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BioID Performed on Golgi Enriched Fractions Identify C10orf76 as a GBF1 Binding Protein Essential for Golgi Maintenance and Secretion

2019; Elsevier BV; Volume: 18; Issue: 11 Linguagem: Inglês

10.1074/mcp.ra119.001645

1535-9484

Calvin J. Chan, Roberta Le, Kaylan Burns, Khadra Ahmed, Étienne Coyaud, Estelle Laurent, Brian Raught, Paul Melançon,

S100 Proteins and Annexins

The Golgi-specific Brefeldin-A resistance factor 1 (GBF1) is the only large GEF that regulates Arf activation at the cis-Golgi and is actively recruited to membranes on an increase in Arf-GDP. Recent studies have revealed that GBF1 recruitment requires one or more heat-labile and protease-sensitive protein factor(s) (Quilty et al., 2018, J. Cell Science, 132). Proximity-dependent biotinylation (BioID) and mass spectrometry from enriched Golgi fractions identified GBF1 proximal proteins that may regulate its recruitment. Knockdown studies revealed C10orf76 to be involved in Golgi maintenance. We find that C10orf76 interacts with GBF1 and rapidly cycles on and off GBF1-positive Golgi structures. More importantly, its depletion causes Golgi fragmentation, alters GBF1 recruitment, and impairs secretion. Homologs were identified in most species, suggesting its presence in the last eukaryotic common ancestor. The Golgi-specific Brefeldin-A resistance factor 1 (GBF1) is the only large GEF that regulates Arf activation at the cis-Golgi and is actively recruited to membranes on an increase in Arf-GDP. Recent studies have revealed that GBF1 recruitment requires one or more heat-labile and protease-sensitive protein factor(s) (Quilty et al., 2018, J. Cell Science, 132). Proximity-dependent biotinylation (BioID) and mass spectrometry from enriched Golgi fractions identified GBF1 proximal proteins that may regulate its recruitment. Knockdown studies revealed C10orf76 to be involved in Golgi maintenance. We find that C10orf76 interacts with GBF1 and rapidly cycles on and off GBF1-positive Golgi structures. More importantly, its depletion causes Golgi fragmentation, alters GBF1 recruitment, and impairs secretion. Homologs were identified in most species, suggesting its presence in the last eukaryotic common ancestor. The Golgi functions as the central organizing organelle and resides at the center of the secretory pathway (1Gosavi P. Gleeson P.A. The function of the golgi ribbon structure - an enduring mystery unfolds!.Bioessays. 2017; 3910.1002/bies.201700063Crossref PubMed Scopus (33) Google Scholar). The central Golgi stacks receive newly synthesized proteins from the ER-Golgi intermediate compartment (ERGIC) 1The abbreviations used are:ERGICER-Golgi intermediate compartmentTGNtrans-Golgi networkArfsADP-ribosylation factorsBFABrefeldin ABioIDProximity-dependent biotinylationC10orf76Chromosome 10 predicted ORF of 76 KDaDMEMDulbeco's modified Eagle's mediumGBF1Golgi-specific Brefeldin-A resistance factor 1GEFGuanine nucleotide Exchange FactorIFimmunofluorescenceWBwestern blotting. 1The abbreviations used are:ERGICER-Golgi intermediate compartmentTGNtrans-Golgi networkArfsADP-ribosylation factorsBFABrefeldin ABioIDProximity-dependent biotinylationC10orf76Chromosome 10 predicted ORF of 76 KDaDMEMDulbeco's modified Eagle's mediumGBF1Golgi-specific Brefeldin-A resistance factor 1GEFGuanine nucleotide Exchange FactorIFimmunofluorescenceWBwestern blotting. and functions in the modification, sorting, and trafficking of this newly synthesized cargo through to the trans-Golgi network (TGN). Once there, proteins can then be secreted or trafficked to other membrane compartments including endosomes, lysosomes, and the plasma membrane. Over one third of the proteins