. 21. Mellacheruvu, D. et al. The CRAPome: a & Gstaiger, M. Quantitative interaction prot- contaminant repository for affi nity purifi ca- eomics using mass spectrometry. Nat Methods tion-mass spectrometry data. Nat Methods 10, 6, 203-5 (2009). 730-6 (2013). 10. Yadav, L. et al. Systematic Analysis of Human 22. Shannon, P. et al. Cytoscape: a software envi- Protein Phosphatase Interactions and ronment for integrated models of biomo- Dynamics. Cell Syst 4, 430-444.e5 (2017). lecular interaction networks. Genome Res 13, 11. Herzog, F. et al. Structural probing of a protein 2498-504 (2003). phosphatase 2A network by chemical cross- 23. Yao, Z. et al. A Global Analysis of the Receptor linking and mass spectrometry. Science 337, Tyrosine Kinase-Protein Phosphatase Interac- 1348-52 (2012). tome. Mol Cell 65, 347-360 (2017). 12. Jennebach, S., Herzog, F., Aebersold, R. & 24. St-Denis, N. et al. Phenotypic and Interaction Cramer, P. Crosslinking-MS analysis reveals RNA Profi ling of the Human Phosphatases Identi- polymerase I domain architecture and basis of fi es Diverse Mitotic Regulators.Cell Rep 17, rRNA cleavage. Nucleic Acids Res 40, 5591-601 2488-2501 (2016). (2012).

11 bioRxiv preprint doi: https://doi.org/10.1101/264788; this version posted February 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

An integrated approach to comprehensively map the molecular context of proteins

25. Varjosalo, M. et al. The protein interaction 37. Fernandez-Ayala, D.J. et al. Expression of the landscape of the human CMGC kinase group. Ciona intestinalis alternative oxidase (AOX) in Cell Rep 3, 1306-20 (2013). Drosophila complements defects in mitochon- 26. Rubin, D.M. & Finley, D. Proteolysis. The protea- drial oxidative phosphorylation. Cell Metab 9, some: a protein-degrading organelle? Curr Biol 449-60 (2009). 5, 854-8 (1995). 38. Szibor, M. et al. Broad AOX expression in a 27. Kachur, T.M. & Pilgrim, D.B. assembly, genetically tractable mouse model does not maintenance and degradation in muscle: disturb normal . Dis Model Mech 10, Role of the chaperone UNC-45 in myosin thick 163-171 (2017). filament dynamics. Int J Mol Sci 9, 1863-75 39. Hakkaart, G.A., Dassa, E.P., Jacobs, H.T. & Rustin, (2008). P. Allotopic expression of a mitochondrial 28. Livneh, I., Cohen-Kaplan, V., Cohen-Rosen- alternative oxidase confers cyanide resistance zweig, C., Avni, N. & Ciechanover, A. The life to human cell respiration. EMBO Rep 7, 341-5 cycle of the 26S proteasome: from birth, (2006). through regulation and function, and onto its 40. Kean, M.J., Couzens, A.L. & Gingras, A.C. Mass death. Cell Res 26, 869-85 (2016). spectrometry approaches to study mammalian 29. Zhu, L. et al. CellWhere: graphical display of kinase and phosphatase associated proteins. interaction networks organized on subcel- Methods 57, 400-8 (2012). lular localizations. Nucleic Acids Res 43, W571-5 41. Choi-Rhee, E., Schulman, H. & Cronan, J.E. (2015). Promiscuous protein biotinylation by Escheri- 30. Varjosalo, M. & Taipale, J. Hedgehog: functions chia coli biotin protein ligase. Protein Sci 13, and mechanisms. Dev 22, 2454-72 3043-50 (2004). (2008). 42. Schultz, P. et al. Molecular structure of human 31. Adachi, A. et al. Golgi-associated GSK3beta TFIIH. Cell 102, 599-607 (2000). regulates the sorting process of post-Golgi 43. He, Y. et al. Near-atomic resolution visualization membrane traffi cking. J Cell Sci 123, 3215-25 of human transcription promoter opening. (2010). Nature 533, 359-65 (2016). 32. Taelman, V.F. et al. Wnt signaling requires 44. Lolli, G., Lowe, E.D., Brown, N.R. & Johnson, L.N. sequestration of glycogen synthase kinase The crystal structure of human CDK7 and its 3 inside multivesicular endosomes. Cell 143, protein recognition properties. Structure 12, 1136-48 (2010). 2067-79 (2004). 33. Gross, J.C., Chaudhary, V., Bartscherer, K. & 45. Yankulov, K.Y. & Bentley, D.L. Regulation of Boutros, M. Active Wnt proteins are secreted CDK7 substrate specifi city by MAT1 and TFIIH. on exosomes. Nat Cell Biol 14, 1036-45 (2012). Embo j 16, 1638-46 (1997). 34. Huotari, J. & Helenius, A. Endosome matura- 46. Giglia-Mari, G. et al. A new, tenth subunit tion. Embo j 30, 3481-500 (2011). of TFIIH is responsible for the DNA repair 35. Woodman, P.G. Biogenesis of the sorting syndrome trichothiodystrophy group A. Nat endosome: the role of Rab5. Traffi c 1, 695-701 Genet 36, 714-9 (2004). (2000). 47. Drapkin, R. et al. Dual role of TFIIH in DNA 36. Andjelkovic, A. et al. Diiron centre in excision repair and in transcription by RNA Ciona intestinalis alternative oxidase abolish polymerase II. Nature 368, 769-72 (1994). enzymatic activity and prevent rescue of 48. Shiekhattar, R. et al. Cdk-activating kinase cytochrome oxidase defi ciency in ies.fl Sci Rep complex is a component of human transcrip- 5, 18295 (2015). tion factor TFIIH. Nature 374, 283-7 (1995).

12 bioRxiv preprint doi: https://doi.org/10.1101/264788; this version posted February 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Liu et al.

49. Rossignol, M., Kolb-Cheynel, I. & Egly, J.M. Author Contributions Substrate specificity of the cdk-activating M.V. and X.L conceived the study and designed kinase (CAK) is altered upon association with experiments. M.V., X.L., K.S. and F.T. performed TFIIH. Embo j 16, 1628-37 (1997). experiments and data analysis. M.V., X.L., K.S. and 50. Maeshima, K. et al. The physical size of tran- F.T. participated in manuscript preparation. M.V. scription factors is key to transcriptional regu- and X.L. wrote the manuscript. lation in chromatin domains. J Phys Condens Matter 27, 064116 (2015). Acknowledgments 51. Lariviere, L. et al. Model of the Mediator middle We thank Sini Miettinen for technical assistance module based on protein cross-linking. Nucleic and Drs. Tiina Öhman and Salla Keskitalo for criti- Acids Res 41, 9266-73 (2013). cal reading and comments on the manuscript. 52. Tsai, K.L. et al. Subunit architecture and func- This study was supported by grants from the tional modular rearrangements of the tran- Academy of Finland (nos. 288475 and 294173), scriptional mediator complex. Cell 157, Sigrid Jusélius Foundation, University of Helsinki 1430-44 (2014). Three-year Research Grant, Biocentrum Helsinki, Biocentrum Finland, HiLIFE and Instrumentarium 53. Tsai, K.L. et al. Mediator structure and rear- Research Foundation. rangements required for holoenzyme formation. Nature 544, 196-201 (2017). Competing fi nancial interests The authors declare no competing financial interests.

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An integrated approach to comprehensively map the molecular context of proteins

ORF MAC-tag N-MAC-Tag BirA* HA StrepIII ORF insert Bait StrepIII BirA* C-MAC-Tag ORF insert StrepIII HA BirA* Bait Flp-In Recombination Prey

Strep-Tactin® Sepharose

AP-MS Select and expand isogenic cell clones BioID +Tetracycline +Tetracycline & Biotin

Bait Bait

Collection of cells and sample preparation

Mild lysis Harsh lysis

Single-step streptavidin purification Bait Bait

LC-MS/MS analysis

Bait Bait Bioinformatics

MS-microscopy Protein complex components Transient or proximal and stoichiometrics interactions

Figure 1: MAC-tag-based workfl ow for isolation and identifi cation of protein complexes, protein-protein interactions and molecular context. Gateway compatible MAC-tag destination vectors containing StrepIII, HA and BirA* were designed to allow the gene of interest either C- or N-terminal tagging. The expression vector can then be transfected into Flp-In 293 T-REx to establish the transgenic stably and inducible expressing isogenic cell lines. For the AP-MS and BioID analysis approaches, the cell line is separated into two cultures, BioID cells receiving addition of 50 μM biotin in their culture medium. In the following protein extraction process, optimized lysis and affi nity purifi cation condi- tions for both analysis approaches were used. The interacting proteins were then analyzed by quantitative mass spectrometry and high confi dence interaction proteins (HCIPs) were inferred via stringent statistical fi ltering. This integrated workfl ow allows laborless generation of cellular material for analyses, and results in integrated view of the formed protein complexes, protein-protein interactions and detailed molecular context defi nition.

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Liu et al.

Ribosome Exosomes

RS6 HSP7C Nuclear Envelope Plasma Membrane

LMNA EZRI Nucleolus Endoplasmic Reticulum

FBRL CALX Mitochondria Early Endosome

AIFM1 EEA1 Lysosome Late Endosome

LAMP1 RAB9A Proteasomes Recycling Endosome

PSA1 RAB11A Centrosome Peroxisomes

TBG1 CATA Chromatin Cis-

H31 GOGA2 Trans-Golgi Apparatus

TBA1A TGON2

- Biotin + Biotin + Biotin Bait Protein Bait Protein Biotinylated Proteins

Figure 2: Fluorescence microscopy analysis of the bona fi de cellular localization markers and their interac- tors. The 18 subcellular localization markers fused with MAC-tag were visualized by immunofl uorescence staining using Alexa Fluor -488 labeled anti-HA immunostaining (green), their in vivo biotinylated interactors with Alexa Fluor -594 streptavidin (red), and cell nuclei with DAPI (blue) (Scale bar was 10μm).

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An integrated approach to comprehensively map the molecular context of proteins

a c

2,000

PSMG3 PSMG1

MYH14 1,500 PSMG2 PSMG4 MYO1D MYO6

PSA7 PSB1 PSME2 1,000 MYO1B MYO1C PSA3 PSA4 2 17 17 MYH10 PSA1 PSA2 POMP PSB6 PSA1 PSB2 PSA5 500 ECH1 # of Interactions PSB4 PSB3 AIMP2 PSME1 GLO2 YES PSB7 ARF4 AP-MS 0 RBM28 SC16A GNAI3 HYOU1 TMOD3 GRPE1 BioID AP-MS BioID

BCCIP NOSIP d SENP1 PSIP1 AP-MS Interaction

b IMA1 GRPE1 PIAS2 IMB1 BioID Interaction YTHD3 RAD50 IMA7 PTN1 IMA6 WRIP1 FBRL

DPY30 AKP8L IMA5 Prey-Prey 400 IMA3 PPIL4 PARP1 ITB1 LBR IMA4

GNL3 BAF ARF RFA3 PSMD7 LMNB2 LMNB1 RAE1L C1QBP Interaction P53 F169A ZKSC4 300 RFA2 PSMD6 LAP2B EMD HDGF LMNA LAP2A TOIP1 ZN691 NUP37 LMNA NUP43 NU133 ZKSC3

NUP98 MAN1 SEH1 ALKB5 CMTR1 200 ELYS MYSM1 EMC1 PGAM5 HMBX1 RFA1 UBP7 NUP50 GLO2 NEUA E1B55 NU107 27 13 83 PIMT KBTB6 ZN724 EMC2 LYRIC VRK2 SCFD1

TPR TFAM NU160 SEC13 NU155 HDGR2 DHX40 100 STIM1 GPKOW TYDP2 NU153 LANC1 P121A CX057 DDX54 LEMD2 SC16A NUP85 DNJC8 LMAN1

SSBP ZKSC1 PCY1A CCD47 KI67 CATIN RBM33 SPT5H PPIL2 DHX30 ACD11 FEN1 K1143 ACSL3 NSMA3 ARI3A # of Interactions/Bait 0 AP-MS

SRPRA YKT6 VAPA AN32E AP-MS BioID P121C SRPRB BioID EDF1 CHCH2 RPC3

PGRC2 LSG1

SMHD1 CX056 WDR70

LTV1 WDR36 DDX50 NOL10 NOL8 NOC4L DIEXF REPI1 GPTC4 e ESF1 NOL9 HEAT1 Centrosome NU188 RM47

NOM1

SAS10

PWP2

DDX21 (TBG1) WDR43 AATF TSR1 KNOP1 ZC21A BRX1 BMS1 PFD5 TCPW RSBN1 DHX37 DPYL5 TM263 PDCL3 POP1 ELOA1 UBP36 HP1B3 MBB1A TBA1C T11L2 BYST NOG2 SDA1 ABT1 GLYR1 CEP41 DDX24 DPYL2 DDX10 RL8 PFD4 TF3C3 DIM1 DPYL3 TBG1 ZKSC8 DDX31 TBL3 CTCF HN1 RFC4 RPP30 PESC RL26L DPYL1 PFD1 ECHA RFC3 RL13A TBA1A RFC1 RL18 RL3 HIG1A WDR3 CAMP2

RL19

AP3M1 RCL1 TBA1B RT34

GRIP1 LAS1L PFD3 NOP14 TBAL3 AT1B3 IF2B3 RL32 TXND9 LONP2 ACD11 ARL1 MPP10 PTGR3 HSDL2 DPYL4 TTC4 RT12 RL10A ISOC1 TYSD1 GBB2 NOP56 RL31 NPA1P NUD12 ACOX1 RS23 RL17 RS14 ARF RS9 PWP1 RL26 RS3A DHRS4 ECHP

PECR PEX5

PLPL8 HMGCL Microtubule RL36 NOC2L RT16 RRP15 RBM19 RM11 RT18B RLP24 RPOM NOL12 DECR2 ACACB TEX10 CATA HYES NVL PK1IP

PRKRA FANCI HACL1 NLTP NUP93 NPM3 IMP3

(TBA1A) SSF1 ESYT2 UTP20 OCTC G6PD SMC1A RL1D1 MOV10

GSTK1 Nucleolus THIK SMC3 PARN Z286A

VWA8 RLA1 NMNA1 ECI2

ADAS PEX14 RLA2 RPF2

ACOT8 ACOX3 PAHX GNPAT RLA0 NO66 ARF4 (FBRL) STAG2 INT1 BAP18 UBR7 MGAP SHCBP P66A NPM NOL6 SP20H KI18B TF3C2 UBE2O UT14A UBP48 RPA49 PSMG4 NELFB ANM1 PB1 PSMG2 DDX47 RS17 DHX33 PSME1 P66B NP1L4 TAD2B PSB7 BBX YTDC2 PSB2 WAPL STRBP KAT2A RL35A CCD86 PSMG3 POGZ TDRD3 PSB6 DHX40 APC16 FUBP3 MEPCE GZF1 Peroxisome ZBT10 RPA2 RL7 TMOD3 DHB4 RSBNL RENT1 PSB4 LMBL3 PSA1 TAF2 ALG13 NUCL WDR12 PSME2 ZFP91 SRFB1 PSA5 ARID2

Z512B ZCHC3 RL13 MAK16 POMP ZMYM4 LAR1B PSA4 EHMT2 (CATA) RIF1 GAR1 RL12 GNL3 PSA7 FBRL PSA3 NOLC1 ZN830 PSMG1 PSB1 RNH2B GNAI3 F120A RL4 WDR18 UIMC1 NCBP1 SMU1 RS6 MTA3 NOP58 NOP2 UTP23 PDS5A OTUD4

KIFC1 UTP18 RL5 LLPH TAF9 NH2L1

NUSAP IF2B2 GRWD1 ELF2 NP1L1 SPB1 RL28

PCGF6 ZN850 RD23A NUD19 TAF5 RL29 EBP2 Proteasome RPAC1 COIL RGAP1 U3IP2 ECH1 RPA1 RBBP5 DKC1 MCCA DNJA1 CSDE1 IPO7 RL14

APC1 HELLS NUMA1 ECHB IMP4

ADNP

(PSA1) MDC1 RRP1 RM24

ZN644 HDGR2 MYSM1 CAF1B NEUA IF6 Ribosome RT27 ZN691 COX15 EHMT1 MAVS MRRP1 UBP30 MIC27 CATIN ACOT1 C1TM RL37A H1X T126B SCMC2 CAF1A MIRO2 TTC19 HDGF MICU2 CL073 CHD8 ZKSC4 RS8 S39A7 AKAP1 ATPF1 CDC16 P33MX ATD3B LAP2A COA6 RS13 NOC3L COA7 DMAP1 AN32E RT31 LACTB F136A CCD58 CSTF3 (RS6) FEN1 RL7A DDX55 P5CR2 COX19 SMHD1 UBN1 AKP8L RT22 IMA5 TIM50 CHD3 PARP1 OMA1 PSIP1 RS4X NGDN DNJC8 NDUA9 RT24 BRD7 H31 DDX51 RT23 RT26 RS24 NFIA SIM12 CLPP PTCD3 RL30 PELP1 RAD18 YMEL1 NDUA5 BCCIP BPTF RL11 LN28B

NUCG HCD2 DDX56 RT25 CC137 PDS5B DDX52

ELYS RT10 NEPRO COX17 TF3C1 GPKOW PPIL4 MICU1 UBP7 NUP43 SURF6 RT05 RL7L ASF1B K1143 CMTR1 PCID2 TACO1 GRSF1 RT15 KIF23 SEH1 RL36A MA7D1 KBTB6 EXOSX PUM3 SSBP RL18A RRS1 HIG2A ZMYM2 RL6 BOP1 P121A CEBPZ RM28 ALKB5 H33 UBXN4 IP6K1 CHMP7 GRM1A NPL4 CYC CLPB RN185 MSPD2 WRIP1 HMGX4 TOIP2 CX057 CLMN CBX1 MACOI DNJC1 ACAD9 TIM10 ZKSC3 MMGT1 NDC1 CDC23 IMB1 UBE2N SARNP TEX2 TIDC1 CA043 RPC3 APC5 GPAT4 PPIL2 OSBL8 ATP5E TOM22 HIRA SND1 NUP85 EGLN1 TF3C4 C1QBP SYIM CMC4 Chromatin BLM NFXL1 LMNA RBGPR CX056 ALR KMT2B ATD3A LMNB2 TMED8 MSH6 RL39 F134B LYAR WDR70 ASF1A SRP68 TAPT1 NDUBB KAD2 RAE1L AT2A2 SENP2 DNLI3 REXO4

SENP1 KI67 CCHL ZN281 RINT1 OCAD1 (H31) RRBP1 PREB TAF6 IMA1

TRRAP ACBD5 NDUA8 NUP50 MIC19 EMC7 PRI1 NU153

ZSC21 NOSIP WDR44 NDUV1 AAAS NDUF4 YES NASP PCY1A NSMA3 LSG1 MYO6 TF3C5 P121C CHCH2 LMNB1 MIA3 ACOT2 MYO1B RFX5 MS18A NDUAD NU133 SPT5H TYDP2 AIFM1 PSB3 TAF6L CALX ATRX IMA4 F169A GPAT3 ATPO PIAS2 E2AK3 CMC2 SC16A IMA3 NUP37 MORC2 EDF1 HYOU1 IMA6 IMA7 MSH2 NU155 ANO6 IF2M EMC3 RT28 MEN1 NU160 BUD31 NUP98 INF2 RBM28 TAF4 ETFB STIM2 PDIP2 RFC5

RFXAP GSCR2 VAS1 CUX1 STX18 TDRKH ODO2 NOP16 HMGB3 GLO2 DEK NAT10 NSDHL PPM1G CS025 HEM6 T2FA AFG32 DHX30 RAD21 DDX54 RFC2 KRI1 RB3GP UB2J1 MED14 RRP12 SAM50 PHB CDC27 Nuclear Envelope NOG1 Endoplasmic Reticulum AQR SPCS2 T2EB CHTOP Z780A GLYM HMGB2 RRP1B TIM8B RNH2A DNMT1 UBE4A KIF4A M4K5 TYY1 RBM34 WIZ BRD2 NSUN2 XRCC6 TAF1 PHLP TIM13 P5CR1 XRCC5 T2FB TBP MSH3 CLGN

Mitochondria REEP5 EMC8 HAX1 QCR1 (LMNA) MXRA7 ADCY9 (CALX) SPCS3 TACC1 NDUS8 SDHA SNP29 TXTP UFL1 BAP31 MIA40 CLPX RT29 CAMLG CDCA3 TACC2 NU205 SOAT1 DNM1L PPIF RPN1 KAP0 NCK1 HMOX2 SERPH

(AIFM1) NDUS1 MIC60 EMD HTRA2 ATPG DDX18 ZCCHV DSG2 RABL3 STX5

ATPA TRIA1 UNC5B

RHG01 MRT4 SCO2 AT5F1 VEZA QCR2 NDUS5 MK67I OST48 CG050 DDX27 TRM1L ECM29 COX5A TIM8A S4A7 ZN512 RFA1 AT2B1 RRP5 OPA1 NDUV2 MAGD2 EEA1 VIP2 UBR4 PGRC1 NDUS2 HXK1 NIP7 RPN2 SRP72 KRR1 SC24A NDUS6 ATP5J ARI4B SC24B RUFY2 ARI3A NKRF MTX2 CX6B1 LMAN1 WDR6 STAU1 RRP8 NDUS3 COX41 SCFD1 DLG1 NIBL1 TOIP1 ITB1 ERAL1 KCRU DEN4C OSBL9 FCHO2 P85A CMC1 THIO SYAP1 EHBP1 MAN1 CI072 PEAK1 PGRC2 ATP5H COX6C LBR YKT6 CC124 OSB11 ATP5L CY1 ECD NOTC2 QCR6 TOM70 GOGA3 GLSK ATPB SMCR8 LETM1 AT1A1 LIN7C NDUBA TOM40 DHE3 COX2 NU107 LAP2B ELP3 ESYT1 STT3A TRPM7 EarlyRAE2 Endosome GCP60 CC115 NUMB GRPE1 SNAA TM209 RFIP1 CK5P3 CSKP VRK2 RFIP5 BASP1 PURA CTND1 WDR41 EMC1 HGS CYFP2 LYRIC CHP1 SWP70 DDRGK RTN4 RABX5 SC23B TFAM NSF TM230 CCD47 TMX1 FERM2 CBPD EMC2 TIM44 LRC59 VAPB MYH10 MYH14 SC23A AKA12 SCRIB DCTN4 AUP1 CNBP STIM1 ACSL3 (EEA1) JPH1 RAB11A TM199 SRPRA MYO1C PDZD8 CLCC1 MYO1D BASI COPE RBGP1 UBP11 SEC63 SAHH2 ANKL2 RABE1 FA21A VATF CKAP4 IF2A RAB5C S12A2 FND3A SYK IRS4 TPPC9 KTN1 RFA2 ACTY DVLP1

AL3A2 TRI13 STRUM CDKAL DD19A KIF5C PDIP3

TPC10 SC22B DIP2A EMC4 DJC11 STBD1 RENR IF2B CALU MLF2 STT3B GORS2 ETFA

SRPRB BAG2 RGPA1 S6A15 UBP47 SEPT8' DOCK7 ZC3HF ERBIN VAPA Recycling Endosome ENAH MPRI NEB2 PTN1 ARFG1 S11IP EFHD1 TM55B NBEA HPS3 PHAR4 TB22A SMAP2 DEP1B STON2 HM13 WDR20 SRBS1 STAM1 STX12 RRAGA GCC1 ZNT5 BORC5 PALM2 NCOA7 AP2M1 EIF2A UBP32 LCAP SNX27 RUFY3 SIR2 PK3C3 AR13B KI16B BORC7 TB182 ARFG2 RBM14 RUFY1 VPS45 ANR27 RLGPB NISCH PARG VPS8 RAB1C VP13C CA198 KS6C1 TECR LRBA PACE1 BORC8 (RAB9A) PTN23 R3HCL PI3R4 ZFY16 M21D2 UBIA1 E41L1 S29A1 GOGA5 PKHM3 DP13A 4F2 GRIN1 GCC2 KXDL1 F135A SNX6 K1468 VAMP8 VAMP3 SH24A SNP23 DEN6A TFR1 CYFP1 OSB10 ABCE1 SCAM1 STX7 BIG3 SNX3 F219A ZFYV9 TRIPB RRAGC LZTL1 EPHA2 RAB9A DYST AP1M1 RAB6A TPPC3 K1211 HOOK3 GTR1 RCN1 CYLD WDR11 TPC6B MIO MPRIP SEM4C SHLB2 VP13B KIF1A RBNS5 LTOR2 CF132 BI2L1 MON2 RHG35 ADDG TNIK SRP14 VPS16 LTOR5 COBL1 PPR21 RFA3 XPOT SYFB NCKP1 GOLP3 RAI14 ABCD3 SHSA2 ODP2 PKN2 RAE1 AT133 ANM7 TARA PAK4 PDXD1 TAB1 RICTR NR3L1 EZRI ABI1 SPE39 RGPA2 MARK2 AAAT ARP10 SCYL2

LIPB1 RFTN1 PLSI ARFG3 VP33A TBCD5 BORC6 ZNT6 DIP2B LSR DSC3 BMP2K SAR1A SEPT6' SNX29 ITSN2 CNNM3 URFB1 PTK7 ABCF2 PACN2 LTOR3 ABI2 TBC23 SNTB1 LTOR1 Late Endosome P55G TPPC1 AP1AR PTN13 TR112 BET1L STX8 MTMR1 KLC4 WASH7 ZDH20 ZDHC5 DSC2 ASAP1 NUMBL GGA3 GAB1 VATD CLCN7 SAHH3 SNX30 PKHA1 LIMC1 RAB10 AP2A1 HACD3 ABR DPM1 (RAB11A) NDRG1 RAB7A LAMP1 GORAB AKAP2 OCRL PLCB1 SNAPN CDC42 ANK3 VPS54 VATA VATB2

