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A Versatile Lentiviral Delivery Toolkit for Proximity-dependent Biotinylation in Diverse Cell Types

2018; Elsevier BV; Volume: 17; Issue: 11 Linguagem: Inglês

10.1074/mcp.tir118.000902

ISSN

1535-9484

Autores

Payman Samavarchi‐Tehrani, Hala Abdouni, Reuben Samson, Anne‐Claude Gingras,

Tópico(s)

Neuroscience and Neural Engineering

Resumo

Proximity-dependent biotinylation strategies have emerged as powerful tools to characterize the subcellular context of proteins in living cells. The popular BioID approach employs an abortive E. coli biotin ligase mutant (R118G; denoted as BirA*), which when fused to a bait protein eNAbles the covalent biotinylation of endogenous proximal polypeptides. This approach has been mainly applied to the study of protein proximity in immortalized mammalian cell lines. To expand the application space of BioID, here we describe a set of lentiviral vectors that eNAble the inducible expression of BirA*-tagged bait fusion proteins for performing proximity-dependent biotinylation in diverse experimental systems. We benchmark this highly adaptable toolkit across immortalized and primary cell systems, demonstrating the ease, versatility and robustness of the system. We also provide guidelines to perform BioID using these reagents. Proximity-dependent biotinylation strategies have emerged as powerful tools to characterize the subcellular context of proteins in living cells. The popular BioID approach employs an abortive E. coli biotin ligase mutant (R118G; denoted as BirA*), which when fused to a bait protein eNAbles the covalent biotinylation of endogenous proximal polypeptides. This approach has been mainly applied to the study of protein proximity in immortalized mammalian cell lines. To expand the application space of BioID, here we describe a set of lentiviral vectors that eNAble the inducible expression of BirA*-tagged bait fusion proteins for performing proximity-dependent biotinylation in diverse experimental systems. We benchmark this highly adaptable toolkit across immortalized and primary cell systems, demonstrating the ease, versatility and robustness of the system. We also provide guidelines to perform BioID using these reagents. Understanding the functional relationships between proteins is essential for gaining mechanistic insight into their biological roles. Proteins can engage in stable or dynamic direct interactions, or can participate in indirect interactions mediated through molecules such as other proteins or nucleic acids. Mass spectrometry (MS)-based proteomics approaches have played an integral role in assessing such interactions (1Gingras A.C. Gstaiger M. Raught B. Aebersold R. ANAlysis of protein complexes using mass spectrometry.NAt. Rev. Mol. Cell Biol. 2007; 8: 645-654Crossref PubMed Scopus (558) Google Scholar). For example, biochemical fractionation followed by MS can be employed to detect protein complexes that co-fractionate (2Stasyk T. Huber L.A. Zooming in: fractioNAtion strategies in proteomics.Proteomics. 2004; 4: 3704-3716Crossref PubMed Scopus (180) Google Scholar, 3HavugimaNA P.C. Hart G.T. Nepusz T. Yang H. Turinsky A.L. Li Z. Wang P.I. Boutz D.R. Fong V. Phanse S. Babu M. 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More frequently, MS is coupled with affinity purification (AP) of a selected protein of interest (bait) in a technique commonly referred to as AP-MS 1The abbreviations used are:AP-MSaffinity-purification coupled to mass spectrometryBirA*E. coli biotin ligase harboring a R118G mutationVP16herpes simplex virus (HSV) virion protein 16rTetRreverse tetracycline repressorrtTAreverse tetracycline-controlled transactivator, rTetR fused to VP16PGKphosphoglycerate kinase 1 promotersEF1ashort form of elongation factor-1 alpha (EF1a), promoterMEFmouse embryonic fibroblastMCSmultiple cloning siteNLSnuclear localization sequence. 