encoded by the human genome are trafficked through the Golgi complex, and consequently, any mutations negatively affecting these processes can impair cellular function and cause disease (2Potelle S. Klein A. Foulquier F. Golgi post-translational modifications and associated diseases.J. Inherit. Metab. Dis. 2015; 38: 741-751Crossref PubMed Scopus (22) Google Scholar). ER-Golgi intermediate compartment trans-Golgi network ADP-ribosylation factors Brefeldin A Proximity-dependent biotinylation Chromosome 10 predicted ORF of 76 KDa Dulbeco's modified Eagle's medium Golgi-specific Brefeldin-A resistance factor 1 Guanine nucleotide Exchange Factor immunofluorescence western blotting. ER-Golgi intermediate compartment trans-Golgi network ADP-ribosylation factors Brefeldin A Proximity-dependent biotinylation Chromosome 10 predicted ORF of 76 KDa Dulbeco's modified Eagle's medium Golgi-specific Brefeldin-A resistance factor 1 Guanine nucleotide Exchange Factor immunofluorescence western blotting. Vesicle formation at the Golgi requires the recruitment of coat proteins, a process regulated by ADP-ribosylation factors (Arfs) (3Jackson C.L. Bouvet S. Arfs at a glance.J. Cell Sci. 2014; 127: 4103-4109Crossref PubMed Scopus (87) Google Scholar). Arfs belong to a family of small GTPases whose regulatory effects occur through cycling between GTP and GDP bound states. Arf guanine nucleotide exchange factors (GEFs) promote this nucleotide exchange reaction, activating Arfs by exchanging GDP for GTP. Only in their active GTP-bound form are Arfs able to interact with their effectors, including coat proteins, and lipid-modifying enzymes, which are required for vesicle formation, budding, and transport. The time and location of Arf activation is coordinated by their associated ArfGEFs (4Wright J. Kahn R.A. Sztul E. Regulating the large Sec7 ARF guanine nucleotide exchange factors: the when, where and how of activation.Cell Mol. Life Sci. 2014; 71: 3419-3438Crossref PubMed Scopus (46) Google Scholar). At the cis-Golgi, the only large Arf-GEF able to catalyze Arf nucleotide exchange reactions is the Golgi-specific BFA Resistance Factor 1 (GBF1) (5Manolea F. Claude A. Chun J. Rosas J. Melançon P. Distinct functions for Arf guanine nucleotide exchange factors at the golgi complex: GBF1 and BIGs are required for assembly and maintenance of the golgi stack and trans-golgi network, respectively.Mol. Biol. Cell. 2008; 19: 523-535Crossref PubMed Scopus (82) Google Scholar). The activity of GBF1 must be regulated to ensure proper maintenance of Golgi structure and direct trafficking of cargo between the ERGIC and Golgi stacks. Loss of GBF1 activity leads to Golgi collapse and ultimately, cell death (6Citterio C. Vichi A. Pacheco-Rodriguez G. Aponte A.M. Moss J. Vaughan M. Unfolded protein response and cell death after depletion of brefeldin A-inhibited guanine nucleotide-exchange protein GBF1.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 2877-2882Crossref PubMed Scopus (80) Google Scholar). The nucleotide exchange reaction must occur on membranes and requires the recruitment and association of both the ArfGEF and its Arf-GDP substrate. In addition to its role at the Golgi, GBF1 has been reported to function at multiple sites, from endosomes (7Gupta G.D. Swetha M.G. Kumari S. Lakshminarayan R. Dey G. Mayor S. Analysis of endocytic pathways in Drosophila cells reveals a conserved role for GBF1 in internalization via GEECs.PloS One. 2009; 4: e6768Crossref PubMed Scopus (55) Google Scholar) to mitochondria (8Walch L. Pellier E. Leng W. Lakisic G. Gautreau A. Contremoulins V. Verbavatz J.M. Jackson C.L. GBF1 and Arf1 interact with Miro and regulate mitochondrial positioning within cells.Sci