WIPI4 FA83B ANK2 T106B VPS18 S19A1 WASF2 UTRO TJAP1 EF2K DJB11 DHCR7 ADDB RADI NHRF1 GRAP1 BAIP2 NMT1 ADDA DIC LSM1 SHC1 RIC1 M3K7 P85B CTNB1 MOES OGA VTI1B ICAL CAPON SRGP2 MARK3 PLST COBL RABL6 BCR ROCK2 ELP1 SVIL TAU CADH2 MTMR5 S12A6 1C07 PDIA4

ITSN1 TGON2 GGA1 DP13B VANG1 TM87A TPC12 S39AE K319L KCNB2

RAB21 RELL1 S38A2 DNJC7 SDCG3 METH ARHG2 ARHGA LRP8 DPH2 HS105 TPPC5 VAT1

TPC2L RPAP1 RPGF6 CPNE8 SPY4 GAK PICAL DPH1 HUWE1 Plasma Membrane S39AA TPC11 VP33B Lysosome TRIM9 PP6R3 GOGA4 F91A1 S38A1 SYNRG TANC1 EDRF1 AP1B1 STEA3 GOPC S12A4 WNK1 KCC2D GALT2 (EZRI) KDM2B LRC49 MPP7 TB22B (LAMP1) PAXI1 COG1 AP4E1 WDCP CLH1 UBN2 ATP7A FA60A OCLN VPS51 MTMR6 HPBP1 HSP72 Trans-Golgi HSP7C TPGS1 AT2C1 STRN4 L2GL1 KDIS TPPC4 ATX10 AAKG1 STRP1 EFNB1 GOGB1 TPPC8 ZFP30 S23IP FYV1 BAG5 IRAK1 GOGA2 DAB2 ZN658 EPN4 SNX5 PHOCN CCD53 CHIP HSP74 (TGON2) RT35 FLOT2 ZNT1 STRN3 DMXL1 PP6R1 NCOR2 HS71L USO1 TMF1 SNX1 TPC13

DVL2 TLE1 FA21C RPB2 KMT2D S20A2 AP3B1 VAMP2 BUB1B CLCA ML12A KDM6A LAT1 AP3D1 WDR7 STRN NCOR1 DIAP1 ASNS MOT1 STX16 AP2B1 DJC13 BAG3 CXA1 STAM2 HAUS5 HS74L CN37 1A03 AP4S1

RAB1A

S12A7

RAB14

BIG2

TPD52

EPN1 EPHA4

UH1BL Exosome ARFP1 Cis-Golgi ILVBL AGFG1 F234B CTR2 WASH3 VAMP7 RCAS1 MRP SCAM3 (HSP7C) SC6A8 SURF4 (GOGA2) VDAC2 TPD54 MUC18 SPY1 UBP8 AP1G1 ATP7B STX6 P4HA1 STX10 TPC2B SNX2 VAMP4 A4 PPR37 GOSR1 AP2A2 ZFPL1 EPN2 SNX4 Novel Interaction Known Interaction

Figure 3: Generation of physical and functional PPIs interaction networks of bona fi de cellular markers. The 18 localization markers we subjected to our integrated analysis, resulting in identifi cation of 679 interac- tions from the AP-MS and 2118 interactions from BioID analysis. (a) The distribution of the number of known (blue) and novel (red) interactions within 18 bona fi de subcellular organelle/structure markers, illustrate the need for systematic analyses. (b) The distribution of the number of interactions per localization marker by AP-MS or BioID purifi cation approach shows similar distribution of connectivity as other publications using these approaches individually. (c, d) The protein-protein interaction network and molecular context for proteasome organelle marker (PSA1) and nuclear envelope (LMNA). The HCIs that were identifi ed from AP-MS (green line) and BioID (yellow line) are shown together with the known prey-prey interactions (dashed grey line). The nodes are color-coded based on the localization rank obtained from the CellWhere database (key: dark green = primary cellular localization for the corresponding protein, light green = possible localization, grey = diff erent or localiza- tion assigned for the protein). The Venn diagram highlights the complementary nature of the AP-MS and BioID approaches. (e) The reference molecular context map for the 18 subcellular organelles/structures. The unique high-confi dence interactors from the BioID analysis are arranged in a circle around the corresponding localization marker and the shared interactors are shown with corresponding colors representing multiple localizations. Preys with more than four subcellular localizations are shown in white color. The novel interactions are shown in pink edges and the known interactions with blue.

16 bioRxiv preprint doi: https://doi.org/10.1101/264788; this version posted February 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Liu et al.

a *

Q:Query PSM; R: Reference PSM Q C R C:Common proteins. k:total number of reference proteins Interaction Context m: 14 cellular compartments

bcd NUC

NE PER SURF6 CE152 OFD1 SC16A LANC1 GLO2 DPM1 MIT PM NOG1 DDX54

MIA3 TF3C3 SPB1 HYOU1

SC16A RRP12 INCE HS90B MIC PRO CDC37 ZFP30 RIF1 RBM28 AURKB 0.50 0.75 AURKB

SUGP2 MYH14 NAT10 CP131 LYS CEN

CE192 MBB1A ARF DDX18 GRPE1 INCE NIM1 HS90A LYAR

GOL CHR NU205 DHX37 CDC37 DDX24 BOREA BIRC5 EXO END ER

NUC

EHBP1 TM87A ARFG2 UNC5B VANG1 PACE1 NE PER ZNT5 M21D2 COG1 RELL1 RGP1 HUWE1 GOSR2 TFG SCYL2

KCNB2 ZNT6 SC16A PCH2 DDX54 VAMP7 SNAG VTI1A CYFP2 SNAG SC24B ATD3B CG050 SCAM3 ZN512 NU188 FANCI MIC60

STRBP HACD3 IF2A TJAP1

SC31A SNX3 MIT PM AHSA1 WDR11 STAU1 NOP16 RIC1 EPHA2 VPS51 STT3A RRBP1 AT2A2 SRPRB SPY4 LMAN1 BCCIP RRP1B BAF SC22B IF2B NUMB DDX18 SC22B

TEX10 CNBP RAB5C VPS45 F91A1 PDXD1 WAC2A VDAC2 YKT6 HM13 LAT1 ARFP1 ARFG3 F135A ECHA CANT1 EPHA4

RAB9A TMF1 NBEA SERPH DDX24 LRBA STX5 VAMP8 KDIS BASI

DYST RABL3 AGFG1 RM47 MPRI SYEP EPN4 SNX6 RABE1 ECH1 ERLN2 NSUN2 TRIPB JPH1 TFR1 SC23B VP13B CXA1 SC24A NIP7 TIM44 STEA3 CASP WDR6 K319L RT18B CALU HS71A YKT6 OCLN STAM2

ACOT9 MRRP1 GOGA4 AKP8L MIC PRO TECR PDIP3 HYOU1 ARHGA

GLO2 LYAR DMXL1 DHX30 ATPG DIC GSCR2 GRPE1 BET1 TPPC8 SMC3 BET1

UBR4 LSR GOGA5 STX12 0.50 0.75 UBIA1 RBM28 LCAP DHCR7 SAR1A RPN2

TRM1L ARHG2 GORAB SC23A BIG1 ITM2C

MAN1 SEC63 GCC2 SMC2 SYAP1 VTI1A ESYT2 ZCCHV SYIC GO45 S12A4 MON2 S23IP SMC1A S6A15 RBM34 TPC11 FLOT2 SEC13 STX7 GOGA3 PESC ZFY16 SYDC MK67I TR10B GOGA2 LYS CEN TB22B VAPB XPOT GCP60 GOSR1 SCFD1 RRP8 NU205 SYLC RABL6 DD19A STX16 STX10 4F2

NU155 GCC1 MRT4 DPM1 FYN VAPA SYK EMD RRP5 GORS2 GGA3 DDX52 GOSR2 CBPD S38A2 STX7 LIS1 S4A7 RAB6A

NKRF SCFD1 ZFPL1 APBA2 AIMP1 UBP8 DHE3 BET1L AT2B1 VAMP3 NOG1 CLCN7 USO1 STX6 SNP23 1A03 COPG1 RCN1 SNP23

SURF6 SNAA SRPRA DP13A CLPX GOSR1

GTR1 CN37 HGS DDX56

TXTP NSF GOL CHR ARF4 GOPC S12A2 ARF6 S39AE A4 STX5 ATPO RBM14 SC16A SNAA TMM33 STT3B

GNAS2 NUP93 TFAM RRP12 NSF NOTC2 GOGB1 CKAP4 OCRL KRR1 GBF1 EXO END DSG2 STAM1 SPB1 ER

NUC

NE PER NU205 PNISR MTA3 SUGP2 TPR

EHMT1 HDGR2 MAZ MIT PM DDX56 NOG2 SC16A MED17 RPA1 CD11B CTR9 CDC37 TAF4 MAT1

PWP1 TAF2 CCNH ERCC2 RBM28 ELOA1

RRP1B TAF5 TF2H5 TF3C1 TF2H3 RIF1 LYAR CCNL1 DDX10 TF2H3 TF2H1 YTHD3 MIC PRO TF3C5 T2EA T2H2L MGAP CDK7 TAF6 0.50 0.75 CDK7 ZFP30 TF3C3 TF2H2 ECH1 SPB1 ERCC5 TF2H5 ERCC3 T2H2L TF2H4 CCNL2 T2EB TF2H1 LYS CEN DDX52 RL39

PCGF6 TAF1 ERCC5 TF2H2 NOG1 CCNH RT12 ZN281 ELL CDC37 TF2H4 NAT10 ERCC2 RPA2 MAT1 ERCC3 GOL CHR MED31 PRI1

LAS1L KI67 ACD11 PESC COIL CNBP SURF6 EXO END ER

NUC

ZSC21 BCCIP NE PER MED13 SBNO1 ESF1 MED16 MED24 MED22 MED4 PESC FANCJ MED11 MED21 EHMT1 RAD18 NU205

PPM1G ISY1 CX057 NADAP MIT PM MED17 MED6 ZN281 CDC37

MED16 KIF4A MED22 MED4 MTA3 LAS1L MED17 MED6 MAT1 MED15 MED30

MED13 MED30

TAF6L MED11 MED25 HDGF ZEP1 NACC1 PAF1 T2FA MED23 MED25

MED24 RPC3 TADA3 MED21

AQR SYF1 TF3C4 MBIP1 MED15 MED14 MED7 MED14 MIC PRO ZN131 MTBP WDHD1 TF3C1 YETS2 ZN830 MED23 MED8

CDK8 CDK8 MED27 MED8 ZZZ3 KAT2B 0.50 0.75 MED7 MED9 MFAP1 WIZ CTR9 PNISR

MED27 WDR61 WDR5 MED12 MED20 MED18 CD11B DPOA2 CSR2B CCNC LYS CEN DDX10 MED20 MED18 SPB1 DPOLA KAT2A TAD2A MED31 MED19 MSH2 TP53B MED31 MED12

MD13L MED10 NOG2 SUGP2 IMA5 MDC1 MED29 PRI2 MD12L MED28 MED1 CCNC MED9

PPIL2 T2EB ARI4B MGAP RIF1 T2EA MD13L MED19 BRE1A PRI1 GOL CHR TF3C3 PCID2 WAPL SC16A MED10

ZMYM4 MYSM1 CDC37 MED29 MD12L MED28 P4R3A ELOA1 MED1 TF3C5 EXO END ER

NUC

NE PER CP131 KDIS CP110 BI2L1 MAZ CADM1

WDR62 F193B

MIT PM CC138 SC16A HAUS5 LTV1 SPAG5 ALMS1 SC16A AXIN2 FRAT1

HAUS4 MACF1 PEX5 ATG2B AXIN1 GSKIP TRAK2 DYST SWP70 DLG5 CLAP2 PP2BA MA7D3 DCP1A NU205 MIC PRO IPP2 E41L3

STON1 ECHB GSK3B 0.50 0.75 SDCG3 GSK3B CDC37 NBR1 CE350 PRC2C OFD1 EDC3 CE152 KAP1 RBM33 LYS CEN PRUNE FR1OP

KANK2 APC ECD ERBIN PRUNE CDC37 KAPCA KAP0

VP13D TRAK1 LAR1B KAP0

CRBG3 CTNB1 GSKIP KAPCB PP6R3 GOL CHR RL39 AXIN1

TPGS1 PP6R1 KAPCA LRC49 DCP1B AKA11 EXO END ER

NUC

NE PER MAN1 UH1BL RAE1 LANC1 SC16A RBM33 EMD DDX52 MIT PM GLO2 VAMP3 TPR ODP2

VPS8 PGTA ABCD3 DP13B

ARF MPRI PIMT GNL3 MIC PRO NOG1 PGTB2 RAB5A RAB5A 0.50 0.75 DDX54 RBNS5

S6A15 RAE2 UBP11 SYK

LYS CEN NU205 OSBL9

GRPE1 DDX24 VP33A DP13A TCAL1 YTHD3 RAE1

GOL CHR F135A VPS18 PGTA PGTB2 LCAP VPS45 RAE2 UBP11 EXO END ER

Figure 4: The reference molecular context map allows molecular level identifi cation of cellular localization profi les for a protein of interest. (a) The schematic overview of the MS-microscope to assess the queried protein localization using our reference interaction context. (b) The polar plot shows the location of query protein observed by MS-microscopy. Each sector represents one subcellular location defi ned by our reference database. The color assigned to each of the localization is based on the annotation frequency (Pink: 0-0.5; Yellow: 0.5-0.75; Green: 0.75-1). (c,d) The PPIs interaction network obtained from BioID and AP-MS are shown separately. The localization of prey proteins was verifi ed by Cellwhere database. Node color scheme coordinates the observation localization from Figure 4 b.

17 bioRxiv preprint doi: https://doi.org/10.1101/264788; this version posted February 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

An integrated approach to comprehensively map the molecular context of proteins

ab NUC

RM16 RM10 NE PER RM04 RM02 RM01 RM55 GRP75 RM50 CDCA3 SYNM DHTK1 TACO1 FMC1 RM28 TR61B FAD1 RM52 HINT2 THNS1 RM21 NT5D2 SUV3 RM51 GCDH HIBCH DUT AL1B1 MRS2 PREY HEM1 ACOT1 CK5P1 IDH3B MIT PM RM49 CISY PDK1 RM24 VWA8 GATA TIDC1 SYMM SYDM ECH1 GRSF1 SYHM RM47 IPYR2 MGME1 ECHB PDK3 RM45 SYIM CQ080 GLYM IF2M PLPL8 SYTM RRP12 CLPP SSBP AASS MPPA LYRM7 LPPRC CH60 TMLH ECHM RM44 ABHDA NO66 EFGM DPOG2 CECR5 NSUN2 OXSM TIM16 SYCM CG050 NUD19 MIMIT THG1 ATPF2 XPP3 MRRP1 RM53 DDX18 THIM RM41 CN37 GLSK SCO1 RF1M SYVM NDKM ATD3B ACOT9 TRM5 GATB RT30 RT29 NAKD2 ETFB RT11 RT27 RM43 COQ3 PAPD1 RF1ML UQCC2 SYK MAOM RM19 ACS2L RT34 RT05 CIA30 MIC PRO PPCEL RT02 TRUA ACADM HS71A SYWM RT23 RT16 MMAB DPOG1 HEMH TR112 RT24 RT22 SYYM G45IP RM11 NDUF5 AKIP RT07 ECI2 NDUF4 IDH3A ODBA MRM2 PTCD3 RT14 MASU1 MUTA PRDX3 CF203 RT33 RT18B 0.50 0.75 SDHF4 MTDC ACADV MPPB RM20 KAD3 KAD4 RT18A RT10 P5CR2 OAT RM37 P5CR1 RNZ2 RT18C RT28 AL4A1 LYPL1 ACOT2 RT06 RM42 RT35 PDIP2 GATC RBM28 SYG CAF17 CH10 PNPT1 RM46 RT31 RT25 RM14 ANGE2 ACON ES1 ZNT9 RT36 RT26 ECHA UQCC1 EFTS PDE12 RT09 PHB2 ATPF1 LYS CEN HSDL2 NDUF7 RT63 HCD2 RM17 CHTOP C1TM ACDSB PTCD1 ALDH2 SYSM RM03 SPRY4 ODO1 SETX GTPB3 ODP2 MTEF3 TIM13 TRM11 ARGI2 DLDH BCS1 ECSIT GRPE1 RM22 RM09 DCXR GLO2 SUCA TIM14 NFS1 NIPS1 CLYBL CH082 ACAD9 DNJA3 GLRX5 PREP RT4I1 PPIF ODO2 RM48 AL7A1 PDPR NT5D3 ADT1 PHB DHX30 RM39 GUF1 NDUF3 NUBPL STML2 SSDH PTPM1 NNTM LONM RM18 DHB8 OXA1L TIM44 RM23 TRAP1 S2540 ADT2 LETM1 DDX54 SCOT1 COASY CLPX RM27 NUDT8 THIL RM12 MAIP1 SUCB1 SUCB2 GOL CHR SMC1A ERAL1 SYLM SHC1 AFG32 RM32 HCDH ETFA SYAM MRM3 THEM4 SLIRP LYRM4 ABCB7 RM38

RM33 RM15 RM35 RM40 EXO END RM30 ICT1 RM13 ER AOX

NDUBA NDUS4 NDUA2

NDUA9 NDUV2 NDUV1 NDUS5

NDUS6 ACPM NDUAD NDUS2 ATP5L ATP5J ATPG

COX2 COX41 NDUA5 NDUA7 NDUS7 NDUS3 QCR7 UCRI ATPA AT5F1 ATPB

NDUAC NDUS1 NDUA6 NDUS8 SDHA SDHB QCR2 QCR1 COX5A COX5B ATPO ATP5H ATPD

c 100% 90% 80% 70% 60% 50% 40% 30% 20% 10%

identified/total 19/44 2/4 4/10 4/19 9/19

d

60

40

20

0 Average component abundance Complex Complex Complex Complex Complex I II III IV V

Figure 5: Transgenic alternative oxidase (AOX) localized to the inner mitochondrial membrane facing to the and locating proximal to Complex II. (a) The AOX from Ciona Intestinalis, was introduced to human cells and the MS-microscopy assigns the AOX to localize to mitochondria. (b) The BioID approach identifi es 333 interactions of which 93.1% (310) were mitochon- drial (green), 0.3% (1) was peroxisomal (pink) and 6.6% (22) were unassigned (grey), based on CellWhere database. (c) Total of 38 of the interactors were components of the mitochondrial respiratory chain complexes (Complex I- V, key: color gradient indicates the percentage of proteins of the each individual complexes identifi ed). (d) The average component abundance shows that the AOX associates most with the Complex II, which is in agreement with the AOX suggested functional role.

18 bioRxiv preprint doi: https://doi.org/10.1101/264788; this version posted February 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Liu et al.

a b c CDK7 CDK8 MED13 CCNH MED12 MED12 ERCC3 MAT1 MED14 ERCC2 MED14 MED1 MED1 TFIIH1 MED24 TFIIH4 MED13 TFIIH2 MED17 MED15 MED24 MED23 MED17 TFIIH3 CCNC MED27 MED23 MED27 MED15 MED4 CDK8 MED16 MED6 MED16 MED6 MED8 MED19 MED4 MED19 MED7 MED25 ERCC5 MED21 MED20 MED20 MED22 MED10 MED29 MED29 MED7 MED10 CCNC MED8 MED22 MED25 MED11 MED9 MED11 MED30 MED28 MED31 MED18 MED28 MED30 BioID Bait Normalized PSM TFIIH5 BioID Bait Normalized PSM MED18 MED9 BioID Bait Normalized PSM MED31 MED21 AP-MS Bait Normalized PSM AP-MS Bait Normalized PSM AP-MS Bait Normalized PSM

d e MED10 MED19 CDK7 MED7 MED31 MED21 MED6 MED9 MED13 MED8 MED12 ERCC2 MED44 MED11 MED22 ERCC3 TFIIH2 MED17 MED1 CNCC MED18 MED14ED14 MED20 CDK8 TFIIH5 MED15 TFIIH3 TFIIH4 MED16 f g

CDK7 1.0 CCNH MED21 MED4 MED8 ERCC2 MED27 0.75 MED16 MED15 MAT1 CCNC ERCC3 TFIIH1 0.50 TFIIH4 0.25 TFIIH2 MED24 TFIIH3

Relative Distance to CDK8 Cor=0.95 ERCC5 0 TFIIH5 0 0.5 1.0 1.5 0 0.25 0.50 0.75 1.0 Reletive Distance to CDK7 Relative Distance to MED13

Figure 6: Integration of MAC-tag data allows characterizing of interaction distances within a protein complex. (a-c) Distance based topology of protein complexes. The APMS and BioID data was blotted based on the bait normalized prey abundances and the correlated data was used to derive interaction distances for CDK7 and the TFIIH complex, as well as for CDK8 and MED13 with the Mediator complex. The CDK7 formed CAK-complex components are shown in grey and the Mediator complex components assigned to the Head (magenta), the Middle (cyan) and the Tail (green) are color-coded. (d-e) The derived interaction distances for CDK7, CDK8 and the MED13 are fi tted into EM derived complex structures and suggested fi tted interaction surface is shown in green dashed line ellipses. The color-coding in e corresponds with the b, c). (f) Relative distances for bait protein and the other complex components can be calculated. (g) The calculated relative distances derived from the integrated APMS and BioID data results to extremely high correlation (c=0.95) for CDK8 and MED13, two neighboring units in the Mediator kinase module.

19 bioRxiv preprint doi: https://doi.org/10.1101/264788; this version posted February 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

An integrated approach to comprehensively map the molecular context of proteins

Strep-tagIII a Strep-tagIII

C-MAC-Tag N-MAC-Tag

b c Biotin 0102030 40 50 Marker – Biotin + Biotin 250KD 150 100 75 Fusion Protein

50 HA 37

25 20

15 Alexa Fluor® 10 594-Streptavidin

Alexa Fluor® 790-Streptavidin

d CDK7 Strep/HA vs MAC-Tag (-Biotin) CDK7 Myc-BirA* vs MAC-Tag (+Biotin)

80 100

40 50 StrepIII-HA BirA*-Myc 0 Cor=0.88 Cor=0.95 0

0 30 60 90 120 0 25 50 75 100 MAC-Tag MAC-Tag

e CDK8 Strep/HA vs MAC-tag (-Biotin) CDK8 Myc-BirA* vs MAC-tag (+Biotin) 125

100 200 75

50 100

StrepIII-HA 25 BirA*-Myc

Cor=0.990 Cor=0.97 0 0 50 100 150 200 250 0 50 100 150 200 MAC-Tag MAC-Tag

Supplementary Figure 1: Features of the MAC-tag. (a) Plasmid maps are graphical representation of the MAC-tag vectors. (b) The western blot shows biotinylated proteins of MAC-tagged GFP cell lysate with biotin concentration gradient (0-50 μM) in culture medium by Alexa Fluor®594-conjugated streptavidin. (c) Immunofl uorescence analysis shows no detectable biotinylation in untreated sample and signifi cant activation of the biotinylation with 10 μM biotin and peaking at 50 μM concen- tration (anti-HA: green; Alexa Fluor®594-conjugated streptavidin: red; DAPI: blue. Scale bar: 10 μm). (d-e) There is strong correlation between MAC-tag AP-MS (no biotin) & StrepIII-HA and MAC-tag BioID (with biotin) & BirA*-Myc. A scatter plot shows 95% confi dence interval of correlation between MAC-tag data and the data from StrepIII-HA & BirA*-Myc alone.

20 bioRxiv preprint doi: https://doi.org/10.1101/264788; this version posted February 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Liu et al.