1The abbreviations used are:AP-MSaffinity-purification coupled to mass spectrometryBirA*E. coli biotin ligase harboring a R118G mutationVP16herpes simplex virus (HSV) virion protein 16rTetRreverse tetracycline repressorrtTAreverse tetracycline-controlled transactivator, rTetR fused to VP16PGKphosphoglycerate kinase 1 promotersEF1ashort form of elongation factor-1 alpha (EF1a), promoterMEFmouse embryonic fibroblastMCSmultiple cloning siteNLSnuclear localization sequence.. In that set-up, an affinity reagent specific to the bait protein (e.g. an antibody specific to the bait or an epitope tag fused to the bait) is used to enrich it from a cellular lysate alongside its interaction partners, which are subsequently identified by MS (4Dunham W.H. Mullin M. Gingras A.C. Affinity-purification coupled to mass spectrometry: basic principles and strategies.Proteomics. 2012; 12: 1576-1590Crossref PubMed Scopus (205) Google Scholar, 5Ho Y. Gruhler A. Heilbut A. Bader G.D. Moore L. Adams S.L. Millar A. Taylor P. Bennett K. Boutilier K. Yang L. Wolting C. DoNAldson I. Schandorff S. ShewNArane J. Vo M. Taggart J. Goudreault M. Muskat B. Alfarano C. Dewar D. Lin Z. Michalickova K. Willems A.R. Sassi H. Nielsen P.A. Rasmussen K.J. Andersen J.R. Johansen L.E. Hansen L.H. Jespersen H. Podtelejnikov A. Nielsen E. Crawford J. Poulsen V. Sorensen B.D. Matthiesen J. Hendrickson R.C. Gleeson F. Pawson T. Moran M.F. Durocher D. Mann M. Hogue C.W. Figeys D. Tyers M. 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To overcome these challenges and to limit the detection of spurious post-lysis interactions, in vivo proximity-dependent labeling approaches have been introduced in the past 5 years (e.g. (9Roux K.J. Kim D.I. Raida M. Burke B. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells.J. Cell Biol. 2012; 196: 801-810Crossref PubMed Scopus (1234) Google Scholar, 10Hung V. Zou P. Rhee H.W. Udeshi N.D. Cracan V. SvinkiNA T. Carr S.A. Mootha V.K. Ting A.Y. Proteomic mapping of the human mitochondrial intermembrane space in live cells via ratiometric APEX tagging.Mol. Cell. 2014; 55: 332-341Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar)). Using these approaches, a bait protein of interest is fused to an enzyme and expressed in a physiologically-relevant system where the addition of an enzymatic substrate leads to covalent biotinylation of proteins located near the bait (11Rees J.S. Li X.W. Perrett S. Lilley K.S. Jackson A.P. Protein neighbors and proximity proteomics.Mol. Cell Proteomics. 2015; 14: 2848-2856Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 12Kim D.I. Roux K.J. Filling the void: proximity-based labeling of proteins in living cells.Trends Cell Biol. 2016; 26: 804-817Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). In the case of the BioID approach described here, a mutant form of biotin ligase catalyzes the activation of exogenously-supplied biotin to the reactive intermediate, biotinoyl-5′-AMP (13Xu Y. Beckett D. Kinetics of biotinyl-5′-adenylate synthesis catalyzed by the Escherichia coli repressor of biotin biosynthesis and the stability of the enzyme-product complex.Biochemistry. 1994; 33: 7354-7360Crossref PubMed Scopus (62) Google Scholar). The abortive BirA* enzyme, which harbors a R118G mutation, displays a reduced affinity for the activated biotin molecule. Biotin-AMP thus diffuses away from the bait and can covalently modify epsilon amine groups of lysine residues on nearby proteins (14Choi-Rhee E. Schulman H. CroNAn J.E. Promiscuous protein biotinylation by Escherichia coli biotin protein ligase.Protein Sci. 2004; 13: 3043-3050Crossref PubMed Scopus (158) Google Scholar, 15CroNAn J.E. Targeted and proximity-dependent promiscuous protein biotinylation by a mutant Escherichia coli biotin protein ligase.J. Nutr. Biochem. 