Rep. 2018; 8: 17121Crossref PubMed Scopus (17) Google Scholar). Recent in vitro evidence argues for the existence of protein-based membrane-associated component(s) that facilitate GBF1 binding and recruitment to Golgi membranes (9Quilty D. Chan C.J. Yurkiw K. Bain A. Babolmorad G. Melancon P. The Arf-GDP-regulated recruitment of GBF1 to Golgi membranes requires domains HDS1 and HDS2 and a Golgi-localized protein receptor.J. Cell Sci. 2018; 132: piiGoogle Scholar). However, because of the transient nature of GBF1's interaction with the membrane, the identification of these interacting proteins has proven rather challenging. Genetic screens performed in yeast as well as traditional immunoprecipitation assays have had some success in identifying GBF1 interactors, including GMH1 and p115 (10Garcia-Mata R. Sztul E. The membrane-tethering protein p115 interacts with GBF1, an ARF guanine-nucleotide-exchange factor.EMBO Rep. 2003; 4: 320-325Crossref PubMed Scopus (61) Google Scholar, 11Chantalat S. Courbeyrette R. Senic-Matuglia F. Jackson C.L. Goud B. Peyroche A. A novel Golgi membrane protein is a partner of the ARF exchange factors Gea1p and Gea2p.Mol. Biol. Cell. 2003; 14: 2357-2371Crossref PubMed Scopus (45) Google Scholar). However, neither protein was revealed to be involved in regulating GBF1 recruitment. Because of the highly dynamic nature in which GBF1 cycles on and off Golgi membranes, a sensitive technique is required to capture these interactors. Here, we use the proximity-dependent biotinylation method (BioID) on enriched Golgi fractions to identify the GBF1 local interactome, which likely consists of transient, weak and/or poorly soluble GBF1 complexes. The BioID approach relies on the use of an abortive biotin ligase, BirA*, that when appropriately tagged to a protein of interest, allows for the irreversible biotinylation of proximal proteins in vivo (12Roux 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 (1251) Google Scholar, 13Kim 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, 14Kim D.I. Jensen S.C. Roux K.J. Identifying protein-protein associations at the nuclear envelope with BioID.Methods Mol. Biol. 2016; 1411: 133-146Crossref PubMed Scopus (15) Google Scholar). When expressed in live cells, supplementation of exogenous biotin will promote the activity of BirA* and the conjugation of biotin to primary amines (e.g. lysine side chains) on proteins surrounding the bait (15Roux K.J. Kim D.I. Burke B. BioID: a screen for protein-protein interactions.Curr. Protoc. Protein Sci. 2013; 74 (Unit 19 23)Crossref PubMed Scopus (154) Google Scholar). These proximal proteins can then be isolated by streptavidin affinity purification and identified by mass spectrometry. The coupling of BioID with Golgi enrichment allowed our focus on the identification of Golgi-localized proteins. Using this method, we identified a previously uncharacterized peripheral Golgi protein, C10orf76 (also referred to as ARMH3 by NCBI) that interacts with GBF1 and appears to be involved in GBF1 recruitment, Golgi maintenance, and protein secretion. Cells were maintained in Dulbeco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 100 μg/ml penicillin and 100 μg/ml streptomycin at 5% CO2 and 37 °C. BFA was purchased from Sigma-Aldrich (St-Louis, MO) and dissolved in DMSO at 1 mg/ml. Doxycycline was purchased from Fisher Scientific (Ottawa, Canada) and dissolved in UltraPure distilled water (Invitrogen) at 1 mg/ml. Puromycin was purchased from Gibco and dissolved in UltraPure distilled water at 10 mg/ml. Sequa-brene was purchased from Sigma-Aldrich and dissolved in UltraPure distilled water (Invitrogen) at 8 mg/ml. Biotin was purchased from Sigma-Aldrich and dissolved in