KAD2 AFG32 AKP8L SPB1 TEX2 MTX2 TDRKH CGT MMGT1 ADCY9 SURF6 PDIP2 PLD3 CLCA TACC1 NFXL1 MPPA CMC1 NDUS5 ATPG SCRB1 S6A15 TMED8 ISOC1 PTGR3 THIO ATP5L CMC2 VDAC2 NPL4 GLSK TRIA1 TAPT1 CLGN MAN1 RENR ZCCHV DECR2 HSDL2 T106C DDX54 ACBD5 LYRIC DHB4 ADAS ERAL1 LETM1 MIC19 GPAT3 ANKL2 EMC4 PDIP2 ANO6 VRK2 SNP29 DNM1L 1C07 ODP2 RRP12 CHMP7 ADA15 PCYXL DIP2A LAP2B CHP1 DHE3 OCAD1 RL32 CLPB T126B MRRP1 ADAM9 BASI EMD UBXN4 AL3A2 GCP60 NDUBB ATPF1 INF2 OCTC ECH1 NDUF4 MIC60 MPRI TOIP1 AT1A1 NUD19 BASI COX15 CAMLG GORS2 SC16A NDUS5 SRPRA DHX30 NDUAD NDUA5 MYO1B KTN1 SYAP1 SYIM SAM50 NSDHL QCR6 RT28 COX17 DJC11 SOAT1 SRP72 GPAT4 ESYT2 PRC2C POTEJ NUD19 LONP2 GNPAT HAX1 EFTU TIM8B CYC S61A1 MBTP1 TM230 FND3A BASP1 SPCS3 ACSL3 SPCS2 KCRU SYIM STT3A ICMT COPG2 AT1B3 S4A7 RABL3 AAAS UB2J1 F169A ERAL1 SERPH MYO6 CDKAL PTN1 GSTK1 HMGCL GRM1A NOP16 COPE SYDM SCO2 COX41 TIDC1 DPY30 WDR1 MIC60 CHTOP EMC8 TIM8A MIRO2 ATP5H PHB UBE4A TM199 SNP29 OPA1 HTRA2 BGLR TYSD1 DHB4 MIA40 NDUA9 SEC63 SC16A CX6B1 HIG2A S12A2 CLPX DNJA1 CKAP4 SWP70 EMC2 PHB CNNM3 EHBP1 WDR44 TOM70 ATPG PON2 COPB2 PGRC1 AT5F1 RHG01 CMC2 CS025 SAHH2 AT2A2 COX6C THIO TACO1 MIA40 LIMA1 NDUBA QCR1 SSRA WWOX B4GT3 VAS1 EMC3 CBPD VAT F TRI13 REEP5 PAHX HYES MLF2 AT5F1 TIM8A LBR LRC59 SC23B CX6B1 ARF4 TOM40 CY1 MICU1 PGAM5 4F2 HACL1 OGFD3 ECHP TOIP2 PDZD8 OSB11 1A03 ATPG CLMN NSMA3 UFL1 GBG12 LIMA1 ECH1 SCO2 E2AK3 SND1 MXRA7 HXK1 QCR2 NDUV2 CDCA3 LG3BP DHRS4 ADAS KAD2 NTPCR CALX AIFM1 MAN1 MOGS TOIP2 SCFD1 CATA ODO2 AIFM1 HAX1 MBRL AT2B1 EMC1 M4K5 P5CR1 PTCD3 RM12 CALX ITB1 S12A2 GBB2 CATA ATPA SAM50 VAPA ESYT1 GSCR2 STBD1 SNAA PGAM5 NDUA8 ACAD9 DDX18 DSG2 DHX40 G6PD NDUAD GLYM SIM12 ATPO CC115 ATPO NDUS2 F134B PHLP TIM8B TIM13 RENR LCAP VDAC2 TECR MS18A ALR RT26 DHB12 JPH1 MSPD2 BAP31 CHCH2 IF2M RT24 PGRC2 MAVS SDHA CHP1 CLCC1 PGRC2 HYOU1 TIM10 HCD2 T106B SSRG RTN4 ACD11 NLTP NDUAD LBR CLGN STT3B MIA3 ACOT2 COX5B NDUS3 OMA1 GNL3 MAGT1 H6ST2 RPN2 ECI2 RT22 UBP30 TM209 MIC60 XRCC1 COX5A NDUV1 STT3B DNJC1 IP6K1 OST48 NU160 ATD3B TPR SENP2 ESYT1 CLPX HS71A CCHL P5CR2 DDRGK PPIF NDUS1 NDUS8 GXLT1 NUD12 ACOX3 CLPP ATP5J AGAL ERLN1 GNAI3 KAP0 VAPB MYO1B LONP2 HMOX2 VEZA CC124 SRPRB NCK1 CMC4 F136A RAE2 RPN2 COA6 TIM13 RRBP1 ATPG RFA2 LAP2B EMC7 STX5 NSF NICA VAS1 ECHA STT3A CCD47 CK5P3 RB3GP PECR PEX14 ATP5E TOIP1 TIM44 COA7 ATP5H ANAG CDCA3 NU155 TOM40 RFA3 ATD3A NDUS6 CC50A OST48 S6A15 NU133 CLPB DYR RPN1 LSG1 RBGPR PEX5 RFA1 UBE2N P33MX ECE1 RT23 TOM22 NDC1 SMCR8 AUP1 GRPE1 NUD19 LRC59 EGLN1 NUP98 TACC2 MLF2 MIRO2 DYL1 MACOI NUP37 NU107 LACTB HEM6 COX15 AKAP1 ENTP6 GLO2 ATPB TIM50 CKAP4 OSBL8 CA043 RN185 YKT6 AKA12 RT31 ECHB TRPM7 SC24A LMF2 EMC1 COX2 NDUA8 REXO4 CI072 SC24B MLF2 ACOX1 SSBP MICU2 ERLN2 TGFR1 RINT1 RN185 DIC FERM2 PGRC1 PLPL8 ACOT8 KTN1 RPN1 GRSF1 ACOT1 NAT10 UNC5B LMAN1 LIMA1 SC23A STIM2 NUCG MIC27 MYO1D TMX1 TTC19 DJC11 ITB1 TYSD1 COX19 C1TM HYOU1 WDR41 VWA8 THIK CCD58 SCMC2 NEMP1 STIM1 LANC1 PREB ACOX1 C1TM ATD3B ETFB SRPRB GBB1 OSBL9 CL073 ETFA RRBP1 DVLP1 VAPA AIFM1 TTC19 YMEL1 COX2 CALX SC22B STX18 CATA

15 28 122 76 31 157 5 729

CC124 SAHH3 UBP47 NKRF RRP8 IMA5 FERM2 DOCK7 LAS1L RT31 MPP10 RT22 VPS45 PALMD RD23A NOL9 SYAP1 EFHD1 MAZ LARP1 PIMT F135A GRPE1 PTCD3 DDX50 PDIP3 MYH13 IF2B3 PESC PHAR4 TB182 NOP16 NCBP1 RT23 RBM28 UT14A NOG1 TAB1 NIBL1 E41L1 RT06 RT34 SPB1 SNTB1 SYFB TARA RRP12 RPA2 ARF YTDC2 SRP72 TPPC1 DP13A PTN23 PGAM5 ICAM5 STAU1 NOC4L SPB1 PARG CA198 SC16A RD23A IF2B2 RCL1 LTV1 RM47 SRGP2 R3HCL MPRIP HGS CLPX RENT1 RL26L ABT1 DDX27 DDX21 SC16A SC16A SIR2 FA21A ESF1 DDX31 ADT2 GRIN1 CTND1 ARFG1 STRUM ASAP1 SH24A DDX52 DIM1 UBE2O GAR1 NP1L1 LAS1L RS18 RL26 8-Sep ANK2 REPI1 GSCR2 AKAP2 ARP10 MOES RT12 RT07 TDRD3 NOLC1 EF2K ELP1 MAGD2 DDX54 F207A COBL1 PEAK1 VIP2 S6A15 SE1L2 MACD2 EIF2A SRBS1 RABE1 NUMB STRBP TBL3

RL13A NPA1P TSR1 WIPI4 BCR PWP1 TF3C5 SURF6 ALG13 TPPC9 LIN7C ARF RT27 F120A ZN512 NAT10 RT34 RT12 ACTN1 PKN2 F135A TR112 RHG35 RFC1 DDX47 ZFR PTCD3 ZN850 MBB1A ZC3HF SRP68 FUBP3 WDR3 DIP2B TPC10 AATF RT26 ADDG LYAR PLSI KLC4 LA TF3C2 CTCF REXO4 ZCHC3 NH2L1 PALM2 EZRI RADI KRI1 DHX30 EEA1 HDAC1 EZRI RT18B RS11 FBRL EEA1 KIF5C RUFY1 TBR1 HS71A ANM7 NUMBL LZTL1 CYLD FBRL DDX18 ANM1 GPTC4 UTP18 SCRIB ABR MYO6 RAI14 DDX24 PHAX HELLS ZCCHV NOG2 RS9

NDRG1 COBL NEB2 PACN2 SAS10 PWP1 MOES DIEXF U3IP2 EPHA2 CSKP ANM1 NOP58 HEAT1 NU188 DDX24 RL8 ANK3 ABCF2 SVIL UTRO U3IP2 NH2L1 SRFB1 WDR36 NOL10 RRP1B EHBP1 OGA DLG1 NHRF1 GNL3 RRP5 PWP2 COIL NOL8 NOP58 TRHY DDX18 CYFP2 PDIP2 DDX54 ECD ERBIN SWP70 MAZ NOTC2 BI2L1 ELOA1 BYST ZFYV9 RUFY2 PCBP2 DDX6 K1211 DYST CC124 DHX37 LAR1B RS14 RADI TAU ENAH PABP4 NOP56 NSUN2 RPA49

AHNK AKA12 CF132 6-Sep CYFP1 ITSN2 LIMC1 NU205 WDR43 RBM34 RPA1 RL10A NP1L4 SYK AP2A1 LBR RT05 M3K7 ARF4 DKC1 MTMR1 NCKP1 RRP12 LYAR KRR1 CSDE1 RPAC1 UBP36 RL31 TRM1L RENT1 SEC13 OCRL ICAL C1QBP MPRI ZFY16 GOGB1 TIE1 NU107 TNIK DDX10 RL18 DHX30 RT23 NOM1 RS23 RS3A RL26 NMT1 TIM44 WASF2 FCHO2 KI67 RL19 LSM1 ABI1 SHC1 KNOP1 IPO7 RS6 LYAR STAU1 PLST GAB1 RL39 NCBP1 ABCE1 ECM29 RL32 RS13 BMS1 RL3 PAK4 ELP3 RBM33 RFA3 RT17 TF3C3 OTUD4 RL32 GLO2 KIF5C SHLB2 TF3C1 NOP14 DDX56 EEA1 EZRI MYO1B SNX6 ABI2 FBRL SRP72 NOP56 RL17

6 116 23 3 125 27 22 119

RBM28 DDX18 UBN1 BCCIP DMAP1 ABL2 AP3D1 ZN658 HMGX4 EHMT1 TF3C4 DDX31 DJC11 TPGS1 ARI4B PTCD3 SP16H CYFP1 ATD3C TRRAP DDX24 GAK ARHG2

TF3C5 NOG1 UBN2 GLO2 ENAH E1B55 CLPX RM28 TF3C3 NUSAP DJC13 HUWE1 RAD18 APC1 RGAP1 PDS5B PCGF6 TAF6L ZMYM2 MBB1A KIFC1 LRC49 ECH1 APC5 POP1 RIR2 AF1Q MORC2 ARI3A SDCG3 RPGF6 PSMD4 KCMF1 SMC3 UIMC1 BAG4 STMN1 MLF2 BAG5 MYO1D RT07 NASP ZN512 LBR NASP NIP7 EHMT2 TBG1 TLN1 MEN1 SMHD1 UBR7 CYFP2 MAGD2 PESC MAN1 CAF1A ZFP91 UBR4 USO1 RM12 CAF1B DDX52 Z512B IRS4 RPAP1 KDM2B GOGA3 HMGB3 KMT2B GNAS2 PDLI5 CAF1B ZN281 BBX ZBT10 RAD21 BRD7 DNJC7 METH AL3A2 IRAK1 DJC13 BAG5 HP1B3 NAT10 NELFB LBR RNH2A HELLS DHX37 CHD8 ZKSC4 DEN4C PPM1G ZN830 MLF2 TRIM9 HS74L TXTP MOGS RT23 ESPL1 MAGD2 DPH2 ANKH1 ASF1B AQR STAU1 RT26 BRX1 KAT2A DDX54 DDX52 ML12A MYH10 TONSL RBBP4 CUX1 APC16 RT34 RT09 CSDE1 DPYL2 TANC1 P85A ASF1A Z780A ECHA PRI1 DDX54 TF3C2 ATX10 PSMD6 ARHGA DPH2 FA60A XRCC6 AN32E ATX10 ETFA

DNJC8 SP20H PTCD3 LAP2B KI67 NEUA CE170 IF4G3 GOGA2 MYH14 XRCC5 MTA3 UBR4 PP6R1 COG1 NCOR2 T22D3 CLCA DPH1 DVL2 RRP12 PARP1 SHCBP KRI1 HP1B3 RT22 KNOP1 FEN1 HSP7C HAUS5 DIAP1 MSH3 LMBL3 SMC3 KDM6A H31 HSP7C DNJC9 H31 ASF1A TAF4 INT1 RFC4 STAG2 HS105 WDCP HPBP1 GOGA2 MYSM1 PSIP1 DNJC7 KLC2 HS71L BRD2 P66B DPH1 ATX2 ECD RPB2 IPO5 IPO4 RFXAP RFC2 BAP18 MGAP ARI3A GOGA5 SC16A KI18B ADNP HMGB2 P66A EDRF1 HS71L MED14 UBP48 E2AK1 HSP72 RFC1 PB1 ARGL1 HSP76 NCOR1 CATIN CHD3 RL32 RT05 CDC27 ZMYM4 ASNS CLCA KIF4A POGZ HS74L INO1 WNK1 PRC2C ELP3 PP6R3 HYOU1 STRN4 RL3 ZN644 CDC23 TAD2B UBR7 RBBP7 H4 BAG3 SDA1 CDC16 HAUS5 CLH1 PHOCN WIZ DEK ZN691 TAF2 AAKG1 HS105 GRPE1 MYO1C RFC5 ZSC21 CTCF WAPL DIP2A WNK1 CBX1 CLH1 T2FB RFC3 RIF1 RSBN1 LAP2A HDGR2 ASNS ST134 HPBP1 CLCB LARP1 BUD31 TSR1 TBP RNH2B BUB1B PP6R1 GORS2 STRN RRP12 RT35 MSH2 MEPCE S12A2 RPAP1 C1QBP DNLI3 ZKSC8 WDR6 RBM28 TRIPB BAG2 TAF1 UBR4 RFX5 EMD MDC1 RSBNL PAXI1 HDGF NFIA DYL1 CHIP ASF1B KIF23 HSP74 WDR6 IRS4 TYY1 PSB3 ARID2 ARI4B ARFP1 DPYL5 HSP72 MSH6 SMU1 DVLP1 BAG2 DNMT1 GLYR1 UBR4 MYO1B TLE1 GNL3 RT27 BLM PDS5A T2EB RL8 RBBP5 TAF9 RPP30 NUMA1 CNN3 KCMF1 T2FA SARNP PWP1 SMC1A ELF2 KCC2D CHIP ZFP30 KMT2D RL13A TAF5 PDIP2 HAT1 EDRF1 SYDC RBM34 ELOA1 SCRIB BAG3 S23IP STRP1 CAF1A ATRX NOG2 TAF6 KRR1 ARMC1 PPP6 HSP74 STRN3 HIRA CSTF3 RPB2 GOGA2 H31 H33 TF3C1 BPTF HSP7C

2 243 26 11 182 40 29 29

RABL3 AAAT FLOT2 SCRIB IMA6 CDKAL FCHO2 WRIP1 TOIP1 HYOU1 GRPE1 IMA3 ZCCHV S20A2 IMA5 LAP2B SC16A ARF4 STT3A T106B ZFY16 NOTC2 SENP1 IMA4 MAN1 DPY30 RBM28 PSMG2 LSR S12A2 BCCIP IMA5 S19A1 AT2B1 SURF6 LMAN1 CAPON RELL1 LRBA PGRC2 MOT1 PSMG4 RBM28 AT1B3 DNJC3 SPT5H IMA7 BASP1 VPS45 TM199 NUMB NUP50 LBR RFTN1 PTK7 ADDA PGRC1 NCKP1 EMC2 CMTR1 DDX54 EMD TM87A TRIPB ADDB VAPA RLGPB CN37 VRK2 ZKSC4 LAP2B ITB1 AGAL DJB11 HDGR2 P121A PSB2 VANG1 AR13B EMD RPN2 MARK2 MTMR5 DSG2 ITB1 GLO2 NUP50 ECH1 PSB4 PSA3 GNAI3 EMC1 STT3B ACD11 LMNB2 CX057 ZKSC3 EMC4 MPRI SRPRA RGPA2 SC16A PSB4 PSB1 DEP1B RENR TFR1 ECE1 FERM2 RAB10 NISCH OCLN SSBP DHX30 AIMP2 PSMG1 MPP7 CKAP4 DSG2 STIM1 MYSM1 ZN691 RPC3 NUP85 LRC59 PTN13 PSME1 PSB6 NU205 ROCK2 SCFD1 EPN4 PDIA4 STIM1 K1143 STEA3 NIBL1 STAM2 AKA12 RBM33 PSMD7 ALKB5 PPIL2 WDR70 RAE1L CCD47 HYOU1 LEMD2 RAD50 LBR CNNM3 OST48 HAX1 VP33B TOIP1 TPPC5 AP3D1 ACSL3 ARI3A PSA4 PSB3 RGPA1 LAT1 CBPD PTK7 CYFP2 GLO2 PSME2 PSA5 YES TM209 DP13B SNX5 PLCB1 PCY1A LMNB2 DSC3 F91A1 DSC2 BAIP2 PIMT GNL3 LYRIC LAP2A CPNE8 SC24B EDF1 AN32E

VAT1 PKHA1 4F2 TRPM7 DJC16 RPN1 UNC5B RICTR ANKL2 SNX6 CXA1 SC24A LMAN1 LBR IMA7 AKP8L KBTB6 ELYS TFR1 KTN1 LMNA PSA1 LAMP1 CDCA3 LAMP1 DDRGK LMNA P121C LSG1 PSMG1 PSA1 PSA4 TMX1 DSC3 PDZD8 LIPB1 IRS4 TRI13 LANC1 PSMD6 KI67 PTN1 ASTN1 1C07 VEZA K319L TPC12 SNP29 TMX1 VRK2 DHX40 NU160 PSB7 PSA5 NBEA YKT6 GRPE1 MYO1D STBD1 SPY4 TPC13 CTND1 SMHD1 SRPRB SC16A PSMG3 1A03 CADH2 1C07 S6A15 TPPC8 VATF FEN1 PPIL4 KCNB2 SCAM1 AL3A2 DP13A TPR LMNB1 EMC1 C1QBP ZDHC5 TPPC3 TPC11 PGAM5 YTHD3 PSMG2 PSA7 LAP2B ARF WDR41 MARK3 GRP78 DJB11 RL39 STX12 P55G SRPRA NOSIP PO210 RN149 RPN2 4F2 WDR6 SNX1 NEUA MAN1 ITB1 NU153 NR3L1 CLCC1 EMC2 PTN1 DLG1 PAK4 FBRL CBPD DYST PSB2 PSA7 E1B55 P53 TYDP2 PSIP1 L2GL1 YES MYO6 TMOD3 PSB1 MYO1C 1A03 EPHA2 TECR CHP1 STX16 ACSL3 ZN724 EMD CATIN UBP7 FND3A P85B ADA10 PDIA4 DEN4C CADH2 TNIK ZKSC1 UBP7 DNJC8 PIAS2 PSA2 ESYT1 LIN7C CTNA1 RAI14 LYRIC NSF AP3B1 RFA1 MYH14 PSB7 NR3L1 FKB10 GPKOW NU155 PARP1 NU133 TARA CC115 SNP23 OST48 PSB3 F135A SRPRB S38A2 ABI1 YKT6 LMNB1 S38A1 TB22B HGS SCFD1 TFAM BAF VPS51 CTNB1 P85A CCD47 PGRC2 HDGF NUP37 T106B GOPC RFA1 PEAK1 VAMP3 PSMG4 PSA3 HYOU1 MYH10

ADDG JPH1 VAPB GORS2 CX056 NUP43 SDF2L TGFR1 CNNM3 SC22B SEC63 BI2L1 STX7 CK5P3 HMBX1 RFA3 UBIA1 STX5 CSKP NSMA3 NUP98 LCAP GOGB1 MYO1B PSMG3 VAPA IMA1 PVR MET KDIS GOGA3 SEC13 RFA2 POMP MAN1 RHG01 LAP2A CHCH2 SEH1 POMP CTNB1 ZNT1 SNX3 F169A IMB1 LAMP1 S4A7 CDC42 GCP60 LMNA NU107 PSA1 Novel Interaction

20 21 188 27 13 83 2 17 17 Known Interaction PREY-PREY

MK67I ZCCHV PKHM3 DDX56 RRS1 SNX29 BIG3 DDX21 RS17 RGPA1 RLGPB RLA1 MBB1A HPS3 DYST Interaction GLO2 RBM28 RT06 RL29 RS9 BORC5 RAE2 PURB IMB1 SYK RRBP1 EBP2 RL13 VP33A PTN1 TSR1 GNL3 PSB4 RLP24 SRP68 FANCI AKP8L RM47 SPB1 MIA40 RT31 IPO7 RBM19 GRPE1 BORC6 RRP12 DDX54 4F2 RRP5 RABX5 RL1D1 NOP2 HS71A ECI1 NKRF DHX33 RRP1B NU205 SRP72 LLPH CI072 SRP14 ESYT1 TFR1 DCTN4 RLA2 NUCL KRI1 RBGP1 ECH1 CG050 IF2B NOG1 KRR1 SRPRB STAU1 RS14 RL26 RM13 RBM28 RL39 LTV1 CLCC1 VAPB SNX3 GCP60 DHX30 TIM44 RT02 RM21 KI67 NOL8 IMP4 RL26 ELOA1 RM28 DDX24 DDX54 RL37A TFAM GRPE1 RM11 DDX31 REXO4 GRPE1 RBM28 TIM44 ECH1 PURA BRX1 GNL3 RRP5 NIP7 IMP3 LN28B HP1B3 RL10A UBIA1 VP13C LTOR5 RPF2 DIM1 AR13B RUFY3 WDR43 RT34 WDR41 SCAM1 REXO4 RL39 RM11 GLO2 TRM1L EMD PDIP3 RAE2 UBP11 PELP1 CCD86 NOP16 RL12 NOP16 STRUM TXTP MK67I GLO2 NU188 HS71A HUTH LA MBB1A RT29 RL35A SYK S11IP ESF1 RT31 RS23 RT16 SAR1A NUP93 RFIP5 SERPH BRX1 FBRL NOM1 RRP15 DDX27 CEBPZ MIO MIC60 RM43 RRBP1 MAK16 RL26L BASP1 NBEA NMNA1 RT15 RBM34 STT3A NOG1 CNBP MOV10 PCID2 EMD RL17 DD19A VAMP3 ZN512 OSB11 DDX18 RRAGA NOG1 RRP12 REPI1 MPP10 RT05 EBP2 NOL10 PK1IP NOG1 BAF BPL1 DIEXF AATF SDA1 NPM3 STT3A SMCR8 RL28 RLA0 UTP23 RFC3 RRBP1 PDIP3 CG050 SERPH RAB1C SMC3 S39A7 GSCR2 NOL6 PWP1 ATPG ARF4 CALU RRP1B CC137 WDR3 NSUN2 RL30 UCHL1 RM27 DDX51 TBL3 GTR1 BORC7 RAB9A NOC3L MRT4 ZN512 RRP12 RAB9A ZN512 GORS2 RAB11A NGDN WDR12 LCAP ATPG RS6 RS6 RT34 RT22 RT05 TRM1L PWP2 RL31 RAB11A STT3B SPB1 BASP1 SDHA ODP2 RUFY1 GTR1 RRP12 NU205 ZCCHV RCL1 NEPRO POP1 NOC4L BORC8 RTN4 RRP8 ACTY RL7L NOL9 SNX6 RAE1 H1X RL14

MCCA PUM3 RPOM LACTB NAT10 GRWD1 DEP1B SMC3 RFIP1 CG050 STAU1 KXDL1 RL26L NDKB RSBN1 WDR36 RT27 RRP8 UTP20 RL7A NISCH VATD REXO4 DHX37 STAU1 NUP93 RM28 RT18B EXOSX RPP30 RBM28 GOGA5 LTOR2 SCAM1 TPC10 GLYR1 TEX10 ZFYV9 S6A15 VAMP3 IF2A LBR PTCD3 GZF1 HEAT1 DHX30 LYAR DHX30 NOP14 RL32 SC16A HYOU1 MA7D1 RT18B PDZD8 RAB7A K1468 RAB6A CLPX GSCR2 NO66 RL36A SURF6 RFA2 NU205 SSF1 RRP1 RL36 RL18 HYOU1 NSUN2 KNOP1 RT22 STAU1 RT27 NOL12 IF6 CBPD TM55B DPM1 F135A SMC1A RRAGC SAS10 DDX18 RL3 RT10 ZKSC8 RL8 IF2B3 CALU TBCD5 RL13A RS13 FA21A LANC1 1433G ZFY16 RBM14 RM37 RT29 ABT1 BMS1 RFC1 PESC MPRI VATA NKRF NOP16 RABE1 DDX27 ECHB RS3A LANC1 TPM4 RS12 RT07 KRR1 LAS1L DHX30 STX8 DDX52 RM24 DDX10 RT25 PARN WDR18 ABCD3 VATB2 NIP7 OSBL9 KRI1 NOC2L BYST RL11 TRIPB LTOR1 SURF6 RT26 RFC4 SC16A NAT10 KRI1 RT09 DDX18 LYAR TF3C3 NOG2 CLCN7 LYAR C1QBP PTCD3 RL18A EPHA2 SNAPN RFA1 SAFB2 RT23 LRBA STX7 ACACB PRKRA DDX50 RL7 ARF4 NSF NPA1P BCCIP EMD XPOT RFA3 SNAA LTOR3 MRT4 TPPC3 AT2A2 BOP1 NVL RL19 ARF4 UBP36 RBM34 RRP1B CKAP4 VAPA RRP1B RT15 TXTP SPB1 GRPE1 GSCR2 RAB5C ECHA NPM RS8 KIF1A STX12 CTCF RS4X GRPE1 TECR TPPC9 ECH1 WASH7 HP1B3 BOP1 RRBP1 RL4 RRP5 RAB6A DD19A RBM14 IF2B RT25 GPTC4 RS24 RCN1 SNP23 VATF DDX55 RL6 RAB9A RAB11A RS6 Z286A RL5 NOP56 Primary localization Possible 6 2 131 5 2 80 22 30 196 localization