2005; 16: 416-418Crossref PubMed Scopus (53) Google Scholar). Because these proximity partners are covalently marked, maintaining protein-protein interactions during lysis and purification is not necessary, and harsh lysis conditions can be employed to maximize solubilization of all cellular structures. Subsequent recovery of the biotinylated proteins via streptavidin affinity purification followed by MS allows identification of the labeled proteins (9Roux K.J. Kim D.I. Raida M. Burke B. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells.J. Cell Biol. 2012; 196: 801-810Crossref PubMed Scopus (1234) Google Scholar, 12Kim D.I. Roux K.J. Filling the void: proximity-based labeling of proteins in living cells.Trends Cell Biol. 2016; 26: 804-817Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Importantly, the inclusion of proper negative controls in the experimental design (to model both endogenously biotinylated proteins, such as the mitochondrial carboxylases, as well as promiscuous biotinylation resulting from expression of an abortive BirA* enzyme) enables the use of computational tools initially developed for AP-MS (e.g. (16Teo G. Liu G. Zhang J. Nesvizhskii A.I. Gingras A.C. Choi H. SAINTexpress: improvements and additioNAl features in Significance ANAlysis of INTeractome software.J. Proteomics. 2014; 100: 37-43Crossref PubMed Scopus (291) Google Scholar, 17Mellacheruvu D. Wright Z. 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Cell Biol. 2012; 196: 801-810Crossref PubMed Scopus (1234) Google Scholar), BioID has since been employed to uncover new components of signaling pathways (18Couzens A.L. Knight J.D. Kean M.J. Teo G. Weiss A. Dunham W.H. Lin Z.Y. Bagshaw R.D. Sicheri F. Pawson T. WraNA J.L. Choi H. Gingras A.C. Protein interaction network of the mammalian Hippo pathway reveals mechanisms of kiNAse-phosphatase interactions.Sci. SigNAl. 2013; 6: rs15Crossref PubMed Scopus (313) Google Scholar) and their enzyme targets (19Coyaud E. Mis M. Laurent E.M. Dunham W.H. Couzens A.L. Robitaille M. Gingras A.C. Angers S. Raught B. BioID-based identification of Skp Cullin F-box (SCF)beta-TrCP1/2 E3 ligase substrates.Mol. Cell. Proteomics. 2015; 14: 1781-1795Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), to describe the protein composition of structures such as the centrosome, primary cilia (20Gupta G.D. Coyaud E. Goncalves J. Mojarad B.A. Liu Y. Wu Q. Gheiratmand L. Comartin D. Tkach J.M. Cheung S.W. Bashkurov M. Hasegan M. Knight J.D. Lin Z.Y. Schueler M. Hildebrandt F. Moffat J. Gingras A.C. Raught B. Pelletier L. A dyNAmic protein interaction landscape of the human centrosome-cilium interface.Cell. 2015; 163: 1484-1499Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, 21Firat-Karalar E.N. Rauniyar N. Yates 3rd, J.R. Stearns T. Proximity interactions among centrosome components identify regulators of centriole duplication.Curr. Biol. 2014; 24: 664-670Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), focal adhesions (22Dong J.M. Tay F.P. Swa H.L. GuNAratne J. Leung T. Burke B. Manser E. Proximity biotinylation provides insight into the molecular composition of focal adhesions at the NAnometer scale.Sci. SigNAl. 