serum-free DMEM at 1 mm. The cell lines used in this study include HeLa cells (ECACC; Sigma-Aldrich, 93031013), HEK293 cells (ATCC, CRL-1573), HeLa cells stably expressing Enhanced GFP (EGFP)-tagged GBF1 (9Quilty D. Chan C.J. Yurkiw K. Bain A. Babolmorad G. Melancon P. The Arf-GDP-regulated recruitment of GBF1 to Golgi membranes requires domains HDS1 and HDS2 and a Golgi-localized protein receptor.J. Cell Sci. 2018; 132: piiGoogle Scholar), and Flp-In T-Rex HeLa cells containing a tetracycline operator regulated BirA*-FLAG-GBF1 or BirA*-FLAG transgene (16Tighe A. Staples O. Taylor S. Mps1 kinase activity restrains anaphase during an unperturbed mitosis and targets Mad2 to kinetochores.J. Cell Biol. 2008; 181: 893-901Crossref PubMed Scopus (142) Google Scholar). Isolation of the tetracycline inducible BirA*-FLAG-GBF1 (16Tighe A. Staples O. Taylor S. Mps1 kinase activity restrains anaphase during an unperturbed mitosis and targets Mad2 to kinetochores.J. Cell Biol. 2008; 181: 893-901Crossref PubMed Scopus (142) Google Scholar) or BirA*-FLAG HeLa cells involved Flip-In T-REx and Gateway cloning systems (Invitrogen). First, PCR amplified full-length GBF1 was introduced into a Gateway pENTRY vector using a TOPO cloning kit by Invitrogen. The GBF1 gene cassette was then transferred from the pENTRY plasmid into the pcDNA5-pcDEST-BirA-FLAG-N-ter vector obtained from Dr. Anne-Claude Gingras (Lunenfeld-Tanenbaum Research Institute, Toronto, Canada) using the LR clonase enzymes. The pcDNA-pcDEST-BirA-FLAG-N-term vector with and without the GBF1 gene cassette was cotransfected with pOG44 into HeLa T-Rex Flp-In cells obtained from Dr. S. Taylor (University of Manchester, Manchester, United Kingdom). Stable cell populations containing BirA*-FLAG-GBF1 or BirA-FLAG were selected for using 150 μg/ml hygromycin over a two-week period. Tetracycline regulated expression of the transgene in hygromycin resistant cells was confirmed by treatment with 0.1 μg/ml doxycycline followed by immunoblotting for the FLAG-tagged proteins (17Claude A. Zhao B.P. Melançon P. Characterization of alternatively spliced and truncated forms of the Arf guanine nucleotide exchange factor GBF1 defines regions important for activity.Biochem. Biophys. Res. Commun. 2003; 303: 160-169Crossref PubMed Scopus (13) Google Scholar) (See supplemental Fig. S1). Molecular biology manipulations were performed as per manufacturer's instructions. The following primary antibodies were used for IF experiments: mouse anti-FLAG (Rockland, Limerick, PA, at 1:100), mouse anti-GBF1 (clone 25) (BD Bioscience; 1:1000), rabbit anti-giantin (1:2500) (From Dr. Edward K.L. Chan; University of Florida Health, Jacksonville; 1:2000), mouse anti-p115 (clone 7D1) (from Dr. Gerry Waters through the late Dr. Dennis Shields; 1:1000), sheep anti-TGN46 (AbD Serotec; 1:1000), mouse anti-β COP (M3A5) (Sigma-Aldrich; 1:250). The following primary antibodies were used for immunoblotting experiments: mouse anti-FLAG (Rockland; 1:10,000), rabbit anti-GBF1 (9D4) (17Claude A. Zhao B.P. Melançon P. Characterization of alternatively spliced and truncated forms of the Arf guanine nucleotide exchange factor GBF1 defines regions important for activity.Biochem. Biophys. Res. Commun. 