URFB1 SHSA2 ZNT6 F91A1 STEA3 PPR21 ZDH20 ZFY16 DEN4C VP13B TJAP1 TECR T11L1 SC16A HPBP1 PGAM5 RBM33 RELL1 RABL3 DNJA3 GHC1 TTC4 T11L2 UNC5B RIC1 ALG1 UBR4 NUP93 TRIPB DHB4 RFOX2 VPS8 L2GL1 KRI1 S39AE TM87A ARFG2 PACE1 K319L GCP6 GCP5 DP13B GCC2 GNL3 RCN1 PK3C3 UBP32 F219A DSG2 ARL1 ESYT2 PWP1 DDX54 CPNE8 KI16B STX12 BAG5 TBB2B VANG1 STX5 ANR27 DHX30

SNX29 RBM14 LTOR1 FCHO2

RUFY1 SNX30 MPCP PDIP3 LBR SMCR8 NSUN2 OZF HLAH SFXN4 NTPCR ATD3B NIBL1 MPP7 SMAP2 NOG1 FA83B GOGA3 SPB1 RLGPB TPC11 ZCCHV NTPCR GCP2 HP1B3 FANCI M21D2 PDXD1 NBEA SCYL2 NFXL1 CHAP1 SCFD1 NAT10 GRAP1 OCRL ZNT5 RRP12 GRIP1 AP3M1 RBNS5 WDR20 RABL6 AR13B NU205 LAT1 ARF4 FACR1 HIG1A HN1 TCPG TPC12 WASH7 DHB12 ATPG NOTC2 TECR VPS16 VAMP3 WDR11 DYL1 TBCD5 MIC60 TM230 AT2C1 SPB1 PTN23 FERM2 OCLN ITSN1

AP2M1 TPPC5 EFNB1 CYFP2 GOGA5 TPC6B SC23A GOLP3 STT3B MON2 SC23B TBAL3 CEP41 ARF STON2 FA21A HIG1A PYR1 CI072 STRUM ALG1 T11L2 SNX2 EPN4 S38A2 DAB2 PEAK1 RABE1 TCPD TCPE S61A1 4F2 NDRG1 AP1AR RS27L TBA1C CMC2 BAG5 CALU COPE TPPC9 ATP7A S6A15 ECHA KS6C1 VEZA STAM1 GORAB RRP1B RT35 OSBL9 SNAA BAG2 TBB6 VP33A GOGB1 DPYL4 CDCA3 ARFG3 LRP8 TMF1 SC16A PFKAL VAT1 ARFP1 NCOA7 HM13 EHBP1 CALU M2OM GCC1 FLOT2 ARL1 COPB2 SAR1A TB22A DEN6A S20A2 VAMP8 VAMP2 NUP93 DPYL3 PI3R4 SERPH DEP1B HOOK3 RS27 PFD1 SEC13 AT2A2 TBC23 OSB11 DLG1 RHG01 TXND9 SERPH SEM4C S38A1 RAB1C SMC1A NSF AP2B1

TBA1B LYAR AKA12 OSB10 KCNB2 SCRIB AP1B1 PDCD5 MLF2 GCP60 RM11 WDR41 UBP8 AT1B3 GALT2 COPD AT133 ABCE1

PGAM5 MOT1 DDX56 SYAP1 PICAL RAB14 RBM28 AP1M1 RAB1A OST48 SP1 PFD2 LONM BAG2 SPE39 S12A2 GGA3 BORC6 TGON2 GOGA4 TPD52 TBA1A ABI2 ARF4 TGON2 VP33B PTN1 TBG1 EMD RL32 WDR6 EPHA4 AFG32 PFKAM VPS54 SAHH3 DYST MOT1 TCPB TCPZ CKAP4 COPB HACD3 TPC2B TBA1A KLC4 GORS2 ITB1 RT29 DPM1 TXTP VPS18 SNX27 BASP1 CTR2 TBG1 GOPC RM37 VAPA CN37 ARAF TBB2A TM263 TBA1C BMP2K VPS45 S29A1 AGFG1 SC24B RAB7A CAMP2 SMC3 F135A TPC2L RPN2 VAMP7 ESYT1 ATP7B TB22B NUMB BET1L SC24A CMC2 PFD3 DPYL1 BASI COPG2 LTOR3 PACN2 SPY4 P4HA1 SNAPN CLCN7 PFD5 DIC NUD19 F234B

TPR PGRC1 LIN7C LMAN1 ADT3 DJB11 TCPA SMC3 S12A6 STX8 VTI1B ZFYV9 VAPB MRP TIM13 GCP3 BAG2 S12A4 GOSR1 T106B AT2B1 RFA3 CYFP1 DDX18 VPS51 RAB21 VAMP4 STT3A TPC10 MTMR6 SYNRG SNX3 SNX4 DIC YMEL1 TCPQ PFD6 LCAP MUC18 DPYL2 PDCL3 ITSN2 SC6A8 TCPH TPPC4 CCD53 TPD54 CBPD 1A03 COPA DP13A VATA HYOU1 LBR GSCR2 TPPC1 YKT6 VDAC2 ODP2 TFR1 TPPC8 STAM2 DHB4 RT12 RFA2 EPN1 GAK S39AA ATPG SCAM3 CSKP P85A TIM21 PFD5 TCPW TIM44 TCPH TCPA K1468 MAN1 DPM1 RBM34 SC16A SYK AP4E1 HYOU1 STX10 RAB6A BCCIP EPHA2 VATD TPPC3 SMC3 CXA1 UBIA1 TIM44 TBAL3 SMC1A DHCR7 VATB2 MZT2B PFD2 SCO2 YTHD3 NISCH S12A7 SURF4 MPRI ECHA TCPE AUP1 TPC13 BIG2 ILVBL GGA1 FYV1 TFR1 UH1BL GTR1 A4 DNJA1 PFD1 TCPQ SYK WDR11 S6A15 NOP16 EPN2 DNJA1 TCPG XPOT RT12 NTPCR AIMP1 KDIS AP2A1 HAX1 EMD SNX6 SNX5 ZFPL1 DPYL5 DMXL1 SNP29 SCO2 TCPD NUMBL SURF6 TCPB ZC21A SMC1A FA21C PGRC1 SPY1 AP2A2 SNX2 PGRC2 TXND9 DNJB6 GCP4 PFD6 WDR7 1A03 SCAM1 PPR37 4F2 ARHGA HS71A PFKAP AP4S1 AT2B1 STX16 USO1 BASI AP3D1 GALT2 RCAS1 SNX1 SNX6 SNP23 SRPRB CTND1

IRS4 AKP8L PFD1 ZNT1 KIF5C ARF4 PFD3 GBB2 ATPG RM12 S4A7 SAHH2 DNJA2 PFKAP PDCD5 CHIP S23IP STX6 COPE AP1G1 ADT2 EFTU PFD4 PFD5 LRBA WWOX SC16A STX7 TCPZ SNX1 AT2A2 HGS EMD AP3B1 LRBA WASH3 NOTC2 EPN4 GOPC TECR TBA1A TBG1 TGON2 GCP60

67 14 23 26 2 6 42 25 330

Supplementary Figure 2: Overview of high-confi dence interactions of 18 bona fi de cellular localization markers. The individual distribution of interactions detected with of AP-MS and BioID approaches with MAC-tagged 18 bona fi de cellular localization markers. The novel interactions are presenting in pink lines and the blue line repre- sents the known interaction. Prey-prey interactions represent via dash lines. The nodes are color-coded based on the localization rank obtained from the CellWhere database (key: dark green = primary cellular localization for the corresponding protein, light green = possible localization, grey = diff erent or localization assigned for the protein). Venn’s diagram compares the number of interactions between AP-MS and BioID methods, overlap showing the number of common interactions.

21 bioRxiv preprint doi: https://doi.org/10.1101/264788; this version posted February 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

An integrated approach to comprehensively map the molecular context of proteins

EEA1 AIFM1 RAB11A RAB9A GOGA2 H31 FBRL RS6 LMNA CALX TBG1 CATA HSP7C EZRI LAMP1 TGON2 TBA1A PSA1 HIS_ACID_PHOSPHAT_1 AIFM1 CATA HSP7C PSA1 RAB11A RAB9A CALX FBRL H31 RS6 GOGA2 EEA1 LMNA TBA1A TBG1 LAMP1 EZRI TGON2 ACYL_COA_DH_1 ARM_REPEAT ACYL_COA_DH_2 ERV_ALR PROTEIN_KINASE_TYR ATPASE_DELTA FTHFS_1 IG_LIKE FTHFS_2 CYTO_HEME_LYASE_1 IG_MHC CYTO_HEME_LYASE_2 CLP_PROTEASE_HIS THIOREDOXIN_2 CLP_PROTEASE_SER COX2 ER_TARGET COX2_CUA COX2_TM THIOREDOXIN_1 COX5B_1 COX5B_2 MIR CYTC GLFV_DEHYDROGENASE CARBOXYPEPT_ZN_1 G_DYNAMIN_1 GED CARBOXYPEPT_ZN_2 G_ERA ETF_BETA LAMP_1 SHMT COPROGEN_OXIDASE FKBP_PPIASE G_TR_2 IF2 CADHERIN_2 ADENYLATE_KINASE PHOSPHAGEN_KINASE CADHERIN_1 PHOSPHAGEN_KINASE_C PHOSPHAGEN_KINASE_N LAMP_3 LETM1_RBD SAM_MT_TRM10 LAMP_2 2FE2S_FER_2 4FE4S_MOW_BIS_MGD VINCULIN_1 COMPLEX1_75K_1 COMPLEX1_75K_2 SEMA COMPLEX1_75K_3 COMPLEX1_49K ENOYL_COA_HYDRATASE COMPLEX1_30K COMPLEX1_51K_1 ATPASE_GAMMA COMPLEX1_51K_2 COMPLEX1_24K 3HCDH NUCLEASE_NON_SPEC APAG S5A_REDUCTASE INSULINASE POTRA WW_DOMAIN_1 FRD_SDH_FAD_BINDING SSB WW_DOMAIN_2 AA_TRNA_LIGASE_I FCP1 DYNEIN_LIGHT_1 UBIQUITIN_CONJUGAT_1 G_DYNAMIN_2 BIOTINYL_LIPOYL HEXOKINASE_1 HEXOKINASE_2 LIPOYL MIRO P5CR NA_NEUROTRAN_SYMP_3 CHCH SOLCAR NA_NEUROTRAN_SYMP_2 ETF_ALPHA DNAJ_1 NA_NEUROTRAN_SYMP_1 4FE4S_FER_1 4FE4S_FER_2 SECY_2 UBIQUITIN_CONJUGAT_2 TUDOR SECY_1 BIOTINYL_LIPOYL ZF_C2H2_AKAP95 YTH AAA ABC_TM1F EF_HAND_2 LIPOYL ZF_CR EF_HAND_1 HIG1 ATPASE_NA_K_BETA_1 ARF CKK TCP1_1 EGF_1 TCP1_2 TCP1_3 EGF_3 TUBULIN FAD_PCMH EGF_2 CATALASE_1 CATALASE_2 PX CATALASE_3 G6P_DEHYDROGENASE LNR HMG_COA_LYASE PYR_CT ATPASE_E1_E2 LON_N LON_PROTEOLYTIC ASX_HYDROXYL LON_SER NUDIX_BOX EGF_CA PNPLA ACYLTRANSF_C_1 ACB_2 ACYLTRANSF_C_2 ADH_SHORT ACB_1 THIOLASE_3 THIOLASE_1 ENTH THIOLASE_2 NUDIX TRBD GRPE ENOYL_COA_HYDRATASE RICIN_B_LECTIN ACTINS_2 ACTINS_ACT_LIKE PH_BEACH ZF_A20 FIP_RBD BEACH ZF_CCHC G10_2 MHD G10_1 HOMEOBOX_1 GOLD SAM_MT_C5 C5_MTASE_2 ZINC_PROTEASE C5_MTASE_1 NET ADAM_MEPRO WAPL PPM_1 DISINTEGRIN_2 ELM2 TBOX_3 INTEGRIN_BETA TBOX_2 BHLH CBS TFIIE_BETA_C TAFH ALPHA_GALACTOSIDASE FATC FAT ATPASE_NA_K_BETA_1 BROMODOMAIN_2 ADD GS DNA_LIGASE_A1 DNA_LIGASE_A2 ZF_RING_2 DNA_LIGASE_A3 DDT IQ CTF_NFI_1 CTF_NFI_2 TH1 ETS_DOMAIN_1 ETS_DOMAIN_2 MYOSIN_MOTOR ETS_DOMAIN_3 CTLH C2 BIN3_SAM HTH_CENPB CALRETICULIN_REPEAT GATA_ZN_FINGER_1 SCD CALRETICULIN_1 ZF_CCHHC ZF_CW CALRETICULIN_2 PRE_SET HMG_BOX_1 PLD TFIID HOMEOBOX_2 FE2OG_OXY RFX_DBD HISTONE_H3_1 LRR HISTONE_H3_2 CHROMO_1 SUBTILASE_HIS GATA_ZN_FINGER_2 BROMODOMAIN_1 SUBTILASE_SER MBT CUT SAM_ICMT DNA_MISMATCH_REPAIR_2 BAH GLYCOSYL_HYDROL_F2_1 CHROMO_2 HMG_BOX_2 GLYCOSYL_HYDROL_F2_2 SAP DUSP GDA1_CD39_NTPASE PI3_4_KINASE_3 KINESIN_MOTOR_1 THIOLASE_1 KINESIN_MOTOR_2 BTB THIOLASE_2 XPG_2 SWIRM THIOLASE_3 XPG_1 PARP_ALPHA_HD DISINTEGRIN_1 PARP_ZN_FINGER_1 PARP_ZN_FINGER_2 UDPGT SCAN_BOX PWWP MSP UBIQUITIN_2 LISH ATPASE_DELTA REKLES ZF_UBR BTB ZF_CXXC SET SRCR_1 FYRC FYRN SRCR_2 POST_SET ZF_PHD_2 ZF_RING_1 ZF_PHD_1 ARID TCP1_1 EPHD SANT TCP1_2 FHA_DOMAIN KRAB TCP1_3 BRCT ZINC_FINGER_C2H2_1 G_TR_1 ZINC_FINGER_C2H2_2 ZF_DAG_PE_1 G_TR_2 GNAT TPR_REGION ADH_SHORT TPR CSA_PPIASE_1 DNAJ_2 CSA_PPIASE_2 HTH_CROC1 DNAJ_1 PINIT ZF_SP_RING PHOSPHOFRUCTOKINASE SAM_MT_RRMJ MATH RANBD1 SOLCAR IF TUBULIN IBB ZF_RANBP2_1 TUBULIN_B_AUTOREG ZF_RANBP2_2 ARM_REPEAT

AAA NUP_C ULP_PROTEASE ZF_CR U_BOX SRP54 ATP_GRASP AMP_BINDING STEROL_REDUCT_1 CPSASE_1 STEROL_REDUCT_2 LEM_LIKE

CPSASE_2 LEM DZF RIBOSOMAL_S27E RNA_POL_D_30KD HTH_LA

RBD YTH COLD_SHOCK OTU ZF_DAG_PE_1 NOP ZF_DAG_PE_2 KH_TYPE_1 UBA RNA_POL_BETA HIG1 MAGE CARBAMOYLTRANSFERASE IMPORTIN_B_NT FIBRILLARIN

DIHYDROOROTASE_1 G_PATCH RIBOSOMAL_L44E

DIHYDROOROTASE_2 PUM_HD RNA_POL_PHAGE_1 GATASE_TYPE_1 RNA_POL_PHAGE_2 COA_CT_CTER MFS COA_CT_NTER CPSASE_1 PCMT RIBOSOMAL_S17E RIBOSOMAL_L35AE ZF_FYVE RIBOSOMAL_L13E RIBOSOMAL_L11 ZINC_FINGER_C2H2_2 RIBOSOMAL_L1E NOL1_NOP2_SUN ZINC_FINGER_C2H2_1 RIBOSOMAL_S8E RIBOSOMAL_S15 ZF_C2H2_AKAP95 RIBOSOMAL_S4E RIBOSOMAL_S24E LEM RIBOSOMAL_L30E_1 RIBOSOMAL_L30E_2 LEM_LIKE RIBOSOMAL_L5 RIBOSOMAL_S5 IF S5_DSRBD RIBOSOMAL_L6E IBB BIOTIN RIBOSOMAL_L24E KRAB RIBOSOMAL_L36E CPSASE_2 HMG_BOX_2 BC ATP_GRASP RANBD1 RIBOSOMAL_L30 PUM HOMEOBOX_2 BRIX S4 ZN_HOOK RIBOSOMAL_L39E RIBOSOMAL_S12 SCAN_BOX RIBOSOMAL_L22 RIBOSOMAL_S4 P53 RIBOSOMAL_L7AE RIBOSOMAL_S3AE SSB RIBOSOMAL_S11 RIBOSOMAL_S6E MATH RIBOSOMAL_L31E RIBOSOMAL_L1 USP_1 RIBOSOMAL_L32E RIBOSOMAL_L19E USP_2 RIBOSOMAL_L18E MI USP_3 RRNA_A_DIMETH SAM_RNA_A_N6_MT ZF_UBR RTC RIBOSOMAL_L24 ZF_ZZ_1 R3H FBOX ZF_ZZ_2 TFIIS_N RIBOSOMAL_L3 RIBOSOMAL_L13 UIM RIBOSOMAL_L2 VWFA G_BMS1 DEAD_ATP_HELICASE Q_MOTIF BAG HELICASE_ATP_BIND_1 IRS_PTB HELICASE_CTER PUA DS_RBD PH_DOMAIN PPR G_CP PROTEIN_KINASE_DOM WD_REPEATS_REGION WD_REPEATS_1 PROTEIN_KINASE_ATP WD_REPEATS_2 3HCDH PROTEIN_KINASE_ST HRDC H15 LIM_DOMAIN_2 DEAH_ATP_HELICASE SAM_MT_TRM1 LIM_DOMAIN_1 S1 G_OBG TPR SAM_MT_RSMB_NOP RRM CLATHRIN_LIGHT_CHN_1 ZF_C2HC_LYAR DDHD CLATHRIN_LIGHT_CHN_2 JMJC PROTEASOME_ALPHA_1 HSP70_3 PROTEASOME_ALPHA_2 PROTEASOME_BETA_1 HSP70_2 PROTEASOME_BETA_2 HSP70_1 TH1 MYOSIN_MOTOR ANK_REPEAT IQ Domains (# of preys contain) Domains (# of preys contain) HECT ADOMET_ACTIVATION CALPONIN_1 B12_BINDING B12_BINDING_NTER CALPONIN_2 HCY PTERIN_BINDING COLD_SHOCK BUB1_N DAD KINESIN_LIGHT GATASE_TYPE_2 CLATHRIN_LIGHT_CHN_1 CHCR CLATHRIN_LIGHT_CHN_2 RASGEF_CAT I_LWEQ RASGEF_NTER B302_SPRY MAGE COS HSP70_1 AH CHCR BAG GATASE_TYPE_2 HSP70_2 HSP70_3 FHA_DOMAIN RA C2_TENSIN SEPARIN PPASE_TENSIN DEP RIBORED_SMALL CBS FH2 WH1 GBD_FH3 CNMP_BINDING_3 SH3 DIX ALDEHYDE_DEHYDR_CYS SH2 ALDEHYDE_DEHYDR_GLU ZF_BBOX SER_THR_PHOSPHATASE DNAJ_2 PPM_2 RNA_POL_BETA ZF_ZZ_1 W2 ZF_ZZ_2 SOHO HP MI NMT_1 NMT_2 TSC22 SIRTUIN WH1 STATHMIN_3 DHR_1 DHR_2 STATHMIN_2 CAP_GLY_1 BAR STATHMIN_1 B5 LIM_DOMAIN_1 U_BOX LIM_DOMAIN_2 FF PDZ FERM_2 FERM_1 FERM_1 TAU_MAP_2 TAU_MAP_1 FERM_2 RPEL FERM_3 DEATH_DOMAIN CAP_GLY_2 G_SEPTIN KH_TYPE_1 FERM_3 WH2 CH ACTININ_1 ACTININ_2 FN3 CH SAM_MT_PRMT MACRO DH_1 KINESIN_LIGHT ACTININ_1 DH_2 WW_DOMAIN_2 ACTININ_2 WW_DOMAIN_1 USP_1 RIBOSOMAL_S15 USP_2 USP_3 S100_CABP HEME_OXYGENASE RINT1_TIP20 TBOX_1 TNASE_2 UBX TBOX_2 VAST ZF_MYND_1 TBOX_3 ZF_MYND_2 MIS18 ANK_REP_REGION TNASE_3 CALRETICULIN_1 AA_TRNA_LIGASE_II CALRETICULIN_2 GUANYLATE_CYCLASE_1 EUKARYOTIC_PORIN GUANYLATE_CYCLASE_2 CNMP_BINDING_1 CHCH CNMP_BINDING_2 CALRETICULIN_REPEAT BRCT RETICULON ZF_RING_1 MIRO ZF_RING_2 ALPHA_KINASE ADENYLATE_KINASE CNH CBM20 G_ERA MTTASE_N MTTASE_RADICAL DHFR_1 TRAM FN3 DHFR_2 SAM_DOMAIN CRAL_TRIO COX2 TSP1 L_LECTIN_LIKE COX2_CUA INTEGRIN_BETA LONGIN COX2_TM ACB_1 ACB_2 INSULINASE GOLD MSP FTHFS_1 CARBOXYPEPT_ZN_1 CARBOXYPEPT_ZN_2 FTHFS_2 C2 PARP_CATALYTIC AA_TRNA_LIGASE_I WWE ZF_C3H1 POTRA SHD RICIN_B_LECTIN WD_REPEATS_2 EPHRIN_RBD_2 EPHRIN_RBD_1 WD_REPEATS_REGION SEMA PI3_4_KINASE_2 WD_REPEATS_1 PI3_4_KINASE_1 SEC7 G_CP AH EUKARYOTIC_PORIN STEROL_REDUCT_1 BPTI_KUNITZ_2 BPTI_KUNITZ_1 STEROL_REDUCT_2 A4_INTRA A4_EXTRA RIBOSOMAL_L32E CS HMA_2 H15 HMA_1 LDLRA_2 HEAT_REPEAT LDLRA_1 TPP_ENZYMES IMPORTIN_B_NT LYSM SURF4 RIBOSOMAL_S9 MARCKS_1 MARCKS_2 HISTONE_H4 PI3K_C2 GAE HISTONE_H3_1 PIK_HELICAL PIPK HISTONE_H3_2 GAT LDLRB RRM GRIP TBC_RABGAP DS_RBD SPR RIBOSOMAL_L39E VHS ENTH RIBOSOMAL_L24 UIM MHD PPR ATPASE_E1_E2 ARFGAP S5_DSRBD EH RHODANESE_3 RIBOSOMAL_S5 CLAT_ADAPTOR_M_1 CLAT_ADAPTOR_M_2 HTH_LA HEAT_REPEAT FE2OG_OXY DEAH_ATP_HELICASE CUE OSBP RIBOSOMAL_S12E BSD ADOHCYASE_1 FRD_SDH_FAD_BINDING ADOHCYASE_2 PKD RIBOSOMAL_L13 MANSC CONNEXINS_1 RIBOSOMAL_S2_1 CONNEXINS_2 MARVEL G_OBG DHHC IG_LIKE BRIX IG_MHC V_SNARE UCH_1 T_SNARE NDP_KINASES PX GAR RIBOSOMAL_S6E EPH_LBD RECEPTOR_TYR_KIN_V_1 SAP RECEPTOR_TYR_KIN_V_2 BEACH TPR_REGION PH_BEACH FN2_1 APAG FN2_2 NA_NEUROTRAN_SYMP_1 RIBOSOMAL_S12 NA_NEUROTRAN_SYMP_2 NA_NEUROTRAN_SYMP_3 HELICASE_ATP_BIND_1 TYR_PHOSPHATASE_PTP TYR_PHOSPHATASE_2 HELICASE_CTER TYR_PHOSPHATASE_1 VWFA DEAD_ATP_HELICASE QOR_ZETA_CRYSTAL SAR1 Q_MOTIF RUN BRO1 ZF_C2HC_LYAR ZF_FYVE ABC_TRANSPORTER_1 NOP ABC_TRANSPORTER_2 AA_TRNA_LIGASE_II UBA VPS9 SUGAR_TRANSPORT_1 UBIQUITIN_2 SUGAR_TRANSPORT_2 MFS RIBOSOMAL_S17 RAB SERPIN RIBOSOMAL_L7AE ER_TARGET ARF SAM_MT_PRMT ATPASE_GAMMA ZINC_PROTEASE DZF ATPASE_ALPHA_BETA EF_HAND_1 FIBRILLARIN EF_HAND_2 PIPLC_X_DOMAIN RIBOSOMAL_S13_1 PIPLC_Y_DOMAIN LAMP_2 RIBOSOMAL_S13_2 LAMP_3 RHO PABC KIND NA_DICARBOXYL_SYMP_1 LON_N NA_DICARBOXYL_SYMP_2 KA1 LON_PROTEOLYTIC LAMP_1 CADHERIN_1 LON_SER CADHERIN_2 THIOREDOXIN_1 ACTINS_2 THIOREDOXIN_2 IRS_PTB ACTINS_ACT_LIKE DDENN DENN G_PROTEIN_GAMMA UDENN SH2 NUDIX PROTEIN_KINASE_ATP PROTEIN_KINASE_ST FAD_PCMH ZF_DAG_PE_2 S5A_REDUCTASE CATALASE_1 RAPGAP CRIB CATALASE_2 AGC_KINASE_CTER IMD CATALASE_3 ZU5 RHOGAP PROTEASOME_ALPHA_1 PH_DOMAIN F_BAR PROTEASOME_ALPHA_2 SH3 PROTEIN_KINASE_TYR PROTEASOME_BETA_1 GUANYLATE_KINASE_1 GUANYLATE_KINASE_2 PROTEASOME_BETA_2 L27 PPASE_MYOTUBULARIN RAB LNR ASX_HYDROXYL GRPE EGF_CA EGF_3 BPL_LPL_CATALYTIC EGF_2 EGF_1 PID PROTEIN_KINASE_DOM PAL_HISTIDASE PDZ LRR 1433_1 ANK_REP_REGION 1433_2 1 20 ANK_REPEAT AP-MS BioID

Supplementary Figure 3: Hierarchical clustering the domain containing of the HCIPs of 18 localization markers. The DAVID bioinformatics recourses 6.8 database (https://david.ncifcrf.gov/) were used for interaction domain containing analysis to generate the value count matrix for clustering (P < 0.01). The color intensities indicate the sum of domain containing count.