2016; 9: rs4Crossref PubMed Scopus (60) Google Scholar), stress granules and P-bodies (23Youn J.Y. Dunham W.H. Hong S.J. Knight J.D.R. Bashkurov M. Chen G.I. Bagci H. Rathod B. MacLeod G. Eng S.W.M. Angers S. Morris Q. Fabian M. Cote J.F. Gingras A.C. High-density proximity mapping reveals the subcellular organization of mRNA-associated granules and bodies.Mol. Cell. 2018; 69: 517-532.e511Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar) and has been used to examine contacts between organelles (24van Vliet A.R. Giordano F. Gerlo S. Segura I. Van Eygen S. Molenberghs G. Rocha S. Houcine A. Derua R. Verfaillie T. Vangindertael J. De Keersmaecker H. Waelkens E. Tavernier J. Hofkens J. AnNAert W. Carmeliet P. Samali A. Mizuno H. Agostinis P. The ER stress sensor PERK coordiNAtes ER-plasma membrane contact site formation through interaction with filamin-A and F-actin remodeling.Mol. Cell. 2017; 65: 885-899.e886Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), to highlight a few examples. Importantly, however, most of the BioID studies have so far been performed in easily-transfectable cell lines, including HEK293, U2OS and HeLa cells. Although these cell systems continue to reveal important biological insight, it is also critical to perform some of these studies in different contexts and model systems that are less amenable to transfection, including primary cells. Although there have now been several reports that have used viral delivery from adenovirus (25Wang Y. Liu L. Zhang H. Fan J. Zhang F. Yu M. Shi L. Yang L. Lam S.M. Wang H. Chen X. Wang Y. Gao F. Shui G. Xu Z. Mea6 controls VLDL transport through the coordiNAted regulation of COPII assembly.Cell Res. 2016; 26: 787-804Crossref PubMed Scopus (22) Google Scholar), lentivirus (26Gimpel P. Lee Y.L. Sobota R.M. Calvi A. Koullourou V. Patel R. Mamchaoui K. Nedelec F. Shackleton S. Schmoranzer J. Burke B. Cadot B. Gomes E.R. Nesprin-1alpha-dependent microtubule nucleation from the nuclear envelope via Akap450 is necessary for nuclear positioning in muscle cells.Curr. 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Burette A.C. Weinberg R.J. Soderling S.H. Identification of an elaborate complex mediating postsyNAptic inhibition.Science. 2016; 353: 1123-1129Crossref PubMed Scopus (162) Google Scholar) systems, these reagents have so far been limited in their range of application to different workflows. Further, there is a lack of demonstration of optimization and benchmarking to facilitate implementation of BioID across various cell types. Our motivation was to expand the ease and breadth of applicability of BioID to include diverse experimental systems by developing and optimizing lentiviral delivery reagents and workflows. To achieve this, we cloned a cassette consisting of BirA* (enabling BioID) and a single FLAG epitope (that can be used both to detect bait expression and to perform AP-MS) (31Hesketh G.G. Youn J.Y. Samavarchi-Tehrani P. Raught B. Gingras A.C. Parallel exploration of interaction space by BioID and affinity purification coupled to mass spectrometry.Methods Mol. Biol. 2017; 1550: 115-136Crossref PubMed Scopus (38) Google Scholar) downstream of a tetracycline-regulatable promoter, enabling inducible gene expression. We inserted this cassette into five lentiviral vector backbones, each harboring different features, including: integrated rtTA (tetracycline-controled transactivator), puromycin selection or mCitrine expression. Each vector is made available as a Gateway-compatible destination vector enabling amino- or carboxy-terminal tagging, and a subset is also available for standard ligase-mediated cloning. We describe the benchmarking of these vectors for performing BioID through lentiviral gene delivery in HeLa cells as well as in mouse and human fibroblasts. The ease and robustness of this approach was compared with BioID performed using stable Flp-In T-REx HeLa cell lines. Importantly, we highlight a major advantage of this approach, which is the overall decrease in experimental timeline (compared with the common practice of stable