2003; 303: 160-169Crossref PubMed Scopus (13) Google Scholar); 1:500), mouse anti-tubulin (Sigma-Aldrich; 1:1000), mouse anti-GM130 (BD Bioscience; 1:250), mouse anti-VDAC1 (Abcam; 1:5000). Streptavidin-cy3 (Invitrogen; 1:1000) was used to detect biotinylated proteins in IF experiments and Alexa Fluor 690 streptavidin (Invitrogen; 1:10,000) was used for detection in immunoblotting experiments, both without a secondary antibody. Secondary antibodies used for IF were all obtained from Invitrogen, used at 1: 1000, and include: Alexa Fluor 488 donkey anti-mouse, Alexa Fluor 647 donkey anti-rabbit, Alexa Fluor 647 donkey anti-mouse, Alexa Fluor 555 donkey anti-sheep. Secondary antibodies used for immunoblotting were all obtained from Invitrogen, used at 1:10,000, and includes: Alexa Fluor 680 goat anti-rabbit, Alexa Fluor 750 goat anti-mouse. Proximity-dependent biotinylation occurred under conditions described in Roux et al., 2013. In brief, tetracycline inducible BirA*-FLAG-GBF1 or BirA*-FLAG HeLa cells were grown in five 15 cm cell culture dishes until 80% confluence. Cells were then treated first with 0.1 μg/ml doxycycline for six hours to allow for expression of BirA*-FLAG proteins, followed by further supplementation with 50 μg/ml exogenous biotin for an additional 24-hour incubation. Cells were then collected by treatment with 0.25% trypsin-EDTA (Gibco, Carlsbad, CA) followed by neutralization with DMEM with 10% FBS, and centrifugation at 298.2 × g for 10 min in a Sorvall RT7 benchtop centrifuge with a RTH-750 swinging bucket rotor. Pelleted cells were then washed in PBS to remove residual medium components and then re-pelleted. For preparation of Golgi-enriched membrane samples pelleted cells were then resuspended in ice-cold homogenization buffer (10 mm Tris-HCl pH 7.6, 0.25 m Sucrose, 150 mm KCl, 1.0 mm MgCl2, 2× complete EDTA-free protease inhibitor (Roche, Mannheim, Germany). Cells were then homogenized by 20 passages through a cell homogenizer (Isobiotech, Heidelberg, Germany) with a 16 μm clearance. Homogenates were centrifuged at 4 °C and 800 × g for 10 min to pellet nuclei and unbroken cells. The supernatant was then adjusted to 1.2 m sucrose and layered in a sucrose gradient for organelle separation by density centrifugation (gradient from top to bottom: 0.25 m, 1.0 m, 1.2 m, 1.3 m, 2 m). Samples were centrifuged at 4 °C and 234116.4 × g for 3 h in an Optima ultracentrifuge with a SW 41 Ti swinging bucket rotor (Beckman Coulter, Missisauga, Canada). Enriched-Golgi fractions were collected between the 0.25 m and 1.0 m sucrose interface, washed with PBS, and pelleted by centrifugation at 4 °C and 163877.9 × g for 20 min in an Optima TLX Benchtop Ultra centrifuge (Beckman Coulter) with a TLA-100.4 rotor. The BirA*FLAG and BirA*FLAG-GBF1 cell pellet and Golgi fractions were resuspended in 10 ml and 5 ml of lysis buffer respectively, (50 mm Tris-HCl pH 7.5, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% Triton X-100, 0.1% SDS, 1:500 protease inhibitor mixture (Sigma-Aldrich), 1:1,000 benzonase nuclease (Novagen) and incubated on an end-over-end rotator at 4 °C for 1 h, briefly sonicated to disrupt any visible aggregates, then centrifuged at 45,000 × g for 30 min at 4 °C. Supernatants were transferred to a fresh 15 ml conical tube. 30 μl of packed, pre-equilibrated Streptavidin-Sepharose beads (GE) were added and the mixture incubated for 3 h at 4 °C with end-over-end rotation. Beads were pelleted by centrifugation at 2,000 rpm for 2 min and transferred with 1 ml of lysis buffer to a fresh Eppendorf tube. Beads were washed once with 1 ml lysis buffer and twice with 1 ml of 50 mm ammonium bicarbonate (pH = 8.3). Beads were transferred in ammonium bicarbonate to a fresh centrifuge tube and washed two more times with 1 ml ammonium bicarbonate buffer. Tryptic digestion was performed by incubating the beads with 1 μg MS-grade TPCK trypsin (Promega, Madison, WI) dissolved in 200 μl of 50 mm ammonium bicarbonate (pH 8.3) overnight at 37 °C. The following morning, 0.5 μg MS-grade TPCK trypsin was added, and beads were incubated 2 additional hours at 37 °C. Beads were pelleted by centrifugation at 2,000 × g for 2 min, and the supernatant