22 bioRxiv preprint doi: https://doi.org/10.1101/264788; this version posted February 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Liu et al.

ab

(Baits)

CATA EEA1 EZRI LAMP1 AIFM1 RAB9A HSP7C H31 FBRL RS6 LMNA CALX TGON2 TBA1A TBG1 RAB11A GOGA2 PSA1

GO:1902965~regulation of protein localization to early endosome CATA GOGA2 PSA1 CALX LAMP1 EZRI H31 FBRL RS6 EEA1 TGON2 LMNA AIFM1 RAB9A HSP7C TBG1 TBA1A RAB11A

GO:1902966~positive regulation of protein localization to early endosome GO:1902946~protein localization to early endosome CATA GOGA2 AIFM1 EZRI EEA1 RAB11A LAMP1 CALX RAB9A TGON2 HSP7C H31 LMNA FBRL RS6 TBA1A TBG1 PSA1 GO:0072329~monocarboxylic acid catabolic process GO:0098632~protein binding involved in cell-cell adhesion GO:0006631~fatty acid metabolic process GO:2000643~positive regulation of early endosome to late endosome transport GO:0019752~carboxylic acid metabolic process GO:1903651~positive regulation of cytoplasmic transport GO:0009062~fatty acid catabolic process GO:0043436~oxoacid metabolic process GO:0098631~protein binding involved in cell adhesion GO:0036010~protein localization to endosome GO:0032787~monocarboxylic acid metabolic process GO:0006082~organic acid metabolic process GO:1903337~positive regulation of vacuolar transport GO:0044712~single-organism catabolic process GO:2000641~regulation of early endosome to late endosome transport GO:0044242~cellular catabolic process GO:0098641~cadherin binding involved in cell-cell adhesion GO:0006635~fatty acid beta-oxidation GO:1903649~regulation of cytoplasmic transport GO:0055114~oxidation-reduction process GO:0006629~lipid metabolic process GO:0045022~early endosome to late endosome transport GO:0030258~lipid modification GO:0098927~vesicle-mediated transport between endosomal compartments GO:0044255~cellular lipid metabolic process GO:0045296~cadherin binding GO:0016042~lipid catabolic process GO:1903335~regulation of vacuolar transport GO:0001676~long-chain fatty acid metabolic process GO:0008360~regulation of cell shape GO:0008610~lipid biosynthetic process GO:0051259~protein oligomerization GO:0050839~cell adhesion molecule binding GO:0061028~establishment of endothelial barrier GO:0046395~carboxylic acid catabolic process GO:0016054~organic acid catabolic process GO:0072665~protein localization to vacuole GO:0044282~small molecule catabolic process GO:0001885~endothelial cell development GO:0033540~fatty acid beta-oxidation using acyl-CoA oxidase GO:0008202~ metabolic process GO:0044877~macromolecular complex binding GO:0022614~membrane to membrane docking GO:0000038~very long-chain fatty acid metabolic process GO:0044283~small molecule biosynthetic process GO:0045446~endothelial cell differentiation GO:0008206~bile acid metabolic process GO:1901029~negative regulation of mitochondrial outer membrane permeabilization involved in apoptotic signaling pathway GO:0044743~intracellular protein transmembrane import GO:0004579~dolichyl-diphosphooligosaccharide-protein glycotransferase activity GO:0065002~intracellular protein transmembrane transport GO:0043484~regulation of RNA splicing GO:0071806~protein transmembrane transport GO:0030855~epithelial cell differentiation GO:0006690~icosanoid metabolic process GO:0055088~lipid GO:0004576~oligosaccharyl activity GO:0022604~regulation of cell morphogenesis GO:0006694~steroid biosynthetic process GO:1901093~regulation of protein homotetramerization GO:0016032~viral process GO:1901091~negative regulation of protein tetramerization GO:0044764~multi-organism cellular process GO:1901094~negative regulation of protein homotetramerization GO:1901090~regulation of protein tetramerization GO:0016758~transferase activity, transferring hexosyl groups GO:0044419~interspecies interaction between organisms GO:0016561~protein import into peroxisome matrix, translocation

GO:0044403~symbiosis, encompassing mutualism through parasitism GO:0008611~ether lipid biosynthetic process GO:0046504~glycerol ether biosynthetic process GO:0016070~RNA metabolic process GO:0016560~protein import into peroxisome matrix, docking GO:0016757~transferase activity, transferring glycosyl groups GO:1901503~ether biosynthetic process GO:0042791~5S class rRNA transcription from RNA polymerase III type 1 promoter GO:0019395~fatty acid oxidation GO:0042797~tRNA transcription from RNA polymerase III promoter GO:0034440~lipid oxidation GO:0007031~peroxisome organization GO:0009304~tRNA transcription GO:0072330~monocarboxylic acid biosynthetic process GO:0016509~long-chain-3-hydroxyacyl-CoA dehydrogenase activity GO:0046394~carboxylic acid biosynthetic process GO:0098781~ncRNA transcription GO:0016053~organic acid biosynthetic process GO:0009303~rRNA transcription GO:0016558~protein import into peroxisome matrix GO:0006732~coenzyme metabolic process GO:0046933~proton-transporting ATP synthase activity, rotational mechanism GO:0006399~tRNA metabolic process GO:0006625~protein targeting to peroxisome GO:0006383~transcription from RNA polymerase III promoter GO:0072663~establishment of protein localization to peroxisome GO:0072662~protein localization to peroxisome GO:0008334~histone mRNA metabolic process GO:0043574~peroxisomal transport GO:0019829~cation-transporting ATPase activity GO:0033539~fatty acid beta-oxidation using acyl-CoA dehydrogenase GO:0045070~positive regulation of viral genome replication GO:0051186~cofactor metabolic process GO:0000154~rRNA modification GO:0006699~bile acid biosynthetic process GO:0006693~prostaglandin metabolic process GO:0009451~RNA modification GO:0006692~prostanoid metabolic process GO:0044769~ATPase activity, coupled to transmembrane movement of ions, rotational mechanism GO:0070542~response to fatty acid GO:0043902~positive regulation of multi-organism process GO:0051289~protein homotetramerization GO:0044085~cellular component biogenesis GO:0036109~alpha-linolenic acid metabolic process GO:0033559~unsaturated fatty acid metabolic process GO:0022853~active ion transmembrane transporter activity GO:0006613~cotranslational protein targeting to membrane GO:0051262~protein tetramerization GO:0070972~protein localization to endoplasmic reticulum GO:0006066~alcohol metabolic process GO:0006790~sulfur compound metabolic process GO:0006412~translation GO:1901615~organic hydroxy compound metabolic process GO:0030520~intracellular estrogen receptor signaling pathway GO:0015077~monovalent inorganic cation transmembrane transporter activity GO:0043043~peptide biosynthetic process GO:0021592~fourth ventricle development GO:0006518~peptide metabolic process GO:0051568~histone H3-K4 methylation GO:0021678~third ventricle development GO:0043604~amide biosynthetic process GO:0043686~co-translational protein modification GO:0022804~active transmembrane transporter activity GO:0043603~cellular amide metabolic process GO:0097485~neuron projection guidance GO:2000059~negative regulation of protein ubiquitination involved in ubiquitin-dependent protein catabolic process GO:1901566~organonitrogen compound biosynthetic process GO:0035966~response to topologically incorrect protein GO:0006986~response to unfolded protein GO:0042625~ATPase coupled ion transmembrane transporter activity GO:0032543~mitochondrial translation GO:0034976~response to endoplasmic reticulum stress GO:0070125~mitochondrial translational elongation GO:0010033~response to organic substance GO:0035967~cellular response to topologically incorrect protein GO:0070126~mitochondrial translational termination GO:0030968~endoplasmic reticulum unfolded protein response GO:0034620~cellular response to unfolded protein GO:0032403~protein complex binding GO:0006415~translational termination GO:0036498~IRE1-mediated unfolded protein response GO:0006414~translational elongation GO:0007029~endoplasmic reticulum organization GO:0032386~regulation of intracellular transport GO:0043624~cellular protein complex disassembly GO:0030433~ER-associated ubiquitin-dependent protein catabolic process GO:0015078~hydrogen ion transmembrane transporter activity GO:0043241~protein complex disassembly GO:0036503~ERAD pathway GO:0060628~regulation of ER to Golgi vesicle-mediated transport GO:0032527~protein exit from endoplasmic reticulum GO:0032984~macromolecular complex disassembly GO:0006487~protein N-linked glycosylation GO:0007005~ organization GO:0071569~protein ufmylation GO:0042626~ATPase activity, coupled to transmembrane movement of substances GO:0022411~cellular component disassembly GO:0090435~protein localization to nuclear envelope GO:0071786~endoplasmic reticulum tubular network organization GO:0010467~ GO:0034975~protein folding in endoplasmic reticulum

GO:0043933~macromolecular complex subunit organization GO:0030970~retrograde protein transport, ER to GO:0016820~ activity, acting on acid anhydrides, catalyzing transmembrane movement of substances GO:1903513~endoplasmic reticulum to cytosol transport GO:0071822~protein complex subunit organization GO:1902235~regulation of endoplasmic reticulum stress-induced intrinsic apoptotic signaling pathway GO:0032781~positive regulation of ATPase activity GO:0019058~viral life cycle GO:1903573~negative regulation of response to endoplasmic reticulum stress GO:0015405~P-P-bond-hydrolysis-driven transmembrane transporter activity GO:0016071~mRNA metabolic process GO:0006984~ER-nucleus signaling pathway GO:0010498~proteasomal protein catabolic process GO:0019080~viral gene expression GO:0030163~protein catabolic process GO:0043161~proteasome-mediated ubiquitin-dependent protein catabolic process GO:0044033~multi-organism metabolic process GO:0009894~regulation of catabolic process GO:0015399~primary active transmembrane transporter activity GO:0042254~ribosome biogenesis GO:0051168~nuclear export GO:0071426~ribonucleoprotein complex export from nucleus GO:0034660~ncRNA metabolic process GO:0022411~cellular component disassembly GO:0006364~rRNA processing GO:0071166~ribonucleoprotein complex localization GO:0043492~ATPase activity, coupled to movement of substances GO:0006999~nuclear pore organization GO:0016072~rRNA metabolic process GO:0046931~pore complex assembly GO:0051292~nuclear pore complex assembly GO:0034470~ncRNA processing GO:0006998~nuclear envelope organization

GO:0006396~RNA processing GO:0006997~nucleus organization GO:0008324~cation transmembrane transporter activity GO:0000972~transcription-dependent tethering of RNA polymerase II gene DNA at nuclear periphery GO:0000184~nuclear-transcribed mRNA catabolic process, nonsense-mediated decay GO:0007077~mitotic nuclear envelope disassembly GO:0000956~nuclear-transcribed mRNA catabolic process GO:0030397~membrane disassembly GO:0051081~nuclear envelope disassembly GO:0022890~inorganic cation transmembrane transporter activity GO:0006402~mRNA catabolic process GO:0006409~tRNA export from nucleus GO:0071431~tRNA-containing ribonucleoprotein complex export from nucleus GO:0006401~RNA catabolic process GO:0051031~tRNA transport GO:0034655~nucleobase-containing compound catabolic process GO:0034504~protein localization to nucleus GO:0000146~microfilament motor activity GO:0006406~mRNA export from nucleus GO:0046700~heterocycle catabolic process GO:0071427~mRNA-containing ribonucleoprotein complex export from nucleus GO:0006405~RNA export from nucleus GO:0044270~cellular nitrogen compound catabolic process GO:0050658~RNA transport GO:0019439~aromatic compound catabolic process GO:0050657~nucleic acid transport GO:0051015~ filament binding GO:0051236~establishment of RNA localization GO:1901361~organic cyclic compound catabolic process GO:0015931~nucleobase-containing compound transport GO:0022613~ribonucleoprotein complex biogenesis GO:0010827~regulation of glucose transport GO:0031047~gene silencing by RNA GO:0042623~ATPase activity, coupled GO:0006413~translational initiation GO:0016458~gene silencing GO:0075733~intracellular transport of virus GO:0006614~SRP-dependent cotranslational protein targeting to membrane GO:1902583~multi-organism intracellular transport GO:1902581~multi-organism cellular localization GO:0045047~protein targeting to ER GO:0046794~transport of virus GO:0017111~nucleoside-triphosphatase activity GO:0072599~establishment of protein localization to endoplasmic reticulum GO:1902579~multi-organism localization GO:0044766~multi-organism transport GO:0042273~ribosomal large subunit biogenesis GO:0051169~nuclear transport GO:0034645~cellular macromolecule biosynthetic process GO:0016925~protein sumoylation GO:0003857~3-hydroxyacyl-CoA dehydrogenase activity GO:0006403~RNA localization GO:0042255~ribosome assembly GO:0051028~mRNA transport GO:0019083~viral transcription GO:0006913~nucleocytoplasmic transport GO:1900034~regulation of cellular response to heat GO:0016462~pyrophosphatase activity GO:0000027~ribosomal large subunit assembly GO:0080135~regulation of cellular response to stress GO:0044744~protein targeting to nucleus GO:0022618~ribonucleoprotein complex assembly GO:0006606~protein import into nucleus GO:0071826~ribonucleoprotein complex subunit organization GO:0017038~protein import GO:0051170~nuclear import GO:0016818~hydrolase activity, acting on acid anhydrides, in phosphorus-containing anhydrides GO:0022607~cellular component assembly GO:1902593~single-organism nuclear import GO:0034622~cellular macromolecular complex assembly GO:0006607~NLS-bearing protein import into nucleus GO:0019054~modulation by virus of host process GO:0065003~macromolecular complex assembly GO:0044068~modulation by symbiont of host cellular process GO:0016817~hydrolase activity, acting on acid anhydrides GO:0071705~nitrogen compound transport GO:0006461~protein complex assembly GO:0050000~ localization GO:0070271~protein complex biogenesis GO:0051701~interaction with host GO:0019048~modulation by virus of host morphology or physiology GO:0034728~nucleosome organization GO:0044003~modification by symbiont of host morphology or physiology GO:0046943~carboxylic acid transmembrane transporter activity GO:0051817~modification of morphology or physiology of other organism involved in symbiotic interaction GO:0034724~DNA replication-independent nucleosome organization GO:0035821~modification of morphology or physiology of other organism GO:0006333~chromatin assembly or disassembly GO:0080134~regulation of response to stress GO:0006464~cellular protein modification process GO:0005342~organic acid transmembrane transporter activity GO:0006336~DNA replication-independent nucleosome assembly GO:0036211~protein modification process GO:0071824~protein-DNA complex subunit organization GO:0006457~protein folding GO:0006626~protein targeting to mitochondrion GO:0043486~histone exchange GO:0006839~mitochondrial transport GO:0051787~misfolded protein binding GO:0072655~establishment of protein localization to mitochondrion GO:0006325~chromatin organization GO:0070585~protein localization to mitochondrion

GO:0043044~ATP-dependent chromatin remodeling GO:0042026~protein refolding GO:0043549~regulation of kinase activity GO:0006974~cellular response to DNA damage stimulus GO:0031647~regulation of protein stability GO:0045295~gamma-catenin binding GO:0001934~positive regulation of protein phosphorylation GO:0006342~chromatin silencing GO:0051129~negative regulation of cellular component organization GO:0006338~chromatin remodeling GO:0032434~regulation of proteasomal ubiquitin-dependent protein catabolic process GO:0016070~RNA metabolic process GO:0001618~virus receptor activity GO:0034723~DNA replication-dependent nucleosome organization GO:0010467~gene expression GO:0006335~DNA replication-dependent nucleosome assembly GO:0090502~RNA phosphodiester bond hydrolysis, endonucleolytic GO:0090501~RNA phosphodiester bond hydrolysis GO:0006334~nucleosome assembly GO:0090305~nucleic acid phosphodiester bond hydrolysis GO:0034645~cellular macromolecule biosynthetic process GO:0046983~protein dimerization activity GO:0031497~chromatin assembly GO:0032774~RNA biosynthetic process GO:0006323~DNA packaging GO:0034654~nucleobase-containing compound biosynthetic process GO:0018130~heterocycle biosynthetic process GO:0065004~protein-DNA complex assembly GO:0019438~aromatic compound biosynthetic process GO:0031072~heat shock protein binding GO:0071103~DNA conformation change GO:0034622~cellular macromolecular complex assembly GO:0009303~rRNA transcription GO:0045814~negative regulation of gene expression, epigenetic GO:0090150~establishment of protein localization to membrane GO:0072657~protein localization to membrane GO:0008092~cytoskeletal protein binding GO:0040029~regulation of gene expression, epigenetic GO:0044085~cellular component biogenesis GO:0061641~CENP-A containing chromatin organization GO:0033365~protein localization to organelle GO:0006605~protein targeting GO:0034080~CENP-A containing nucleosome assembly GO:1902582~single-organism intracellular transport GO:0016032~viral process GO:0000774~adenyl- exchange factor activity GO:0031055~chromatin remodeling at centromere GO:0044764~multi-organism cellular process GO:0034508~centromere complex assembly GO:0072594~establishment of protein localization to organelle GO:0019058~viral life cycle GO:0033554~cellular response to stress GO:0044403~symbiosis, encompassing mutualism through parasitism GO:0060590~ATPase regulator activity GO:0019395~fatty acid oxidation GO:0044419~interspecies interaction between organisms GO:0009057~macromolecule catabolic process GO:0034440~lipid oxidation GO:0044248~cellular catabolic process GO:0019083~viral transcription GO:0030258~lipid modification GO:0019080~viral gene expression GO:0060589~nucleoside-triphosphatase regulator activity GO:0044255~cellular lipid metabolic process GO:0044033~multi-organism metabolic process GO:0042273~ribosomal large subunit biogenesis GO:0016042~lipid catabolic process GO:0070972~protein localization to endoplasmic reticulum

GO:0072599~establishment of protein localization to endoplasmic reticulum GO:0033540~fatty acid beta-oxidation using acyl-CoA oxidase GO:0051087~chaperone binding GO:0043043~peptide biosynthetic process GO:0006631~fatty acid metabolic process GO:0006402~mRNA catabolic process GO:0000184~nuclear-transcribed mRNA catabolic process, nonsense-mediated decay GO:0006629~lipid metabolic process GO:0006401~RNA catabolic process GO:0036094~small molecule binding GO:0044712~single-organism catabolic process GO:0000956~nuclear-transcribed mRNA catabolic process GO:0034655~nucleobase-containing compound catabolic process GO:0006635~fatty acid beta-oxidation GO:0046700~heterocycle catabolic process GO:0044270~cellular nitrogen compound catabolic process GO:0009062~fatty acid catabolic process GO:0019439~aromatic compound catabolic process GO:0030554~adenyl nucleotide binding GO:0019752~carboxylic acid metabolic process GO:1901361~organic cyclic compound catabolic process GO:0000463~maturation of LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) GO:0043436~oxoacid metabolic process GO:0000460~maturation of 5.8S rRNA GO:0072329~monocarboxylic acid catabolic process GO:0016071~mRNA metabolic process GO:0005524~ATP binding GO:0006614~SRP-dependent cotranslational protein targeting to membrane GO:0006082~organic acid metabolic process GO:0006613~cotranslational protein targeting to membrane GO:0045047~protein targeting to ER GO:0044242~cellular lipid catabolic process GO:0006612~protein targeting to membrane GO:0046395~carboxylic acid catabolic process GO:0000470~maturation of LSU-rRNA GO:0017076~purine nucleotide binding GO:0006412~translation GO:0016054~organic acid catabolic process GO:0043604~amide biosynthetic process GO:0036109~alpha-linolenic acid metabolic process GO:0006518~peptide metabolic process GO:0043603~cellular amide metabolic process GO:0000166~nucleotide binding GO:0016050~vesicle organization GO:1901566~organonitrogen compound biosynthetic process GO:0006413~translational initiation GO:0051641~cellular localization GO:0022618~ribonucleoprotein complex assembly GO:0034613~cellular protein localization GO:0071826~ribonucleoprotein complex subunit organization GO:1901265~nucleoside phosphate binding GO:0070125~mitochondrial translational elongation

GO:0070727~cellular macromolecule localization GO:0070126~mitochondrial translational termination GO:0006415~translational termination

GO:0045184~establishment of protein localization GO:0032543~mitochondrial translation GO:0008104~protein localization GO:0042255~ribosome assembly GO:0032549~ribonucleoside binding GO:0006414~translational elongation GO:0033036~macromolecule localization GO:0002181~cytoplasmic translation

GO:0005975~carbohydrate metabolic process GO:0042254~ribosome biogenesis GO:0022613~ribonucleoprotein complex biogenesis GO:0001882~nucleoside binding GO:0044723~single-organism carbohydrate metabolic process GO:0006364~rRNA processing GO:0016072~rRNA metabolic process GO:0009199~ribonucleoside triphosphate metabolic process GO:0034470~ncRNA processing GO:1990542~mitochondrial transmembrane transport GO:0034660~ncRNA metabolic process GO:0006396~RNA processing GO:0035639~purine ribonucleoside triphosphate binding GO:0009119~ribonucleoside metabolic process GO:0000478~endonucleolytic cleavage involved in rRNA processing GO:0000469~cleavage involved in rRNA processing GO:0006839~mitochondrial transport GO:0000479~endonucleolytic cleavage of tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA)

GO:0033365~protein localization to organelle GO:0000466~maturation of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) GO:0032550~purine ribonucleoside binding GO:0000480~endonucleolytic cleavage in 5’-ETS of tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA)

GO:0061024~membrane organization GO:0000447~endonucleolytic cleavage in ITS1 to separate SSU-rRNA from 5.8S rRNA and LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) GO:0044802~single-organism membrane organization GO:0044265~cellular macromolecule catabolic process GO:0010501~RNA secondary structure unwinding GO:0001883~purine nucleoside binding GO:0046907~intracellular transport GO:0000462~maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) GO:0042274~ribosomal small subunit biogenesis GO:0051649~establishment of localization in cell GO:0030490~maturation of SSU-rRNA

GO:0006886~intracellular protein transport GO:0001510~RNA methylation GO:0000154~rRNA modification GO:0032555~purine ribonucleotide binding GO:1902580~single-organism cellular localization GO:0009451~RNA modification GO:0007005~mitochondrion organization

GO:0015031~protein transport GO:0000027~ribosomal large subunit assembly GO:1902582~single-organism intracellular transport GO:0043624~cellular protein complex disassembly GO:0051082~unfolded protein binding GO:0043241~protein complex disassembly GO:0006612~protein targeting to membrane GO:0032984~macromolecular complex disassembly GO:0043413~macromolecule glycosylation GO:0033750~ribosome localization GO:0033753~establishment of ribosome localization GO:0006486~protein glycosylation GO:0000054~ribosomal subunit export from nucleus GO:0097367~carbohydrate derivative binding GO:0071428~rRNA-containing ribonucleoprotein complex export from nucleus GO:0070085~glycosylation GO:0006417~regulation of translation GO:0034471~ncRNA 5’-end processing GO:0006487~protein N-linked glycosylation GO:0000472~endonucleolytic cleavage to generate mature 5’-end of SSU-rRNA from (SSU-rRNA, 5.8S rRNA, LSU-rRNA) GO:0032553~ribonucleotide binding GO:0009101~glycoprotein biosynthetic process GO:0000967~rRNA 5’-end processing