cells), while maintaining data quality and reproducibility. We also outline strategies for lentiviral backbone selection and considerations for experimental design that will assist the community in the use of these reagents. The region internal to the long terminal repeats (LTRs) of the second generation pLVX-Tight-Puro lentiviral backbone (Takara Bio, Clontech, Mountain View, CA) was modified to generate the five different lentiviral transfer vector backbones (Fig. 1A). The orientation of the inducible P-tight promoter, which consists of tetracycline response element (TRE) along with a minimum Cytomegalovirus (CMV) promoter, was reversed to allow for robust transgene expression (32Golding M.C. Mann M.R. A bidirectioNAl promoter architecture enhances lentiviral transgenesis in embryonic and extraembryonic stem cells.Gene. Ther. 2011; 18: 817-826Crossref PubMed Scopus (13) Google Scholar, 33Amendola M. Venneri M.A. Biffi A. VigNA E. NAldini L. CoordiNAte dual-gene transgenesis by lentiviral vectors carrying synthetic bidirectioNAl promoters.NAt. Biotechnol. 2005; 23: 108-116Crossref PubMed Scopus (253) Google Scholar). The phosphoglycerate kinase 1 (PGK) promoter was also replaced with a shorter fragment (227bp) of the stronger elongation factor-1 alpha (EF1a) promoter (denoted as sEF1a). Downstream of these constitutive promoters, various cassettes were inserted to generate the transfer vectors. For the pSTVH5 transfer vector, mCitrine was amplified by PCR from pLM-mCitrine-2A-Sox2 (a gift from Michel Sadelain, Addgene #23242) (34Papapetrou E.P. Tomishima M.J. Chambers S.M. Mica Y. Reed E. Menon J. Tabar V. Mo Q. Studer L. Sadelain M. Stoichiometric and temporal requirements of Oct4, Sox2, Klf4, and c-Myc expression for efficient human iPSC induction and differentiation.Proc. NAtl. Acad. Sci. U.S.A. 2009; 106: 12759-12764Crossref PubMed Scopus (226) Google Scholar), fused to rtTA from PB-CA-rtTA Advanced (a gift from Andras nagy, Addgene #20910) (35Woltjen K. Michael I.P. Mohseni P. Desai R. Mileikovsky M. Hamalainen R. Cowling R. Wang W. Liu P. Gertsenstein M. Kaji K. Sung H.K. NAgy A. piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells.NAture. 2009; 458: 766-770Crossref PubMed Scopus (1444) Google Scholar) using fusion-PCR, and inserted downstream of the sEF1a promoter. For the pSTV6 transfer vector, the PGK-puromycin-2A-rtTA cassette was transferred by restriction cloning from the pLIX-402 vector (a gift from David Root, Addgene #41394) using the MluI/KpnI sites and substituted for the sEF1a-mCitrine-2A-rtTA of the pSTVH5 vector. For design of the pSTVH7 transfer vector, the puromycin resistance cassette from pLVX-Tight-Puro was placed downstream of the sEF1a promoter. For the pSTVH8 transfer vector, the rtTA from the PB-CA-rtTA Advanced vector was amplified and cloned downstream of the sEF1a promoter. Subsequently, the BirA*-FLAG-Gateway fragments from pcDna5-FRT/TO-BirA*-FLAG vectors were excised using HindIII/SphI restriction sites and ligated into the pSTVH5-pSTVH8 lentiviral backbones which were also digested with the same enzymes. Additionally, we constructed a minimal packaging virus (pSTV2) in which the PGK-puromycin cassette from the pLVX-Tight-Puro was removed and blunt-end repaired. The BirA*-FLAG Gateway fragment was digested using HindIII/XhoI cut sites and blunt-end repaired and ligated into pSTV2. We also constructed a non-Gateway version of the pSTV2 vector by using the same cloning strategy as above but using the pcDna5-FRT/TO-BirA*-FLAG-MCS (Multiple cloning sites) vectors (the MCS vectors were a kind gift from Brian Raught) (19Coyaud E. Mis M. Laurent E.M. Dunham W.H. Couzens A.L. Robitaille M. Gingras A.C. Angers S. Raught B. BioID-based identification of Skp Cullin F-box (SCF)beta-TrCP1/2 E3 ligase substrates.Mol. Cell. Proteomics. 