was transferred to a fresh Eppendorf tube. Beads were washed twice with 150 μl of 50 mm ammonium bicarbonate, and these washes were pooled with the first eluates. The sample were lyophilized and resuspended in buffer A (0.1% formic acid). One fifth of the sample was analyzed per MS run. High performance liquid chromatography was conducted using a 2 cm pre-column (Acclaim PepMap 50 mm × 100 μm inner diameter (ID)), and 50 cm analytical column (Acclaim PepMap, 500 mm × 75 μm diameter; C18; 2 μm; 100 Å, Thermo Fisher Scientific, Waltham, MA), running a 120 min reversed-phase buffer gradient at 225 nl/min on a Proxeon EASY-nLC 1000 pump in-line with a Thermo Q-Exactive HF quadrupole-Orbitrap mass spectrometer. A parent ion scan was performed using a resolving power of 60,000, then up to the twenty most intense peaks were selected for MS/MS (minimum ion count of 1000 for activation) using higher energy collision induced dissociation (HCD) fragmentation. Dynamic exclusion was activated such that MS/MS of the same m/z (within a range of 10 ppm; exclusion list size = 500) detected twice within 5 s were excluded from analysis for 15 s. For protein identification, raw files were converted to the .mzXML format using Proteowizard (v3.0.10800; (18Kessner D. Chambers M. Burke R. Agus D. Mallick P. ProteoWizard: open source software for rapid proteomics tools development.Bioinformatics. 2008; 24: 2534-2536Crossref PubMed Scopus (1227) Google Scholar), then searched using X!Tandem (v2013.06.15.1; (19Craig R. Beavis R.C. TANDEM: matching proteins with tandem mass spectra.Bioinformatics. 2004; 20: 1466-1467Crossref PubMed Scopus (1989) Google Scholar) against Human RefSeq Version 45 (containing 36,113 entries). Search parameters specified a parent MS tolerance of 15 ppm and an MS/MS fragment ion tolerance of 0.4 Da, with up to two missed cleavages allowed for trypsin. No fixed modifications were set but oxidation of methionine was allowed as a variable modification. Data were analyzed using the trans-proteomic pipeline (20Pedrioli P.G.A. Trans-proteomic pipeline: a pipeline for proteomic analysis.in: Hubbard S.J. Jones A.R. Proteome Bioinformatics. Humana Press, Springer New York Dordrecht Heidelberg London2010: 213-238Google Scholar) via the ProHits 5.0.2 software suite (21Liu G. Zhang J. Larsen B. Stark C. Breitkreutz A. Lin Z.Y. Breitkreutz B.J. Ding Y. Colwill K. Pasculescu A. Pawson T. Wrana J.L. Nesvizhskii A.I. Raught B. Tyers M. Gingras A.C. ProHits: integrated software for mass spectrometry-based interaction proteomics.Nat. Biotechnol. 2010; 28: 1015-1017Crossref PubMed Scopus (151) Google Scholar). Proteins identified with a iProphet cut-off of 0.9 (corresponding to ≤1% probabilistic FDR (22Nesvizhskii A.I. Keller A. Kolker E. Aebersold R. A statistical model for identifying proteins by tandem mass spectrometry.Anal. Chem. 2003; 75: 4646-4658Crossref PubMed Scopus (3633) Google Scholar) and 2+ unique peptides were analyzed with SAINT Express v. 3.6.1 (23Choi H. Larsen B. Lin Z.Y. Breitkreutz A. Mellacheruvu D. Fermin D. Qin Z.S. Tyers M. Gingras A.C. Nesvizhskii A.I. SAINT: probabilistic scoring of affinity purification-mass spectrometry data.Nat. Methods. 