GO:0009100~glycoprotein metabolic process GO:0034248~regulation of cellular amide metabolic process GO:0000028~ribosomal small subunit assembly GO:0018279~protein N-linked glycosylation via asparagine GO:0000966~RNA 5’-end processing GO:0032559~adenyl ribonucleotide binding GO:0031167~rRNA methylation GO:0018196~peptidyl-asparagine modification GO:0010608~posttranscriptional regulation of gene expression GO:0034976~response to endoplasmic reticulum stress GO:0007000~nucleolus organization GO:0006360~transcription from RNA polymerase I promoter GO:0035967~cellular response to topologically incorrect protein GO:0016074~snoRNA metabolic process GO:0044183~protein binding involved in protein folding GO:0006353~DNA-templated transcription, termination GO:0030968~endoplasmic reticulum unfolded protein response GO:0006363~termination of RNA polymerase I transcription GO:0034620~cellular response to unfolded protein GO:0006362~transcription elongation from RNA polymerase I promoter GO:0006361~transcription initiation from RNA polymerase I promoter GO:0005200~structural constituent of GO:0036498~IRE1-mediated unfolded protein response GO:0007004~telomere maintenance via telomerase GO:0010033~response to organic substance GO:0043933~macromolecular complex subunit organization GO:0033554~cellular response to stress GO:0072657~protein localization to membrane GO:0032446~protein modification by small protein conjugation GO:0007346~regulation of mitotic cell cycle GO:0008514~organic anion transmembrane transporter activity GO:0042776~mitochondrial ATP synthesis coupled proton transport GO:0051726~regulation of cell cycle GO:1902600~hydrogen ion transmembrane transport GO:0040029~regulation of gene expression, epigenetic GO:0045815~positive regulation of gene expression, epigenetic GO:0015985~energy coupled proton transport, down electrochemical gradient GO:0043414~macromolecule methylation GO:0003872~6-phosphofructokinase activity GO:0015986~ATP synthesis coupled proton transport GO:0032259~methylation GO:0006354~DNA-templated transcription, elongation GO:0009205~purine ribonucleoside triphosphate metabolic process GO:0033043~regulation of organelle organization GO:0051301~cell division GO:0003924~GTPase activity GO:0002480~antigen processing and presentation of exogenous peptide antigen via MHC class I, TAP-independent GO:0007049~cell cycle GO:0009144~purine nucleoside triphosphate metabolic process GO:0022402~cell cycle process GO:0000278~mitotic cell cycle GO:0009141~nucleoside triphosphate metabolic process GO:1903047~mitotic cell cycle process GO:0070647~protein modification by small protein conjugation or removal GO:0008443~phosphofructokinase activity

GO:0034975~protein folding in endoplasmic reticulum GO:0051276~chromosome organization GO:0030203~glycosaminoglycan metabolic process GO:0006259~DNA metabolic process GO:0019219~regulation of nucleobase-containing compound metabolic process GO:0006605~protein targeting GO:0097659~nucleic acid-templated transcription GO:0005310~dicarboxylic acid transmembrane transporter activity GO:1903506~regulation of nucleic acid-templated transcription GO:0006022~aminoglycan metabolic process GO:2001141~regulation of RNA biosynthetic process GO:0046128~purine ribonucleoside metabolic process GO:0051171~regulation of nitrogen compound metabolic process GO:0010468~regulation of gene expression GO:0042278~purine nucleoside metabolic process GO:0005525~GTP binding GO:0010605~negative regulation of macromolecule metabolic process GO:0009168~purine ribonucleoside monophosphate biosynthetic process GO:0009892~negative regulation of metabolic process GO:0000819~sister chromatid segregation

GO:0009127~purine nucleoside monophosphate biosynthetic process GO:0098813~nuclear chromosome segregation GO:0007059~chromosome segregation GO:0008509~anion transmembrane transporter activity GO:0009201~ribonucleoside triphosphate biosynthetic process GO:0010629~negative regulation of gene expression GO:0009145~purine nucleoside triphosphate biosynthetic process GO:0018205~peptidyl-lysine modification GO:0018193~peptidyl-amino acid modification GO:0009206~purine ribonucleoside triphosphate biosynthetic process GO:0007062~sister chromatid cohesion GO:0032561~guanyl ribonucleotide binding GO:0006754~ATP biosynthetic process GO:0034968~histone lysine methylation GO:0018022~peptidyl-lysine methylation

GO:1901135~carbohydrate derivative metabolic process GO:0016571~histone methylation GO:0006479~protein methylation GO:0090150~establishment of protein localization to membrane GO:0008213~protein alkylation GO:0019001~guanyl nucleotide binding GO:0015672~monovalent inorganic cation transport GO:0010564~regulation of cell cycle process GO:0031324~negative regulation of cellular metabolic process GO:0055085~transmembrane transport GO:0010604~positive regulation of macromolecule metabolic process GO:1901137~carbohydrate derivative biosynthetic process GO:0009893~positive regulation of metabolic process GO:0019200~carbohydrate kinase activity GO:0006260~DNA replication GO:0015992~proton transport GO:0016569~covalent chromatin modification GO:0006351~transcription, DNA-templated GO:0006818~hydrogen transport GO:0051052~regulation of DNA metabolic process GO:0051011~microtubule minus-end binding GO:0006261~DNA-dependent DNA replication GO:0006811~ion transport GO:0006323~DNA packaging GO:1903508~positive regulation of nucleic acid-templated transcription GO:0046034~ATP metabolic process GO:0045893~positive regulation of transcription, DNA-templated GO:0008202~steroid metabolic process GO:0051173~positive regulation of nitrogen compound metabolic process GO:0043015~gamma-tubulin binding GO:0009167~purine ribonucleoside monophosphate metabolic process GO:0045935~positive regulation of nucleobase-containing compound metabolic process GO:0051253~negative regulation of RNA metabolic process GO:0009126~purine nucleoside monophosphate metabolic process GO:1902679~negative regulation of RNA biosynthetic process GO:0009161~ribonucleoside monophosphate metabolic process GO:1903507~negative regulation of nucleic acid-templated transcription GO:0008017~microtubule binding GO:0045786~negative regulation of cell cycle GO:0009142~nucleoside triphosphate biosynthetic process GO:0006325~chromatin organization GO:0006974~cellular response to DNA damage stimulus GO:0009156~ribonucleoside monophosphate biosynthetic process GO:0071824~protein-DNA complex subunit organization GO:0036503~ERAD pathway GO:0006352~DNA-templated transcription, initiation GO:0015631~tubulin binding GO:2000113~negative regulation of cellular macromolecule biosynthetic process GO:0009124~nucleoside monophosphate biosynthetic process GO:0018394~peptidyl-lysine acetylation GO:0010948~negative regulation of cell cycle process GO:0046129~purine ribonucleoside biosynthetic process GO:0045787~positive regulation of cell cycle GO:0003779~actin binding GO:0042451~purine nucleoside biosynthetic process GO:0006355~regulation of transcription, DNA-templated GO:0065004~protein-DNA complex assembly GO:0008203~ metabolic process GO:1902275~regulation of chromatin organization GO:0042455~ribonucleoside biosynthetic process GO:0043543~protein acylation GO:0006367~transcription initiation from RNA polymerase II promoter GO:0044822~poly(A) RNA binding GO:1902652~secondary alcohol metabolic process GO:0044702~single organism reproductive process GO:0006473~protein acetylation GO:0016125~sterol metabolic process GO:0022414~reproductive process GO:0009163~nucleoside biosynthetic process GO:0000122~negative regulation of transcription from RNA polymerase II promoter GO:0003723~RNA binding GO:0000003~reproduction GO:1901659~glycosyl compound biosynthetic process GO:0090068~positive regulation of cell cycle process GO:0006888~ER to Golgi vesicle-mediated transport GO:0010639~negative regulation of organelle organization GO:2000112~regulation of cellular macromolecule biosynthetic process GO:0003676~nucleic acid binding GO:0006891~intra-Golgi vesicle-mediated transport GO:0098781~ncRNA transcription GO:0010556~regulation of macromolecule biosynthetic process GO:0016482~cytosolic transport GO:0045892~negative regulation of transcription, DNA-templated GO:0006890~retrograde vesicle-mediated transport, Golgi to ER GO:0031328~positive regulation of cellular biosynthetic process GO:0009891~positive regulation of biosynthetic process GO:0019843~rRNA binding GO:0048193~Golgi vesicle transport GO:0051252~regulation of RNA metabolic process GO:0006333~chromatin assembly or disassembly GO:0042147~retrograde transport, endosome to Golgi GO:0006302~double-strand break repair GO:0007034~vacuolar transport GO:0008156~negative regulation of DNA replication GO:1901363~heterocyclic compound binding GO:0032200~telomere organization GO:0098609~cell-cell adhesion GO:0006334~nucleosome assembly GO:0007155~cell adhesion GO:0006306~DNA methylation GO:0006305~DNA alkylation GO:0022610~biological adhesion GO:0006271~DNA strand elongation involved in DNA replication GO:0097159~organic cyclic compound binding GO:0044728~DNA methylation or demethylation GO:0045216~cell-cell junction organization GO:0022616~DNA strand elongation GO:0006928~movement of cell or subcellular component GO:0000722~telomere maintenance via recombination GO:0032392~DNA geometric change GO:0003735~structural constituent of ribosome GO:0016337~single organismal cell-cell adhesion GO:0016073~snRNA metabolic process GO:0006312~mitotic recombination GO:0098602~single organism cell adhesion GO:0006304~DNA modification GO:0043009~chordate embryonic development GO:0006383~transcription from RNA polymerase III promoter GO:0005198~structural molecule activity GO:0006301~postreplication repair GO:0043069~negative regulation of programmed cell death GO:0006289~nucleotide-excision repair GO:0009792~embryo development ending in birth or egg hatching GO:0033045~regulation of sister chromatid segregation GO:0008406~gonad development GO:0060548~negative regulation of cell death GO:0032508~DNA duplex unwinding GO:0003729~mRNA binding GO:0045137~development of primary sexual characteristics GO:0045321~leukocyte activation GO:0009411~response to UV GO:0048870~cell motility GO:1902680~positive regulation of RNA biosynthetic process GO:0006336~DNA replication-independent nucleosome assembly GO:0030515~snoRNA binding GO:0051674~localization of cell GO:0051254~positive regulation of RNA metabolic process GO:0034330~cell junction organization GO:0034724~DNA replication-independent nucleosome organization GO:0010212~response to ionizing radiation GO:2001252~positive regulation of chromosome organization GO:0016477~cell migration GO:0051783~regulation of nuclear division GO:0004004~ATP-dependent RNA helicase activity GO:0007156~homophilic cell adhesion via plasma membrane adhesion molecules GO:0007064~mitotic sister chromatid cohesion GO:0040011~locomotion GO:0034728~nucleosome organization GO:1901796~regulation of signal transduction by p53 class mediator GO:0018193~peptidyl-amino acid modification GO:0006366~transcription from RNA polymerase II promoter GO:0008186~RNA-dependent ATPase activity GO:0000070~mitotic sister chromatid segregation GO:0098742~cell-cell adhesion via plasma-membrane adhesion molecules GO:0007067~mitotic nuclear division GO:0042269~regulation of natural killer cell mediated cytotoxicity GO:0045934~negative regulation of nucleobase-containing compound metabolic process GO:0051053~negative regulation of DNA metabolic process GO:0003724~RNA helicase activity GO:0001912~positive regulation of leukocyte mediated cytotoxicity GO:0031497~chromatin assembly Molecular Function (# of preys involved ) GO:0002715~regulation of natural killer cell mediated immunity GO:0051172~negative regulation of nitrogen compound metabolic process GO:0006275~regulation of DNA replication GO:0031343~positive regulation of cell killing GO:0060249~anatomical structure homeostasis GO:0006338~chromatin remodeling GO:0070035~purine NTP-dependent helicase activity GO:0043067~regulation of programmed cell death GO:0009314~response to radiation

GO:0001910~regulation of leukocyte mediated cytotoxicity GO:0042769~DNA damage response, detection of DNA damage GO:0042795~snRNA transcription from RNA polymerase II promoter GO:0042267~natural killer cell mediated cytotoxicity GO:0009301~snRNA transcription GO:0008026~ATP-dependent helicase activity GO:0007507~heart development GO:0006283~transcription-coupled nucleotide-excision repair GO:1900262~regulation of DNA-directed DNA polymerase activity GO:0001701~in utero embryonic development GO:1900264~positive regulation of DNA-directed DNA polymerase activity GO:0006273~lagging strand elongation GO:0010941~regulation of cell death GO:0042393~histone binding GO:2001251~negative regulation of chromosome organization GO:0002228~natural killer cell mediated immunity GO:0031056~regulation of histone modification GO:0045910~negative regulation of DNA recombination GO:0031341~regulation of cell killing GO:0006266~DNA ligation GO:0016578~histone deubiquitination GO:0031491~nucleosome binding GO:0002708~positive regulation of lymphocyte mediated immunity GO:0051123~RNA polymerase II transcriptional preinitiation complex assembly GO:0002705~positive regulation of leukocyte mediated immunity GO:0070897~DNA-templated transcriptional preinitiation complex assembly GO:0006399~tRNA metabolic process GO:0044409~entry into host GO:0010165~response to X-ray GO:0003682~chromatin binding GO:0046718~viral entry into host cell GO:0000281~mitotic cytokinesis GO:0006298~mismatch repair GO:0051828~entry into other organism involved in symbiotic interaction GO:0006281~DNA repair GO:0006357~regulation of transcription from RNA polymerase II promoter GO:0030260~entry into host cell GO:0010558~negative regulation of macromolecule biosynthetic process GO:0003697~single-stranded DNA binding GO:0051806~entry into cell of other organism involved in symbiotic interaction GO:0071103~DNA conformation change GO:0000280~nuclear division GO:0002706~regulation of lymphocyte mediated immunity GO:0090329~regulation of DNA-dependent DNA replication GO:0048285~organelle fission GO:0003677~DNA binding GO:0034332~adherens junction organization GO:0031327~negative regulation of cellular biosynthetic process GO:0043066~negative regulation of apoptotic process GO:0009890~negative regulation of biosynthetic process GO:0016570~histone modification GO:0001775~cell activation GO:0006368~transcription elongation from RNA polymerase II promoter GO:0003684~damaged DNA binding GO:0002703~regulation of leukocyte mediated immunity GO:0000723~telomere maintenance GO:2000104~negative regulation of DNA-dependent DNA replication GO:0051701~interaction with host GO:0006297~nucleotide-excision repair, DNA gap filling GO:0044786~cell cycle DNA replication GO:0002699~positive regulation of immune effector process GO:0006335~DNA replication-dependent nucleosome assembly GO:0005521~lamin binding GO:0002486~antigen processing and presentation of endogenous peptide antigen via MHC class I via ER pathway, TAP-independent GO:0034723~DNA replication-dependent nucleosome organization GO:0006296~nucleotide-excision repair, DNA incision, 5’-to lesion GO:0070887~cellular response to chemical stimulus GO:0051321~meiotic cell cycle GO:0033683~nucleotide-excision repair, DNA incision GO:0048585~negative regulation of response to stimulus GO:0016874~ligase activity GO:0006303~double-strand break repair via nonhomologous end joining GO:0002682~regulation of immune system process GO:0045944~positive regulation of transcription from RNA polymerase II promoter GO:0043570~maintenance of DNA repeat elements GO:0071310~cellular response to organic substance GO:0000731~DNA synthesis involved in DNA repair GO:0035966~response to topologically incorrect protein GO:0000726~non-recombinational repair GO:0050662~coenzyme binding GO:0051096~positive regulation of helicase activity GO:0006986~response to unfolded protein GO:0000710~meiotic mismatch repair GO:0010216~maintenance of DNA methylation GO:0007010~cytoskeleton organization GO:0051256~mitotic spindle midzone assembly GO:0070003~threonine-type peptidase activity GO:1902589~single-organism organelle organization GO:0075713~establishment of integrated proviral latency GO:0042023~DNA endoreduplication GO:0007017~microtubule-based process GO:0032875~regulation of DNA endoreduplication GO:0044087~regulation of cellular component biogenesis GO:2000278~regulation of DNA biosynthetic process GO:0004298~threonine-type endopeptidase activity GO:0000022~mitotic spindle elongation GO:0042026~protein refolding GO:0051095~regulation of helicase activity GO:0019043~establishment of viral latency GO:0080135~regulation of cellular response to stress GO:0051231~spindle elongation GO:0019886~antigen processing and presentation of exogenous peptide antigen via MHC class II GO:0051255~spindle midzone assembly GO:0004175~endopeptidase activity GO:0007127~meiosis I GO:0002495~antigen processing and presentation of peptide antigen via MHC class II GO:0051103~DNA ligation involved in DNA repair GO:0002504~antigen processing and presentation of peptide or polysaccharide antigen via MHC class II GO:0019042~viral latency GO:0006310~DNA recombination GO:0070011~peptidase activity, acting on L-amino acid peptides GO:0051129~negative regulation of cellular component organization GO:0033044~regulation of chromosome organization GO:0042797~tRNA transcription from RNA polymerase III promoter GO:1900034~regulation of cellular response to heat GO:0042791~5S class rRNA transcription from RNA polymerase III type 1 promoter GO:0016192~vesicle-mediated transport GO:0051983~regulation of chromosome segregation GO:0009304~tRNA transcription GO:0008233~peptidase activity GO:0051276~chromosome organization GO:0010557~positive regulation of macromolecule biosynthetic process GO:0010628~positive regulation of gene expression GO:0006259~DNA metabolic process GO:0016573~histone acetylation GO:0006260~DNA replication GO:0018393~internal peptidyl-lysine acetylation GO:0019904~protein domain specific binding GO:0042276~error-prone translesion synthesis GO:0006281~DNA repair GO:0070987~error-free translesion synthesis GO:0010256~endomembrane system organization GO:0006475~internal protein amino acid acetylation GO:0007063~regulation of sister chromatid cohesion GO:0042803~protein homodimerization activity GO:0007126~meiotic nuclear division GO:0022402~cell cycle process GO:0070979~protein K11-linked ubiquitination GO:0007049~cell cycle GO:1903046~meiotic cell cycle process GO:0051171~regulation of nitrogen compound metabolic process GO:0051567~histone H3-K9 methylation GO:0071897~DNA biosynthetic process GO:0010468~regulation of gene expression GO:0051054~positive regulation of DNA metabolic process GO:0061647~histone H3-K9 modification GO:0007077~mitotic nuclear envelope disassembly GO:0007052~mitotic spindle organization GO:0006289~nucleotide-excision repair GO:0019985~translesion synthesis GO:0031060~regulation of histone methylation GO:0034654~nucleobase-containing compound biosynthetic process GO:0031325~positive regulation of cellular metabolic process GO:0016925~protein sumoylation GO:0032876~negative regulation of DNA endoreduplication GO:0031401~positive regulation of protein modification process GO:0018130~heterocycle biosynthetic process GO:0051338~regulation of transferase activity GO:0032270~positive regulation of cellular protein metabolic process GO:0019438~aromatic compound biosynthetic process GO:0051247~positive regulation of protein metabolic process GO:1901796~regulation of signal transduction by p53 class mediator GO:0031399~regulation of protein modification process GO:0032268~regulation of cellular protein metabolic process GO:0051168~nuclear export GO:0051246~regulation of protein metabolic process GO:0090305~nucleic acid phosphodiester bond hydrolysis GO:0051347~positive regulation of transferase activity GO:1901990~regulation of mitotic cell cycle phase transition GO:1901987~regulation of cell cycle phase transition GO:0098813~nuclear chromosome segregation GO:0044772~mitotic cell cycle phase transition GO:0007059~chromosome segregation GO:0044770~cell cycle phase transition GO:0006998~nuclear envelope organization GO:0051436~negative regulation of ubiquitin-protein ligase activity involved in mitotic cell cycle GO:0051439~regulation of ubiquitin-protein ligase activity involved in mitotic cell cycle GO:0006997~nucleus organization GO:1904667~negative regulation of ubiquitin protein ligase activity GO:0051437~positive regulation of ubiquitin-protein ligase activity involved in regulation of mitotic cell cycle transition GO:0051081~nuclear envelope disassembly GO:0031145~anaphase-promoting complex-dependent catabolic process AP-MS GO:0030397~membrane disassembly GO:0051444~negative regulation of ubiquitin-protein transferase activity GO:1904668~positive regulation of ubiquitin protein ligase activity GO:0051169~nuclear transport GO:1904666~regulation of ubiquitin protein ligase activity GO:0006913~nucleocytoplasmic transport GO:2000060~positive regulation of protein ubiquitination involved in ubiquitin-dependent protein catabolic process GO:0051443~positive regulation of ubiquitin-protein transferase activity GO:0000722~telomere maintenance via recombination GO:0051438~regulation of ubiquitin-protein transferase activity GO:2000058~regulation of protein ubiquitination involved in ubiquitin-dependent protein catabolic process GO:0006284~base-excision repair GO:1903052~positive regulation of proteolysis involved in cellular protein catabolic process GO:0006312~mitotic recombination GO:1903364~positive regulation of cellular protein catabolic process GO:0031397~negative regulation of protein ubiquitination GO:0007084~mitotic nuclear envelope reassembly GO:1903321~negative regulation of protein modification by small protein conjugation or removal GO:0006606~protein import into nucleus GO:0042787~protein ubiquitination involved in ubiquitin-dependent protein catabolic process GO:1903362~regulation of cellular protein catabolic process GO:0044744~protein targeting to nucleus GO:0031396~regulation of protein ubiquitination GO:1903050~regulation of proteolysis involved in cellular protein catabolic process GO:1902593~single-organism nuclear import GO:0002429~immune response-activating cell surface receptor signaling pathway GO:0051170~nuclear import GO:0043488~regulation of mRNA stability GO:0031398~positive regulation of protein ubiquitination GO:0006283~transcription-coupled nucleotide-excision repair GO:0060828~regulation of canonical Wnt signaling pathway GO:0010565~regulation of cellular ketone metabolic process GO:0000731~DNA synthesis involved in DNA repair GO:0034612~response to tumor necrosis factor GO:0017038~protein import GO:0050852~T cell receptor signaling pathway GO:0002758~innate immune response-activating signal transduction GO:0031468~nuclear envelope reassembly GO:0090090~negative regulation of canonical Wnt signaling pathway GO:0000724~double-strand break repair via homologous recombination GO:0030178~negative regulation of Wnt signaling pathway GO:2000027~regulation of organ morphogenesis GO:0000725~recombinational repair GO:0002218~activation of innate immune response GO:0031331~positive regulation of cellular catabolic process GO:0070987~error-free translesion synthesis GO:0035567~non-canonical Wnt signaling pathway GO:0042276~error-prone translesion synthesis GO:0043487~regulation of RNA stability GO:0030177~positive regulation of Wnt signaling pathway GO:0046827~positive regulation of protein export from nucleus GO:0045732~positive regulation of protein catabolic process GO:0038095~Fc-epsilon receptor signaling pathway GO:0006293~nucleotide-excision repair, preincision complex stabilization GO:1903322~positive regulation of protein modification by small protein conjugation or removal

GO:0019219~regulation of nucleobase-containing compound metabolic process GO:0050851~antigen receptor-mediated signaling pathway GO:0071356~cellular response to tumor necrosis factor GO:0006295~nucleotide-excision repair, DNA incision, 3’-to lesion GO:0000209~protein polyubiquitination GO:0006297~nucleotide-excision repair, DNA gap filling GO:1903320~regulation of protein modification by small protein conjugation or removal GO:0060070~canonical Wnt signaling pathway GO:0018205~peptidyl-lysine modification GO:0030111~regulation of Wnt signaling pathway GO:0033209~tumor necrosis factor-mediated signaling pathway GO:0006294~nucleotide-excision repair, preincision complex assembly GO:0001738~morphogenesis of a polarized epithelium

GO:0046825~regulation of protein export from nucleus GO:0009308~amine metabolic process GO:0001736~establishment of planar polarity GO:0042769~DNA damage response, detection of DNA damage GO:0007164~establishment of tissue polarity GO:0006296~nucleotide-excision repair, DNA incision, 5’-to lesion GO:0006521~regulation of cellular amino acid metabolic process GO:0042180~cellular ketone metabolic process GO:0006298~mismatch repair GO:0002479~antigen processing and presentation of exogenous peptide antigen via MHC class I, TAP-dependent GO:0045087~innate immune response GO:0019985~translesion synthesis GO:0042590~antigen processing and presentation of exogenous peptide antigen via MHC class I GO:0033683~nucleotide-excision repair, DNA incision GO:0009968~negative regulation of signal transduction GO:0033238~regulation of cellular amine metabolic process GO:0036297~interstrand cross-link repair GO:0010648~negative regulation of cell communication GO:0002474~antigen processing and presentation of peptide antigen via MHC class I GO:0032774~RNA biosynthetic process GO:0023057~negative regulation of signaling GO:0050658~RNA transport GO:0002223~stimulatory C-type lectin receptor signaling pathway GO:0048585~negative regulation of response to stimulus GO:0050657~nucleic acid transport GO:0038061~NIK/NF-kappaB signaling GO:0051236~establishment of RNA localization GO:0009967~positive regulation of signal transduction GO:0002220~innate immune response activating cell surface receptor signaling pathway GO:0006301~postreplication repair GO:0006952~defense response GO:0060071~Wnt signaling pathway, planar cell polarity pathway GO:0006611~protein export from nucleus GO:0043248~proteasome assembly GO:0006355~regulation of transcription, DNA-templated GO:0090175~regulation of establishment of planar polarity GO:0010647~positive regulation of cell communication GO:1903506~regulation of nucleic acid-templated transcription GO:0044106~cellular amine metabolic process GO:0070192~chromosome organization involved in meiotic cell cycle GO:0023056~positive regulation of signaling GO:0090263~positive regulation of canonical Wnt signaling pathway GO:2001141~regulation of RNA biosynthetic process GO:0030048~actin filament-based movement GO:0006508~proteolysis GO:0000018~regulation of DNA recombination GO:2000026~regulation of multicellular organismal development GO:0006302~double-strand break repair GO:0060429~epithelium development GO:0051248~negative regulation of protein metabolic process GO:0075733~intracellular transport of virus GO:0032269~negative regulation of cellular protein metabolic process GO:0023014~signal transduction by protein phosphorylation GO:1902583~multi-organism intracellular transport GO:0034097~response to cytokine