2015; 14: 1781-1795Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 36Comartin D. Gupta G.D. Fussner E. Coyaud E. Hasegan M. Archinti M. Cheung S.W. Pinchev D. Lawo S. Raught B. Bazett-Jones D.P. Luders J. Pelletier L. CEP120 and SPICE1 cooperate with CPAP in centriole elongation.Curr. Biol. 2013; 23: 1360-1366Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). All vectors were validated through restriction digestion and Dna sequencing of subcloned fragments. The plasmid sequences are available through the Network Biology Collaborative Centre, NBCC, website (maps and sequences available at http://nbcc.lunenfeld.ca/resources). All the Gateway constructs were functionally tested through virus production (See the Testing and Optimization of Lentiviral Vectors section below), infection and immunostaining to assess BirA*-FLAG expression and in vivo biotinylation (See the Immunofluorescence section below). Gateway-compatible entry clones for the genes LMna (Genbank accession number EU832167, encoding lamin A), HIST1H2BG (EU446968, encoding histone H2B), TBP (EU831783, Tata Binding Protein) and TUBB (DQ891742, tubulin beta) were transferred in to the pcDna5-FRT/TO-BirA*-FLAG Gateway destination vector (37Katzen F. Gateway((R)) recombiNAtioNAl cloning: a biological operating system.Expert. Opin. Drug Discov. 2007; 2: 571-589Crossref PubMed Scopus (142) Google Scholar) or the pSTV2-BirA*-FLAG Gateway lentivirus destination vector using LR-Gateway cloning. The pcDna5-based plasmids were used to generate stable cell lines in Flp-In T-REx HeLa cells, as previously described (31Hesketh G.G. Youn J.Y. Samavarchi-Tehrani P. Raught B. Gingras A.C. Parallel exploration of interaction space by BioID and affinity purification coupled to mass spectrometry.Methods Mol. Biol. 2017; 1550: 115-136Crossref PubMed Scopus (38) Google Scholar), whereas the pSTV2 vectors were used to generate lentivirus for infection-mediated gene delivery. Different control vectors were also generated to allow for background subtraction. "No-BirA*" control samples were infected with a lentivirus vector without BirA*-FLAG. EGFP and EGFP fused to SV40 Nuclear Localization Sequence (EGFP-NLS) were transferred from pEntry vectors (pENTR223, Invitrogen) to the pSTV2-N-BirA*-FLAG destination vectors to create the BirA*-EGFP and BirA*-EGFP-NLS controls, respectively. To deliver rtTA into the cell lines not harboring the rtTA transcriptional activator, a lentivirus driving the expression of rtTA from the constitutive UbC promoter, was also used. The cancer cell lines used are HeLa and Flp-In-T-REx HeLa cells (kind gifts from Laurence Pelletier and Arshad Desai, respectively). Normal human foreskin fibroblasts (BJ-5ta) and Mouse Embryonic Fibroblasts (MEF) isolated from E14.5 ROSA26 Neo-Out rtTA mouse strain (38Belteki G. Haigh J. Kabacs N. Haigh K. Sison K. Costantini F. Whitsett J. Quaggin S.E. NAgy A. ConditioNAl and inducible transgene expression in mice through the combiNAtorial use of Cre-mediated recombiNAtion and tetracycline induction.Nucleic Acids Res. 2005; 33: e51Crossref PubMed Scopus (273) Google Scholar) were both kindly provided by Jeffrey Wrana. HEK293T cells (American Type and Tissue Collection, ATCC, Manassas, VA; Cat# CRL-3216) were used for virus production. Although a diverse range of Dna concentrations and transfection conditions are described in the literature, we have systematically optimized the protocol for our vectors and transfection reagents used in this study, with the final version detailed below. Briefly, 1.3 μg of psPAX2 (a gift from Didier Trono, AddGene #12260), 0.8 μg of VSV-G packaging vectors (a gift from Bob