2011; 8: 70-73Crossref PubMed Scopus (484) Google Scholar, 24Teo 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 (294) Google Scholar) to identify high-confidence interactors. The four controls were collapsed to the highest two spectral counts for each hit. Proteins identified with two or more unique peptides and scoring above a Bayesian False Discovery Rate of 1% (see (24Teo 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 (294) Google Scholar) for details on BFDR calculation) were high-confidence proximity interactors. All data are publicly available and have been uploaded to the MassIVE archive (www.massive.ucsd.edu) with ID: MSV000083866. Four BioID runs were conducted on FlagBirA*-GBF1 expressing cells, both on the whole cell lysate and on the Golgi fraction. These four runs consisted of two technical replicates (n = 2) from two biological replicates (n = 2; total n = 4). Four control runs of a BioID analysis conducted on the corresponding fractions on cells expressing the FlagBirA* tag alone were used for comparative purposes. The codon optimized C10orf76 open reading frame was obtained from Dr. Vincent A. Blomen. mCherry-, EGFP-, and FLAG-tagged versions were created by PCR amplification of the C10orf76 open reading frame and subcloning into a mCherry-C1, EGFP-C1, and FLAG-C1 vector, using restriction enzymes SacII and SbfI (for mCherry and EGFP), and SacII and BamHI (for FLAG). EGFP-tagged truncated C10orf76 plasmids were also generated using PCR amplification with the same restriction enzymes and vectors. Fixed-cell imaging experiments were performed with tissue culture cells grown on #1.5 glass coverslips (Fisher Scientific) in 6-well plates. Coverslips were sterilized in 70% ethanol. Transfection of plasmids for transient expression of tagged-proteins were performed on cells grown to ∼60–70% confluency using Lipofectamine 2000 (Invitrogen) according to manufacturer's instructions and cultured for ∼18 h to allow for protein expression. For fixation, cells were washed in PBS warmed to 37 °C and fixed in 4% paraformaldehyde (with 100 μm calcium chloride and 100 μm magnesium chloride in PBS) at 37 °C for 20 min. Cells were then incubated in permeabilization buffer (0.1% Triton X-100 in PBS) to allow for subsequent labeling of intracellular structures by antibodies. Live-cell imaging experiments were performed with cells grown on #1.5 25 mm round glass coverslips (Fisher Scientific) in 6-well dishes. During imaging, coverslips were transferred onto Attofluor cell chambers (Invitrogen) and maintained in CO2-independent DMEM (Gibco Laboratories, Grand Island) supplemented with 10% FBS and maintained at 37 °C. Both live- and fixed-cell imaging were performed using wide-field fluorescence microscopy in a DeltaVision Elite (GE Healthcare, Buckinghamshire, UK) microscope equipped with a front-illuminated sCMOS camera driven by softWoRx 6 (GE Healthcare) using a 60 × 1.4 NA oil objective (Olympus, Richmond Hill, Canada). Before analysis, images were deconvolved in softWoRx6 and processed using FIJI imaging software (National Institutes of Health, Bethesda, MD). Quantification of fixed and live cells images was carried out using Imaris 8 software (Bitplane Scientific Software, South Windsor, CT), similarly to previously published studies (9Quilty D. Chan C.J. Yurkiw K. Bain A. Babolmorad G. Melancon P. The Arf-GDP-regulated recruitment of GBF1 to Golgi membranes requires domains HDS1 and HDS2 and a Golgi-localized protein receptor.J. Cell Sci. 2018; 132: piiGoogle Scholar, 25Quilty D. Gray F. Summerfeldt N. Cassel D. Melancon P. Arf activation at the Golgi is modulated by feed-forward stimulation of the exchange factor GBF1.J. Cell Sci. 2014; 127: 354-364Crossref PubMed Scopus (12) Google Scholar). In brief, three-dimensional surfaces were created around areas of interest (such as GBF1 positive structures) in selected cells by using the surfaces feature in Imaris 8. The average pixel intensity (Int) values in the surveyed regions and the volumes (Vol) of these regions were measured. Average Golgi intensity values were corrected by subtracting for the average cytosolic intensity. Whole-cell intensity was further corrected by subtracting the average intensity of the image