GO:1902581~multi-organism cellular localization GO:0030162~regulation of proteolysis GO:0051603~proteolysis involved in cellular protein catabolic process GO:0015931~nucleobase-containing compound transport GO:0071345~cellular response to cytokine stimulus (Baits) GO:0046794~transport of virus GO:0031349~positive regulation of defense response GO:0045088~regulation of innate immune response GO:0046822~regulation of nucleocytoplasmic transport GO:0051348~negative regulation of transferase activity GO:0043632~modification-dependent macromolecule catabolic process GO:0051252~regulation of RNA metabolic process GO:0000165~MAPK cascade GO:0097659~nucleic acid-templated transcription GO:0019941~modification-dependent protein catabolic process GO:0042176~regulation of protein catabolic process GO:0044766~multi-organism transport GO:0045862~positive regulation of proteolysis GO:1902579~multi-organism localization GO:0045089~positive regulation of innate immune response GO:0009887~organ morphogenesis GO:0000082~G1/S transition of mitotic cell cycle GO:0031400~negative regulation of protein modification process GO:0044257~cellular protein catabolic process GO:0031334~positive regulation of protein complex assembly GO:0009896~positive regulation of catabolic process GO:0006511~ubiquitin-dependent protein catabolic process

GO:0006457~protein folding

GO:0016567~protein ubiquitination

GO:0006403~RNA localization GO:0031347~regulation of defense response GO:0031329~regulation of cellular catabolic process

GO:0000723~telomere maintenance GO:0019221~cytokine-mediated signaling pathway AIFM1 TBG1 EEA1 CALX EZRI LAMP1 TGON2 CATA H31 LMNA FBRL RS6 RAB11A GOGA2 HSP7C PSA1 RAB9A TBA1A GO:0032200~telomere organization GO:0002253~activation of immune response GO:0050662~coenzyme binding GO:0050778~positive regulation of immune response GO:0033044~regulation of chromosome organization GO:0006955~immune response GO:0016616~oxidoreductase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor GO:0060249~anatomical structure homeostasis GO:0044092~negative regulation of molecular function GO:0043086~negative regulation of catalytic activity GO:0016627~oxidoreductase activity, acting on the CH-CH group of donors GO:0071897~DNA biosynthetic process GO:0065009~regulation of molecular function GO:0050790~regulation of catalytic activity GO:0005102~receptor binding GO:2001252~positive regulation of chromosome organization GO:0043085~positive regulation of catalytic activity GO:0048037~cofactor binding GO:0051052~regulation of DNA metabolic process GO:0019220~regulation of phosphate metabolic process GO:0051174~regulation of phosphorus metabolic process GO:0000062~fatty-acyl-CoA binding GO:0034504~protein localization to nucleus GO:0030182~neuron differentiation GO:0040011~locomotion GO:0016614~oxidoreductase activity, acting on CH-OH group of donors GO:0033043~regulation of organelle organization GO:0051258~protein polymerization GO:0010638~positive regulation of organelle organization GO:0032528~microvillus organization GO:0003997~acyl-CoA oxidase activity GO:0022008~neurogenesis GO:0032204~regulation of telomere maintenance GO:0007409~axonogenesis GO:1901681~sulfur compound binding GO:0032206~positive regulation of telomere maintenance GO:0061564~axon development GO:0016477~cell migration GO:0016634~oxidoreductase activity, acting on the CH-CH group of donors, oxygen as acceptor GO:0072594~establishment of protein localization to organelle GO:0022604~regulation of cell morphogenesis GO:0010638~positive regulation of organelle organization GO:0052890~oxidoreductase activity, acting on the CH-CH group of donors, with a flavin as acceptor GO:1904869~regulation of protein localization to Cajal body GO:1903829~positive regulation of cellular protein localization GO:0030838~positive regulation of actin filament polymerization GO:1904871~positive regulation of protein localization to Cajal body GO:0003995~acyl-CoA dehydrogenase activity GO:0000910~cytokinesis GO:1904867~protein localization to Cajal body GO:0022612~gland morphogenesis GO:0050660~flavin adenine dinucleotide binding GO:1990173~protein localization to nucleoplasm GO:1902946~protein localization to early endosome GO:1902965~regulation of protein localization to early endosome GO:0047617~acyl-CoA hydrolase activity GO:1903405~protein localization to nuclear body GO:1902966~positive regulation of protein localization to early endosome GO:0044089~positive regulation of cellular component biogenesis GO:0016289~CoA hydrolase activity GO:1904874~positive regulation of telomerase RNA localization to Cajal body GO:0097581~lamellipodium organization GO:0016628~oxidoreductase activity, acting on the CH-CH group of donors, NAD or NADP as acceptor GO:1904872~regulation of telomerase RNA localization to Cajal body GO:0051270~regulation of cellular component movement GO:0007265~Ras protein signal transduction GO:0016790~thiolester hydrolase activity GO:0090671~telomerase RNA localization to Cajal body GO:0051130~positive regulation of cellular component organization GO:0051674~localization of cell GO:0016853~isomerase activity GO:0090670~RNA localization to Cajal body GO:0051639~actin filament network formation GO:0090672~telomerase RNA localization GO:0048870~cell motility GO:0019166~trans-2-enoyl-CoA reductase (NADPH) activity GO:0035089~establishment of apical/basal cell polarity GO:1904851~positive regulation of establishment of protein localization to telomere GO:0036010~protein localization to endosome GO:0016508~long-chain-enoyl-CoA hydratase activity GO:0070203~regulation of establishment of protein localization to telomere GO:0030859~polarized epithelial cell differentiation GO:0061162~establishment of monopolar cell polarity GO:0008670~2,4-dienoyl-CoA reductase (NADPH) activity GO:0070202~regulation of establishment of protein localization to chromosome GO:0061339~establishment or maintenance of monopolar cell polarity GO:0030033~microvillus assembly GO:0016401~palmitoyl-CoA oxidase activity GO:1904816~positive regulation of protein localization to chromosome, telomeric region GO:0016601~Rac protein signal transduction

GO:0030866~cortical actin cytoskeleton organization GO:0004165~dodecenoyl-CoA delta-isomerase activity GO:1904814~regulation of protein localization to chromosome, telomeric region GO:0070423~nucleotide-binding oligomerization domain containing signaling pathway GO:0070200~establishment of protein localization to telomere GO:0035872~nucleotide-binding domain, leucine rich repeat containing receptor signaling pathway GO:0005545~1-phosphatidylinositol binding GO:0032212~positive regulation of telomere maintenance via telomerase GO:0030865~cortical cytoskeleton organization GO:0030032~lamellipodium assembly GO:0044183~protein binding involved in protein folding GO:1904358~positive regulation of telomere maintenance via telomere lengthening GO:0051056~regulation of small GTPase mediated signal transduction GO:0030042~actin filament depolymerization GO:0017056~structural constituent of nuclear pore GO:0070199~establishment of protein localization to chromosome GO:1902305~regulation of sodium ion transmembrane transport GO:0005487~nucleocytoplasmic transporter activity GO:0032210~regulation of telomere maintenance via telomerase GO:0002753~cytoplasmic pattern recognition receptor signaling pathway GO:0031344~regulation of cell projection organization GO:0005521~lamin binding GO:0070198~protein localization to chromosome, telomeric region GO:0090066~regulation of anatomical structure size GO:0048699~generation of neurons GO:0008139~nuclear localization sequence binding GO:1904356~regulation of telomere maintenance via telomere lengthening GO:0008360~regulation of cell shape GO:0007004~telomere maintenance via telomerase GO:0050767~regulation of neurogenesis GO:0005048~signal sequence binding GO:0032535~regulation of cellular component size GO:2000573~positive regulation of DNA biosynthetic process GO:0030334~regulation of cell migration GO:0042277~peptide binding GO:0010833~telomere maintenance via telomere lengthening GO:0001932~regulation of protein phosphorylation GO:0030031~cell projection assembly GO:0033218~amide binding GO:0007339~binding of sperm to zona pellucida GO:0048568~embryonic organ development GO:0060491~regulation of cell projection assembly GO:0048365~Rac GTPase binding GO:0006278~RNA-dependent DNA biosynthetic process GO:2000145~regulation of cell motility GO:0035036~sperm-egg recognition GO:0051495~positive regulation of cytoskeleton organization GO:0017048~Rho GTPase binding GO:0040012~regulation of locomotion GO:2000278~regulation of DNA biosynthetic process GO:0051960~regulation of nervous system development GO:0005200~structural constituent of cytoskeleton GO:0031334~positive regulation of protein complex assembly GO:0009988~cell-cell recognition GO:0060284~regulation of cell development GO:0032403~protein complex binding GO:1900182~positive regulation of protein localization to nucleus GO:0007417~central nervous system development GO:2000601~positive regulation of Arp2/3 complex-mediated actin nucleation GO:0051015~actin filament binding GO:0031647~regulation of protein stability GO:0032273~positive regulation of protein polymerization GO:0003779~actin binding GO:0051054~positive regulation of DNA metabolic process GO:0045198~establishment of epithelial cell apical/basal polarity GO:1903651~positive regulation of cytoplasmic transport GO:0008093~cytoskeletal adaptor activity GO:0032846~positive regulation of homeostatic process GO:0007016~cytoskeletal anchoring at plasma membrane GO:0051764~actin crosslink formation GO:0016301~kinase activity GO:0008037~cell recognition GO:0036445~neuronal stem cell division GO:0090666~scaRNA localization to Cajal body GO:0055057~neuroblast division GO:0070064~proline-rich region binding GO:0010737~protein kinase A signaling GO:0051973~positive regulation of telomerase activity GO:0034315~regulation of Arp2/3 complex-mediated actin nucleation GO:0000774~adenyl-nucleotide exchange factor activity GO:1901998~toxin transport GO:0048103~somatic stem cell division GO:0051127~positive regulation of actin nucleation GO:0060590~ATPase regulator activity GO:0034502~protein localization to chromosome GO:0007420~brain development GO:0002009~morphogenesis of an epithelium GO:0031072~heat shock protein binding

GO:1903829~positive regulation of cellular protein localization GO:0048729~tissue morphogenesis GO:0038093~Fc receptor signaling pathway GO:0030234~enzyme regulator activity GO:0050821~protein stabilization GO:0002768~immune response-regulating cell surface receptor signaling pathway GO:1900180~regulation of protein localization to nucleus GO:0002764~immune response-regulating signaling pathway GO:0005516~calmodulin binding GO:0002757~immune response-activating signal transduction

GO:0032844~regulation of homeostatic process GO:0022603~regulation of anatomical structure morphogenesis GO:0000146~microfilament motor activity GO:0022414~reproductive process GO:0006468~protein phosphorylation GO:0030898~actin-dependent ATPase activity GO:0016310~phosphorylation GO:0000003~reproduction GO:0006796~phosphate-containing compound metabolic process GO:0003774~motor activity GO:0007338~single fertilization GO:0006793~phosphorus metabolic process GO:0007010~cytoskeleton organization GO:0070016~armadillo repeat domain binding GO:0009566~fertilization GO:0006928~movement of cell or subcellular component GO:0030029~actin filament-based process GO:0051721~protein phosphatase 2A binding GO:1903827~regulation of cellular protein localization GO:0007015~actin filament organization GO:0030036~actin cytoskeleton organization GO:1904951~positive regulation of establishment of protein localization GO:0070003~threonine-type peptidase activity GO:0045197~establishment or maintenance of epithelial cell apical/basal polarity GO:0060341~regulation of cellular localization GO:0008154~actin polymerization or depolymerization GO:0004298~threonine-type endopeptidase activity GO:0032880~regulation of protein localization GO:0061245~establishment or maintenance of bipolar cell polarity GO:0035088~establishment or maintenance of apical/basal cell polarity GO:0004175~endopeptidase activity GO:0070201~regulation of establishment of protein localization GO:0090004~positive regulation of establishment of protein localization to plasma membrane GO:1904377~positive regulation of protein localization to cell periphery GO:0070011~peptidase activity, acting on L-amino acid peptides GO:0072655~establishment of protein localization to mitochondrion GO:1903078~positive regulation of protein localization to plasma membrane GO:0008233~peptidase activity GO:0070585~protein localization to mitochondrion GO:0030041~actin filament polymerization GO:0008064~regulation of actin polymerization or depolymerization GO:0055114~oxidation-reduction process GO:0030832~regulation of actin filament length GO:0019904~protein domain specific binding GO:0051493~regulation of cytoskeleton organization GO:0032787~monocarboxylic acid metabolic process GO:0032956~regulation of actin cytoskeleton organization GO:0019900~kinase binding GO:0006732~coenzyme metabolic process GO:0007399~nervous system development GO:0019901~protein kinase binding GO:0032970~regulation of actin filament-based process GO:0035336~long-chain fatty-acyl-CoA metabolic process GO:0044087~regulation of cellular component biogenesis GO:0046982~protein heterodimerization activity GO:0051186~cofactor metabolic process GO:0051017~actin filament bundle assembly GO:0061572~actin filament bundle organization GO:0019903~protein phosphatase binding GO:0035384~thioester biosynthetic process GO:0032271~regulation of protein polymerization GO:1903827~regulation of cellular protein localization GO:0019902~phosphatase binding GO:0071616~acyl-CoA biosynthetic process GO:0030833~regulation of actin filament polymerization

GO:0006094~gluconeogenesis GO:0034332~adherens junction organization GO:0030165~PDZ domain binding GO:0034330~cell junction organization GO:0019319~hexose biosynthetic process GO:0045216~cell-cell junction organization GO:0015175~neutral amino acid transmembrane transporter activity GO:0098602~single organism cell adhesion GO:0006835~dicarboxylic acid transport GO:1902531~regulation of intracellular signal transduction GO:0046935~1-phosphatidylinositol-3-kinase regulator activity GO:0046364~monosaccharide biosynthetic process GO:0048010~vascular endothelial growth factor receptor signaling pathway GO:0043254~regulation of protein complex assembly GO:0015171~amino acid transmembrane transporter activity GO:0042592~homeostatic process GO:0043009~chordate embryonic development GO:0035014~phosphatidylinositol 3-kinase regulator activity GO:0035337~fatty-acyl-CoA metabolic process GO:0009792~embryo development ending in birth or egg hatching GO:0002252~immune effector process GO:0045294~alpha-catenin binding GO:0019318~hexose metabolic process GO:0035556~intracellular signal transduction GO:0009966~regulation of signal transduction GO:0006006~glucose metabolic process GO:0007166~cell surface receptor signaling pathway GO:0001618~virus receptor activity GO:0044283~small molecule biosynthetic process GO:0010646~regulation of cell communication GO:0004579~dolichyl-diphosphooligosaccharide-protein glycotransferase activity GO:0023051~regulation of signaling GO:0005996~monosaccharide metabolic process GO:0044093~positive regulation of molecular function GO:0004576~oligosaccharyl transferase activity GO:0006790~sulfur compound metabolic process GO:0042325~regulation of phosphorylation GO:0198738~cell-cell signaling by wnt GO:0005047~signal recognition particle binding GO:0006091~generation of precursor metabolites and energy GO:0016055~Wnt signaling pathway GO:1905114~cell surface receptor signaling pathway involved in cell-cell signaling GO:0098632~protein binding involved in cell-cell adhesion GO:0006002~fructose 6-phosphate metabolic process GO:0050776~regulation of immune response GO:0035383~thioester metabolic process GO:0002684~positive regulation of immune system process GO:0098631~protein binding involved in cell adhesion GO:0071310~cellular response to organic substance GO:0006637~acyl-CoA metabolic process GO:0048584~positive regulation of response to stimulus GO:0098641~cadherin binding involved in cell-cell adhesion GO:0070887~cellular response to chemical stimulus GO:0006090~pyruvate metabolic process GO:0002682~regulation of immune system process GO:0045296~cadherin binding GO:0061718~glucose catabolic process to pyruvate GO:0008286~insulin receptor signaling pathway GO:0003333~amino acid transmembrane transport GO:0050839~cell adhesion molecule binding GO:0006735~NADH regeneration GO:0032868~response to insulin GO:0019899~enzyme binding GO:0061621~canonical glycolysis GO:0016337~single organismal cell-cell adhesion GO:0034329~cell junction assembly GO:0030695~GTPase regulator activity GO:0061615~glycolytic process through fructose-6-phosphate GO:0032869~cellular response to insulin stimulus GO:0070201~regulation of establishment of protein localization GO:0008092~cytoskeletal protein binding GO:0061620~glycolytic process through glucose-6-phosphate GO:0071375~cellular response to peptide hormone stimulus GO:0050796~regulation of insulin GO:0050863~regulation of T cell activation GO:0005484~SNAP receptor activity GO:0031589~cell-substrate adhesion GO:0046942~carboxylic acid transport GO:1901653~cellular response to peptide GO:0000149~SNARE binding GO:0006007~glucose catabolic process GO:1903037~regulation of leukocyte cell-cell adhesion GO:0002064~epithelial cell development GO:0019905~syntaxin binding GO:0051130~positive regulation of cellular component organization GO:0006909~phagocytosis GO:0086003~cardiac muscle cell contraction GO:0022804~active transmembrane transporter activity GO:0043623~cellular protein complex assembly GO:1905039~carboxylic acid transmembrane transport GO:0090307~mitotic spindle assembly GO:0051251~positive regulation of lymphocyte activation GO:0008289~lipid binding GO:1903825~organic acid transmembrane transport Biological Process (# of preys involved ) GO:0046785~microtubule polymerization GO:1900076~regulation of cellular response to insulin stimulus GO:0035091~phosphatidylinositol binding

Biological Process (# of preys involved ) GO:0048857~neural nucleus development GO:0051298~centrosome duplication GO:0001775~cell activation GO:0015291~secondary active transmembrane transporter activity GO:0007020~microtubule nucleation GO:0086001~cardiac muscle cell action potential GO:0098901~regulation of cardiac muscle cell action potential GO:0015293~symporter activity GO:0031109~microtubule polymerization or depolymerization GO:0038094~Fc-gamma receptor signaling pathway GO:0060589~nucleoside-triphosphatase regulator activity GO:0031122~cytoplasmic microtubule organization GO:0038096~Fc-gamma receptor signaling pathway involved in phagocytosis GO:0002431~Fc receptor mediated stimulatory signaling pathway GO:0046943~carboxylic acid transmembrane transporter activity GO:0051415~interphase microtubule nucleation by interphase microtubule organizing center GO:0032878~regulation of establishment or maintenance of cell polarity GO:0070830~bicellular tight junction assembly GO:0005342~organic acid transmembrane transporter activity GO:0051418~microtubule nucleation by microtubule organizing center GO:0050865~regulation of cell activation GO:1902850~microtubule cytoskeleton organization involved in mitosis GO:0007043~cell-cell junction assembly GO:0005158~insulin receptor binding GO:0043297~apical junction assembly GO:0031023~microtubule organizing center organization GO:0098911~regulation of ventricular cardiac muscle cell action potential GO:0051020~GTPase binding GO:0015849~organic acid transport GO:0000226~microtubule cytoskeleton organization GO:0002433~immune response-regulating cell surface receptor signaling pathway involved in phagocytosis GO:0030674~protein binding, bridging GO:0051972~regulation of telomerase activity GO:0031532~actin cytoskeleton reorganization GO:0006865~amino acid transport GO:0060090~binding, bridging GO:0007067~mitotic nuclear division GO:0002480~antigen processing and presentation of exogenous peptide antigen via MHC class I, TAP-independent GO:0007098~centrosome cycle GO:0044409~entry into host GO:0008047~enzyme activator activity GO:0022407~regulation of cell-cell adhesion GO:1903046~meiotic cell cycle process GO:0043434~response to peptide hormone GO:0005096~GTPase activator activity GO:0051828~entry into other organism involved in symbiotic interaction GO:0007126~meiotic nuclear division GO:0086004~regulation of cardiac muscle cell contraction GO:0098772~molecular function regulator GO:0051225~spindle assembly GO:0030155~regulation of cell adhesion GO:0051806~entry into cell of other organism involved in symbiotic interaction GO:0017016~Ras GTPase binding

GO:0007051~spindle organization GO:0051249~regulation of lymphocyte activation GO:0031267~small GTPase binding GO:0051321~meiotic cell cycle GO:0046718~viral entry into host cell GO:0030260~entry into host cell GO:0017137~Rab GTPase binding GO:0051297~centrosome organization GO:2001275~positive regulation of glucose import in response to insulin stimulus GO:0050870~positive regulation of T cell activation GO:0015405~P-P-bond-hydrolysis-driven transmembrane transporter activity GO:0051258~protein polymerization GO:0002694~regulation of leukocyte activation GO:0051131~chaperone-mediated protein complex assembly GO:1903039~positive regulation of leukocyte cell-cell adhesion GO:0015399~primary active transmembrane transporter activity GO:0060322~head development GO:0044702~single organism reproductive process GO:0045321~leukocyte activation GO:0042626~ATPase activity, coupled to transmembrane movement of substances GO:0022409~positive regulation of cell-cell adhesion GO:0010557~positive regulation of macromolecule biosynthetic process GO:0035315~hair cell differentiation GO:0016820~hydrolase activity, acting on acid anhydrides, catalyzing transmembrane movement of substances GO:0019953~sexual reproduction GO:0006886~intracellular protein transport GO:0034613~cellular protein localization GO:0043492~ATPase activity, coupled to movement of substances GO:0051128~regulation of cellular component organization GO:0070727~cellular macromolecule localization GO:0061024~membrane organization GO:0008565~protein transporter activity GO:1903333~negative regulation of protein folding GO:0046907~intracellular transport GO:0044703~multi-organism reproductive process GO:0051649~establishment of localization in cell GO:0022853~active ion transmembrane transporter activity GO:0051641~cellular localization GO:0044265~cellular macromolecule catabolic process GO:0033036~macromolecule localization GO:0042625~ATPase coupled ion transmembrane transporter activity GO:0009057~macromolecule catabolic process GO:0015031~protein transport GO:0008104~protein localization GO:0019829~cation-transporting ATPase activity GO:0000278~mitotic cell cycle GO:0045184~establishment of protein localization GO:0046915~transition metal ion transmembrane transporter activity GO:0044802~single-organism membrane organization GO:1903047~mitotic cell cycle process GO:1902580~single-organism cellular localization GO:0030276~ binding GO:0044772~mitotic cell cycle phase transition GO:0070925~organelle assembly GO:0065003~macromolecular complex assembly GO:0015294~solute:cation symporter activity GO:0044770~cell cycle phase transition GO:0060341~regulation of cellular localization GO:0044248~cellular catabolic process GO:0071822~protein complex subunit organization GO:1901981~phosphatidylinositol phosphate binding GO:0022607~cellular component assembly GO:0002478~antigen processing and presentation of exogenous peptide antigen GO:0006461~protein complex assembly GO:0015296~anion:cation symporter activity GO:0070271~protein complex biogenesis GO:0048002~antigen processing and presentation of peptide antigen GO:0098609~cell-cell adhesion GO:0005543~phospholipid binding GO:0019884~antigen processing and presentation of exogenous antigen GO:0048468~cell development GO:0030030~cell projection organization GO:0008324~cation transmembrane transporter activity GO:0019882~antigen processing and presentation GO:0048666~neuron development GO:1990778~protein localization to cell periphery GO:0046873~metal ion transmembrane transporter activity GO:0007346~regulation of mitotic cell cycle GO:0072659~protein localization to plasma membrane GO:1901990~regulation of mitotic cell cycle phase transition GO:0051128~regulation of cellular component organization GO:0005385~zinc ion transmembrane transporter activity GO:1902589~single-organism organelle organization GO:1901987~regulation of cell cycle phase transition GO:0000902~cell morphogenesis GO:0035615~clathrin adaptor activity GO:0070647~protein modification by small protein conjugation or removal GO:0032989~cellular component morphogenesis GO:0000904~cell morphogenesis involved in differentiation GO:0098748~endocytic adaptor activity GO:0032446~protein modification by small protein conjugation GO:1904375~regulation of protein localization to cell periphery GO:0022820~potassium ion symporter activity GO:0007009~plasma membrane organization GO:1903364~positive regulation of cellular protein catabolic process GO:1903076~regulation of protein localization to plasma membrane GO:0015379~potassium:chloride symporter activity GO:1903362~regulation of cellular protein catabolic process GO:1903729~regulation of plasma membrane organization GO:0090002~establishment of protein localization to plasma membrane GO:0005154~epidermal growth factor receptor binding GO:0045732~positive regulation of protein catabolic process GO:0007169~transmembrane receptor protein tyrosine kinase signaling pathway GO:0031331~positive regulation of cellular catabolic process GO:0032880~regulation of protein localization GO:1990459~transferrin receptor binding GO:0006897~endocytosis GO:0036211~protein modification process GO:0007163~establishment or maintenance of cell polarity GO:1990460~leptin receptor binding GO:0090003~regulation of establishment of protein localization to plasma membrane GO:0006464~cellular protein modification process GO:0007155~cell adhesion GO:0015662~ATPase activity, coupled to transmembrane movement of ions, phosphorylative mechanism GO:0042787~protein ubiquitination involved in ubiquitin-dependent protein catabolic process GO:0022610~biological adhesion GO:0030010~establishment of cell polarity GO:0008200~ion channel inhibitor activity GO:0006511~ubiquitin-dependent protein catabolic process GO:0007167~enzyme linked receptor protein signaling pathway GO:0018279~protein N-linked glycosylation via asparagine GO:0016248~channel inhibitor activity GO:0019941~modification-dependent protein catabolic process GO:0018196~peptidyl-asparagine modification GO:0017112~Rab guanyl-nucleotide exchange factor activity GO:0043632~modification-dependent macromolecule catabolic process GO:0006890~retrograde vesicle-mediated transport, Golgi to ER GO:0055080~cation homeostasis GO:0051603~proteolysis involved in cellular protein catabolic process GO:0098771~inorganic ion homeostasis GO:0015377~cation:chloride symporter activity GO:0044257~cellular protein catabolic process GO:0050801~ion homeostasis GO:0022890~inorganic cation transmembrane transporter activity GO:0007264~small GTPase mediated signal transduction GO:0030163~protein catabolic process GO:0043623~cellular protein complex assembly GO:0019869~chloride channel inhibitor activity GO:0031175~neuron projection development GO:0016567~protein ubiquitination GO:0048858~cell projection morphogenesis GO:0015077~monovalent inorganic cation transmembrane transporter activity GO:0006521~regulation of cellular amino acid metabolic process GO:0032990~cell part morphogenesis GO:0048812~neuron projection morphogenesis GO:0017081~chloride channel regulator activity GO:0002479~antigen processing and presentation of exogenous peptide antigen via MHC class I, TAP-dependent GO:0016358~dendrite development GO:0042590~antigen processing and presentation of exogenous peptide antigen via MHC class I GO:0048667~cell morphogenesis involved in neuron differentiation GO:0005537~mannose binding GO:1903337~positive regulation of vacuolar transport GO:0051436~negative regulation of ubiquitin-protein ligase activity involved in mitotic cell cycle GO:1903649~regulation of cytoplasmic transport GO:0070273~phosphatidylinositol-4-phosphate binding GO:0008105~asymmetric protein localization GO:0051439~regulation of ubiquitin-protein ligase activity involved in mitotic cell cycle GO:2000641~regulation of early endosome to late endosome transport GO:0001054~RNA polymerase I activity GO:1904667~negative regulation of ubiquitin protein ligase activity GO:2000643~positive regulation of early endosome to late endosome transport GO:0043087~regulation of GTPase activity GO:0048027~mRNA 5’-UTR binding GO:0033238~regulation of cellular amine metabolic process GO:0043547~positive regulation of GTPase activity GO:0045182~translation regulator activity GO:0051437~positive regulation of ubiquitin-protein ligase activity involved in regulation of mitotic cell cycle transition GO:0007173~epidermal growth factor receptor signaling pathway GO:0038127~ERBB signaling pathway GO:0016779~nucleotidyltransferase activity GO:0031145~anaphase-promoting complex-dependent catabolic process GO:0051336~regulation of hydrolase activity GO:0051345~positive regulation of hydrolase activity GO:0051444~negative regulation of ubiquitin-protein transferase activity GO:0072665~protein localization to vacuole GO:0003899~DNA-directed RNA polymerase activity GO:1904668~positive regulation of ubiquitin protein ligase activity GO:0060627~regulation of vesicle-mediated transport GO:0034062~RNA polymerase activity GO:0090114~COPII-coated vesicle budding GO:1904666~regulation of ubiquitin protein ligase activity GO:0006900~membrane budding GO:0042623~ATPase activity, coupled GO:0006903~vesicle targeting GO:2000060~positive regulation of protein ubiquitination involved in ubiquitin-dependent protein catabolic process GO:0048208~COPII vesicle coating GO:0016887~ATPase activity GO:0002474~antigen processing and presentation of peptide antigen via MHC class I GO:0048207~vesicle targeting, rough ER to cis-Golgi GO:0006901~vesicle coating GO:0017111~nucleoside-triphosphatase activity GO:2000058~regulation of protein ubiquitination involved in ubiquitin-dependent protein catabolic process GO:0048199~vesicle targeting, to, from or within Golgi GO:0051443~positive regulation of ubiquitin-protein transferase activity GO:0051650~establishment of vesicle localization GO:0016462~pyrophosphatase activity GO:0051648~vesicle localization GO:0002223~stimulatory C-type lectin receptor signaling pathway GO:0006888~ER to Golgi vesicle-mediated transport GO:0016818~hydrolase activity, acting on acid anhydrides, in phosphorus-containing anhydrides GO:0051640~organelle localization GO:0038061~NIK/NF-kappaB signaling GO:0048193~Golgi vesicle transport GO:0016817~hydrolase activity, acting on acid anhydrides GO:0060071~Wnt signaling pathway, planar cell polarity pathway GO:0016192~vesicle-mediated transport GO:0051656~establishment of organelle localization GO:0044822~poly(A) RNA binding GO:0002220~innate immune response activating cell surface receptor signaling pathway GO:0010256~endomembrane system organization GO:0051049~regulation of transport GO:0003723~RNA binding GO:0090175~regulation of establishment of planar polarity GO:0016050~vesicle organization GO:0003676~nucleic acid binding GO:0031398~positive regulation of protein ubiquitination GO:0061025~membrane fusion GO:0022406~membrane docking GO:1901363~heterocyclic compound binding GO:0044106~cellular amine metabolic process GO:0006914~autophagy GO:0051438~regulation of ubiquitin-protein transferase activity GO:0016197~endosomal transport GO:0097159~organic cyclic compound binding GO:0016482~cytosolic transport GO:0090263~positive regulation of canonical Wnt signaling pathway GO:0007034~vacuolar transport GO:0004004~ATP-dependent RNA helicase activity GO:0044801~single-organism membrane fusion GO:0001736~establishment of planar polarity GO:0048284~organelle fusion GO:0008186~RNA-dependent ATPase activity GO:0007164~establishment of tissue polarity GO:0006906~vesicle fusion GO:0090174~organelle membrane fusion GO:0003724~RNA helicase activity GO:1903322~positive regulation of protein modification by small protein conjugation or removal GO:0042147~retrograde transport, endosome to Golgi