Weinberg, AddGene #8454) (39Stewart S.A. Dykxhoorn D.M. Palliser D. Mizuno H. Yu E.Y. An D.S. Sabatini D.M. Chen I.S. Hahn W.C. Sharp P.A. Weinberg R.A. NoviNA C.D. Lentivirus-delivered stable gene silencing by RNAi in primary cells.RNA. 2003; 9: 493-501Crossref PubMed Scopus (1002) Google Scholar) and 1.3 μg of transfer vectors encoding the gene of interest (such as pSTV2) are transfected into HEK293T cells (at 85% confluence in a 6-well plate) using the jetPRIME reagent as per manufacturer's recommendations (Polyplus-transfection SA, Illkirch-Graffenstaden, France, Cat# 114–01). After 10 h, the media is replaced with 3 ml of virus production media. Virus production media consists of DMEM supplemented with 5% heat-inactivated Fetal Bovine Serum (Gibco, ThermoFisher Scientific, Waltham, MA) and 50 U/ml Penicillin-Streptomycin solution (Corning, Manassas, VA). Virus is harvested at 36–40 h post media change, and cleared by centrifugation (500 rcf, 5 min) and filtering through a 0.45 μm filter. The viral titer is estimated by co-infecting HeLa cells (at 40% confluence in a 24-well plate) with a range of 25–100 μl of the pSTV2-BirA* tagged bait as well as the rtTA viral supernatant. The next day, the medium is replaced by media containing doxycycline (1 μg/ml; Sigma-Aldrich, St. Louis, MO) and biotin (40 μm; BioBasic, Amherst, New York, Cat# 58–85-5) and the cells are incubated for 24 h. Cells are subsequently fixed and analyzed by immunofluorescence (staining for both the FLAG epitope, the biotinylated proteins and the nuclei by DAPI; see the Immunofluorescence section below) to determine the optimal amount of supernatant that yields 75–85% infection rate. These results provide the infection parameters for scaling up of experiments for BioID followed by mass spectrometry. For all BioID experiments, cells in a single 10 cm dish at ∼35–40% density were infected with the determined amount of virus determined to yield 75–85% infection. For the transfer vectors described here, this corresponded to 750–850 μl of viral supernatant. Cells were then grown until ready for splitting, at which point they were scaled up into 15 cm plates for the BioID experiment. One 15 cm dish was used for each biological replicate. Biological duplicates were prepared for all experiments (alongside negative controls defined below). An aliquot of the cell suspension was also plated onto coverslips for immunofluorescence. Throughout the lentiviral transfer vector design and production, the various plasmids were analyzed by restriction digest and were sequence-validated, followed by functional testing. The functionality of the elements downstream of the constitutive promoters were tested before and after inserting the BirA*-FLAG fragment into the pSTVH5 to pSTVH8 transfer vectors (Fig. 1A). To test the functionality of the rtTA element in our vectors, we co-infected HeLa cells with a lentivirus expressing H2B-mCherry downstream of the tetracycline inducible TRE promoter with our various transfer vectors encoding the rtTA (pSTVH5, pSTV6 and pSTVH8). We next assessed rtTA/doxycycline-dependent expression of H2B-mCherry. The functionality of the selection cassettes was also validated: for pSTVH5, we confirmed expression of mCitrine by fluorescence microscopy. For the vectors harboring the puromycin resistance gene (pSTV6 and pSTVH7), we confirmed cell survival in the presence of puromycin (2 μg/ml) for 3–4 days. The plasmids that tested positive in these assays were then used for cloning the BirA*-FLAG fragments downstream of the P-tight promoter (as described above). The resulting vectors were re-tested by assessing for BirA*-FLAG expression and in vivo biotinylation using immu

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