background. The equation used for determining the fraction of signal at the Golgi = Golgi Vol (Golgi Int-cytosol Int)/cell Vol (cell Int-background Int). Average fold increase of signal at the Golgi is determined as fraction of signal at the Golgi after BFA/fraction of signal at the Golgi before BFA. For FRAP experiments, images were acquired on a Quorum Technologies WaveFX microscope with a Yokagawa CSU 10 spinning disk confocal scan-head and a 60 × 1.42NA oil objective. Imaging was driven by the Perkin Elmer's Volocity program and photobleaching was performed using the Andor iQ3 live cell imaging software. Image analysis and fraction signal at the Golgi was performed and calculated in the same manner as for the live cell experiments described above. Fraction at the Golgi prior to bleaching for both GBF1 and C10orf76 was normalized to 1 for comparison. The average half-life (t1/2) values were determined from the exponential portion of each graph. Cells were grown to about 80% confluency, fixed in Karnovsky (2% paraformaldehyde and 2% glutaraldehyde) and centrifuged at increasing speeds to produce pellets. Secondary fixation was in 1% osmium tetroxide for one hour, followed by staining with 1% uranyl acetate in water for one hour or O/N (All chemicals are from Ted Pella, Inc.). Pellets were dehydrated in increasing concentrations of ethanol and transferred into Epon 812 (EMS # 14120) beams for polymerization. Sectioning was done with UltracutE (Reichert Jung) followed by imaging with a Jeol JEM-2100 transmission electron microscope. Proteins were separated by SDS-PAGE and transferred onto nitrocellulose membrane (GE Healthcare) at 376 mA for two hours in transfer buffer (25 mm Tris-HCl, 190 mm glycine, 20% (v/v) methanol, 2.5% (v/v) isopropanol). Membranes were then blocked in Licor Odyssey Blocking Reagent (Licor Biotechnology, Lincoln, NE) for at least one hour. Blocked membranes were then incubated with primary antibodies then secondary antibodies each in 50% Licor Odyssey Blocking Reagent in PBS. Detection of biotinylated proteins utilized Alexa Fluor 690 streptavidin without a secondary antibody. Membranes were then washed twice in Tris buffered saline with Tween-20 (50 mm NaCl, 0.5% (v/v) Tween-20, 20 mm Tris-HCl pH 7.5) for 10 min each followed by three washes in PBS for 10 min each. Membranes were then scanned on a Licor Odyssey scanner (Licor Biotechnology). Quantitation of immunoblots was performed using Image Studio version 5.0 (Licor Biotechnology). Band intensities were quantified by manually drawing a rectangle around a region of interest, which was then corrected for background using the average pixel intensity in a 3-pixel region along the edges of the drawn rectangle. Quantified intensity values were then exported with Excel worksheets (Microsoft, Redmond) where mean, standard deviations, and normalization calculations were performed. Plasmids encoding shRNAs targeting genes of interest were obtained from the Sigma-Aldrich MISSION shRNA library. Sequences selected for use can be found in supplemental Table S2. Lentiviruses containing the shRNA encoding plasmids were generated by cotransfecting 80% confluent HEK293 cells in 6-well plates with shRNA encoding plasmids and the Sigma Aldrich MISSION Lentiviral Packaging Mix using LipoD293 DNA In Vitro Transfection Reagent (FroggaBio). The secreted viruses were collected over a period of 3 days and filtered with 0.45 μm low protein binding PVDF syringe filter (Millex). Lentiviruses were stored at −80 °C until use. shRNA knockdowns were performed on tissue culture cells transduced with the appropriate shRNA plasmid containing lentiviruses (multiplicity of infection at two) with 8 μg/ml Sequa-brene (Sigma