GO:0007041~lysosomal transport GO:0008026~ATP-dependent helicase activity GO:0009308~amine metabolic process GO:0048278~vesicle docking GO:0031397~negative regulation of protein ubiquitination GO:0006887~exocytosis GO:0070035~purine NTP-dependent helicase activity GO:0007267~cell-cell signaling GO:0001738~morphogenesis of a polarized epithelium GO:0090630~activation of GTPase activity GO:0004386~helicase activity GO:0033209~tumor necrosis factor-mediated signaling pathway GO:0006814~sodium ion transport GO:0032940~secretion by cell GO:0030515~snoRNA binding GO:1903321~negative regulation of protein modification by small protein conjugation or removal GO:0043001~Golgi to plasma membrane protein transport GO:0046903~secretion GO:0034511~U3 snoRNA binding GO:0043488~regulation of mRNA stability GO:0043112~receptor metabolic process GO:0008173~RNA methyltransferase activity GO:0043487~regulation of RNA stability GO:0006812~cation transport GO:0097479~ localization GO:0003735~structural constituent of ribosome GO:0030177~positive regulation of Wnt signaling pathway GO:0030001~metal ion transport GO:0035567~non-canonical Wnt signaling pathway GO:0006904~vesicle docking involved in exocytosis GO:0019843~rRNA binding GO:0006811~ion transport GO:0010565~regulation of cellular ketone metabolic process GO:0006836~neurotransmitter transport GO:0005198~structural molecule activity GO:0055085~transmembrane transport GO:0031396~regulation of protein ubiquitination GO:0006892~post-Golgi vesicle-mediated transport GO:0070180~large ribosomal subunit rRNA binding GO:0090090~negative regulation of canonical Wnt signaling pathway GO:0034498~early endosome to Golgi transport GO:0006623~protein targeting to vacuole GO:0043021~ribonucleoprotein complex binding GO:0050852~T cell receptor signaling pathway GO:0072666~establishment of protein localization to vacuole GO:0015804~neutral amino acid transport GO:1990932~5.8S rRNA binding GO:1903052~positive regulation of proteolysis involved in cellular protein catabolic process GO:0007030~Golgi organization GO:1903320~regulation of protein modification by small protein conjugation or removal GO:0000301~retrograde transport, vesicle recycling within Golgi GO:0008649~rRNA methyltransferase activity GO:0006891~intra-Golgi vesicle-mediated transport GO:0038095~Fc-epsilon receptor signaling pathway GO:0008333~endosome to lysosome transport GO:0070181~small ribosomal subunit rRNA binding GO:0030178~negative regulation of Wnt signaling pathway GO:0000041~transition metal ion transport GO:0017157~regulation of exocytosis GO:0036094~small molecule binding GO:0050851~antigen receptor-mediated signaling pathway GO:0061462~protein localization to lysosome GO:0007017~microtubule-based process GO:0000166~nucleotide binding GO:0042180~cellular ketone metabolic process GO:0030705~cytoskeleton-dependent intracellular transport GO:1901265~nucleoside phosphate binding GO:1903050~regulation of proteolysis involved in cellular protein catabolic process GO:0072583~clathrin-mediated endocytosis GO:0002495~antigen processing and presentation of peptide antigen via MHC class II GO:0032549~ribonucleoside binding GO:0060828~regulation of canonical Wnt signaling pathway GO:0006898~receptor-mediated endocytosis GO:0019886~antigen processing and presentation of exogenous peptide antigen via MHC class II GO:0038093~Fc receptor signaling pathway GO:0001882~nucleoside binding GO:0002504~antigen processing and presentation of peptide or polysaccharide antigen via MHC class II GO:0002758~innate immune response-activating signal transduction GO:0055076~transition metal ion homeostasis GO:0008757~S-adenosylmethionine-dependent methyltransferase activity GO:0045022~early endosome to late endosome transport GO:0071356~cellular response to tumor necrosis factor GO:0098927~vesicle-mediated transport between endosomal compartments GO:0044877~macromolecular complex binding GO:0002218~activation of innate immune response GO:0006896~Golgi to vacuole transport GO:0007033~vacuole organization GO:0033170~protein-DNA loading ATPase activity GO:2000027~regulation of organ morphogenesis GO:0030100~regulation of endocytosis GO:1901185~negative regulation of ERBB signaling pathway GO:0003689~DNA clamp loader activity GO:0000209~protein polyubiquitination GO:0006820~anion transport GO:0034612~response to tumor necrosis factor GO:0002366~leukocyte activation involved in immune response GO:0030554~adenyl nucleotide binding GO:0042059~negative regulation of epidermal growth factor receptor signaling pathway GO:0060070~canonical Wnt signaling pathway GO:0015672~monovalent inorganic cation transport GO:0005524~ATP binding GO:0045089~positive regulation of innate immune response GO:0006873~cellular ion homeostasis GO:0007018~microtubule-based movement GO:0032559~adenyl ribonucleotide binding GO:0030111~regulation of Wnt signaling pathway GO:1903530~regulation of secretion by cell GO:0044212~transcription regulatory region DNA binding GO:0002263~cell activation involved in immune response GO:0009896~positive regulation of catabolic process GO:0001505~regulation of neurotransmitter levels GO:0000975~regulatory region DNA binding GO:0051348~negative regulation of transferase activity GO:0030003~cellular cation homeostasis GO:0002699~positive regulation of immune effector process GO:0001067~regulatory region nucleic acid binding GO:0045862~positive regulation of proteolysis GO:0002697~regulation of immune effector process GO:0061088~regulation of sequestering of zinc ion GO:0003677~DNA binding GO:0045088~regulation of innate immune response GO:0035646~endosome to melanosome transport GO:0002429~immune response-activating cell surface receptor signaling pathway GO:0001821~histamine secretion GO:0000217~DNA secondary structure binding GO:0045576~mast cell activation GO:0042176~regulation of protein catabolic process GO:0002553~histamine secretion by mast cell GO:0000988~transcription factor activity, protein binding GO:0043161~proteasome-mediated ubiquitin-dependent protein catabolic process GO:0002349~histamine production involved in inflammatory response GO:0048757~pigment granule maturation GO:0003712~transcription cofactor activity GO:0031329~regulation of cellular catabolic process GO:0035493~SNARE complex assembly GO:0002441~histamine secretion involved in inflammatory response GO:0003690~double-stranded DNA binding GO:0002768~immune response-regulating cell surface receptor signaling pathway GO:0043299~leukocyte degranulation GO:0003684~damaged DNA binding GO:0031349~positive regulation of defense response GO:0050690~regulation of defense response to virus by virus GO:0007032~endosome organization GO:0010498~proteasomal protein catabolic process GO:1904376~negative regulation of protein localization to cell periphery GO:0008080~N-acetyltransferase activity GO:0010564~regulation of cell cycle process GO:0006895~Golgi to endosome transport GO:0002448~mast cell mediated immunity GO:0003682~chromatin binding GO:0016055~Wnt signaling pathway GO:0034067~protein localization to Golgi apparatus GO:0043566~structure-specific DNA binding GO:0043300~regulation of leukocyte degranulation GO:0198738~cell-cell signaling by wnt GO:0043303~mast cell degranulation GO:0042393~histone binding GO:0010608~posttranscriptional regulation of gene expression GO:0050688~regulation of defense response to virus GO:0098655~cation transmembrane transport GO:0008094~DNA-dependent ATPase activity GO:0002757~immune response-activating signal transduction GO:0046916~cellular transition metal ion homeostasis GO:0098660~inorganic ion transmembrane transport GO:0003713~transcription coactivator activity GO:0009894~regulation of catabolic process GO:0033059~cellular pigmentation GO:0002009~morphogenesis of an epithelium GO:0050792~regulation of viral process GO:0016407~acetyltransferase activity GO:0097576~vacuole fusion GO:1905114~cell surface receptor signaling pathway involved in cell-cell signaling GO:0002275~myeloid cell activation involved in immune response GO:0004402~histone acetyltransferase activity GO:0002764~immune response-regulating signaling pathway GO:0097352~autophagosome maturation GO:0045921~positive regulation of exocytosis GO:0061733~peptide-lysine-N-acetyltransferase activity GO:0002253~activation of immune response GO:0017156~ ion regulated exocytosis GO:0002444~myeloid leukocyte mediated immunity GO:1990837~sequence-specific double-stranded DNA binding GO:0019221~cytokine-mediated signaling pathway GO:0002279~mast cell activation involved in immune response GO:0002039~p53 binding GO:0031400~negative regulation of protein modification process GO:0098662~inorganic cation transmembrane transport GO:0002705~positive regulation of leukocyte mediated immunity GO:0048729~tissue morphogenesis GO:0002274~myeloid leukocyte activation GO:0003714~transcription corepressor activity GO:0051347~positive regulation of transferase activity GO:0002831~regulation of response to biotic stimulus GO:0034212~peptide N-acetyltransferase activity GO:0016079~synaptic vesicle exocytosis GO:0031347~regulation of defense response GO:1903305~regulation of regulated secretory pathway GO:0016747~transferase activity, transferring acyl groups other than amino-acyl groups GO:0045055~regulated exocytosis GO:0050778~positive regulation of immune response GO:0034220~ion transmembrane transport GO:0001075~transcription factor activity, RNA polymerase II core promoter sequence-specific binding involvedcomplex in preinitiation assembly GO:0043903~regulation of symbiosis, encompassing mutualism through parasitism GO:0051726~regulation of cell cycle GO:0051608~histamine transport GO:0016410~N-acyltransferase activity GO:0071345~cellular response to cytokine stimulus GO:0043485~endosome to pigment granule transport GO:1903077~negative regulation of protein localization to plasma membrane GO:0000983~transcription factor activity, RNA polymerase II core promoter sequence-specific GO:0030162~regulation of proteolysis GO:0043302~positive regulation of leukocyte degranulation GO:0060429~epithelium development GO:0010970~establishment of localization by movement along microtubule GO:0004003~ATP-dependent DNA helicase activity GO:0048813~dendrite morphogenesis GO:0034097~response to cytokine GO:0048280~vesicle fusion with Golgi apparatus GO:0000977~RNA polymerase II regulatory region sequence-specific DNA binding GO:0031401~positive regulation of protein modification process GO:0048268~clathrin coat assembly GO:0048013~ephrin receptor signaling pathway GO:0001012~RNA polymerase II regulatory region DNA binding GO:0043476~pigment accumulation GO:0045087~innate immune response GO:0043482~cellular pigment accumulation GO:0003700~transcription factor activity, sequence-specific DNA binding GO:0043086~negative regulation of catalytic activity GO:0006622~protein targeting to lysosome GO:0001071~nucleic acid binding transcription factor activity GO:0000165~MAPK cascade GO:0033003~regulation of mast cell activation GO:0072600~establishment of protein localization to Golgi GO:0000976~transcription regulatory region sequence-specific DNA binding GO:0023014~signal transduction by protein phosphorylation GO:0023061~signal release GO:0000042~protein targeting to Golgi GO:0000400~four-way junction DNA binding GO:0050776~regulation of immune response GO:1903593~regulation of histamine secretion by mast cell

GO:0051338~regulation of transferase activity GO:0099504~synaptic vesicle cycle GO:0000989~transcription factor activity, transcription factor binding GO:0033006~regulation of mast cell activation involved in immune response GO:0002684~positive regulation of immune system process GO:0045117~azole transport GO:0043142~single-stranded DNA-dependent ATPase activity GO:0009887~organ morphogenesis GO:0055069~zinc ion homeostasis GO:0099003~vesicle mediated transport in synapse GO:0043565~sequence-specific DNA binding GO:0032269~negative regulation of cellular protein metabolic process GO:0043473~pigmentation GO:0001919~regulation of receptor recycling GO:0032142~single guanine insertion binding GO:0051248~negative regulation of protein metabolic process GO:0071577~zinc II ion transmembrane transport GO:0022603~regulation of anatomical structure morphogenesis GO:0090161~Golgi ribbon formation GO:0035064~methylated histone binding GO:0097480~establishment of synaptic vesicle localization GO:0032270~positive regulation of cellular protein metabolic process GO:0006882~cellular zinc ion homeostasis GO:0003678~DNA helicase activity GO:0009968~negative regulation of signal transduction GO:0048489~synaptic vesicle transport GO:0043304~regulation of mast cell degranulation GO:0016891~endoribonuclease activity, producing 5’-phosphomonoesters GO:0044092~negative regulation of molecular function GO:0042058~regulation of epidermal growth factor receptor signaling pathway GO:1901184~regulation of ERBB signaling pathway GO:0000403~Y-form DNA binding GO:0051247~positive regulation of protein metabolic process GO:1903335~regulation of vacuolar transport GO:0032137~guanine/thymine mispair binding GO:0010648~negative regulation of cell communication GO:0098876~vesicle-mediated transport to the plasma membrane GO:1903595~positive regulation of histamine secretion by mast cell GO:0032357~oxidized purine DNA binding GO:0023057~negative regulation of signaling GO:0002886~regulation of myeloid leukocyte mediated immunity GO:0032438~melanosome organization GO:0032356~oxidized DNA binding GO:0031399~regulation of protein modification process GO:0006829~zinc II ion transport GO:0080134~regulation of response to stress GO:0048753~pigment granule organization GO:0032138~single base insertion or deletion binding GO:0043307~eosinophil activation GO:0009967~positive regulation of signal transduction GO:0071578~zinc II ion transmembrane import GO:0016893~endonuclease activity, active with either ribo- or deoxyribonucleic acids and producing 5’-phosphomonoesters GO:0043248~proteasome assembly GO:0043320~natural killer cell degranulation GO:0015677~copper ion import GO:0018024~histone-lysine N-methyltransferase activity GO:0007267~cell-cell signaling GO:0099643~signal release from synapse GO:0007269~neurotransmitter secretion GO:0070087~chromo shadow domain binding GO:0006952~defense response GO:0099518~vesicle cytoskeletal trafficking

GO:0006955~immune response GO:0001881~receptor recycling Molecular Function (# of preys involved ) GO:0032135~DNA insertion or deletion binding GO:0099531~presynaptic process involved in chemical synaptic transmission GO:0010647~positive regulation of cell communication GO:0050775~positive regulation of dendrite morphogenesis GO:0016279~protein-lysine N-methyltransferase activity GO:0043085~positive regulation of catalytic activity GO:0002703~regulation of leukocyte mediated immunity GO:0080171~lytic vacuole organization GO:0016278~lysine N-methyltransferase activity GO:0023056~positive regulation of signaling GO:0007040~lysosome organization GO:0051046~regulation of secretion GO:0042054~histone methyltransferase activity GO:0006508~proteolysis GO:0047496~vesicle transport along microtubule GO:0004521~endoribonuclease activity GO:2000026~regulation of multicellular organismal development GO:0090160~Golgi to lysosome transport GO:0031338~regulation of vesicle fusion GO:0031491~nucleosome binding GO:0032268~regulation of cellular protein metabolic process GO:0002278~eosinophil activation involved in immune response GO:0043308~eosinophil degranulation GO:0008134~transcription factor binding GO:0044093~positive regulation of molecular function GO:0002447~eosinophil mediated immunity GO:0006468~protein phosphorylation GO:0072384~organelle transport along microtubule GO:0035639~purine ribonucleoside triphosphate binding GO:0002532~production of molecular mediator involved in inflammatory response GO:0051246~regulation of protein metabolic process GO:0008088~axo-dendritic transport GO:0032550~purine ribonucleoside binding GO:0048584~positive regulation of response to stimulus GO:0016236~macroautophagy GO:0032418~lysosome localization GO:0001883~purine nucleoside binding GO:0016310~phosphorylation GO:0045851~pH reduction GO:0033572~transferrin transport GO:0032555~purine ribonucleotide binding GO:0010605~negative regulation of macromolecule metabolic process GO:0051452~intracellular pH reduction GO:0031324~negative regulation of cellular metabolic process GO:0090382~phagosome maturation GO:0032553~ribonucleotide binding GO:0015682~ferric iron transport GO:0050790~regulation of catalytic activity GO:0072512~trivalent inorganic cation transport GO:0017076~purine nucleotide binding GO:0051453~regulation of intracellular pH GO:0009893~positive regulation of metabolic process GO:0030641~regulation of cellular pH GO:0051082~unfolded protein binding GO:0009892~negative regulation of metabolic process GO:0006826~iron ion transport GO:0019882~antigen processing and presentation GO:0016810~hydrolase activity, acting on carbon-nitrogen (but not peptide) bonds GO:0035556~intracellular signal transduction GO:0002478~antigen processing and presentation of exogenous peptide antigen GO:0031005~filamin binding GO:0007166~cell surface receptor signaling pathway GO:0048002~antigen processing and presentation of peptide antigen GO:0019884~antigen processing and presentation of exogenous antigen GO:0032561~guanyl ribonucleotide binding GO:0009966~regulation of signal transduction GO:0031929~TOR signaling GO:0032006~regulation of TOR signaling GO:0019001~guanyl nucleotide binding GO:0065009~regulation of molecular function GO:0032008~positive regulation of TOR signaling

GO:0010646~regulation of cell communication GO:0010506~regulation of autophagy GO:0036033~mediator complex binding GO:0090383~phagosome acidification GO:0023051~regulation of signaling GO:0006885~regulation of pH GO:0046961~proton-transporting ATPase activity, rotational mechanism GO:0071230~cellular response to amino acid stimulus GO:0006796~phosphate-containing compound metabolic process GO:0043200~response to amino acid GO:0036442~hydrogen-exporting ATPase activity GO:0006793~phosphorus metabolic process GO:0016241~regulation of macroautophagy GO:0071229~cellular response to acid chemical GO:0044769~ATPase activity, coupled to transmembrane movement of ions, rotational mechanism GO:0031325~positive regulation of cellular metabolic process GO:1901699~cellular response to nitrogen compound GO:0019079~viral genome replication GO:0097367~carbohydrate derivative binding GO:0010604~positive regulation of macromolecule metabolic process GO:0048524~positive regulation of viral process GO:0030004~cellular monovalent inorganic cation homeostasis GO:0005525~GTP binding GO:0071417~cellular response to organonitrogen compound GO:0003924~GTPase activity GO:0055067~monovalent inorganic cation homeostasis 1 20 AP-MS BioID BioID

Supplementary Figure 4: Gene Ontology analysis of interaction contexts of 18 localization markers. The DAVID bioinformatics recourses 6.8 database (https://david.ncifcrf.gov/) are used for GO term analysis to generate the value count matrix for clustering with a P-value cut-off of P < 0.01. The hierarchically clustered heatmap including: biology process (a) and molecular function (b). The color intensities indicate the sum of preys of corresponding GO term. Hierarchal clustering was used to generate the cluster dendrogram.

23 bioRxiv preprint doi: https://doi.org/10.1101/264788; this version posted February 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

An integrated approach to comprehensively map the molecular context of proteins

ab

Golgi Lysosome Chromatin Nuclear envelope 186 46 59 20 9 1 142 71

85 23 28 62 2

17 29 3 31 52 15 4 107 ER4 Endosome Nucleolus

ER (CALX) Chromatin (H31) Golgi (GOGA2+TGON2) Nuclear envelope (LMNA) Lysosome (LAMP1) Nucleolus (FBRL) Endosome (EEA1+RAB9A+RAB11A)

Supplementary Figure 5: Inter-relation of diff erent subcellular organelles. Venn’s diagram compares the number of identifi ed HCIPs from diff erent subcellular compartments to the biological relationship between diff erent organelles. (a) BioID identifi ed proteins that are traffi cking among Golgi, lysosome, endosome and ER are shown. (b) BioID identifi ed proteins traffi cking among nucleolus, NE and chromatin are shown.

24 bioRxiv preprint doi: https://doi.org/10.1101/264788; this version posted February 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Liu et al.

AOX

AURKB

BET1

CDK7

CDK8

GSK3b

MED13

RAB5A

– Biotin + Biotin + Biotin Bait Protein Bait Protein Biotinylated Proteins

Supplementary Figure 6: Validation of protein localization with immunofl uorescence. Fluorescence microscopy is applied to observe the MAC-tagged proteins that have been monitored by MS-micros- copy (Fig. 4). These tagged bait proteins are visualized with anti-HA immunostaining (green), DAPI (blue) and the in vivo biotinylated interactors are staining with Alexa Fluor®594-conjugated streptavidin (red), Scale bar: 10 μm.

25