Protein Interaction Profiling of the p97 Adaptor UBXD1 Points to a Role for the Complex in Modulating ERGIC-53 Trafficking
2012; Elsevier BV; Volume: 11; Issue: 6 Linguagem: Inglês
10.1074/mcp.m111.016444
ISSN1535-9484
AutoresDale S. Haines, J. Eugene Lee, Stephen L. Beauparlant, Dane B. Kyle, Willem den Besten, Michael J. Sweredoski, R. L. Graham, Sonja Hess, Raymond J. Deshaies,
Tópico(s)Fungal and yeast genetics research
ResumoUBXD1 is a member of the poorly understood subfamily of p97 adaptors that do not harbor a ubiquitin association domain or bind ubiquitin-modified proteins. Of clinical importance, p97 mutants found in familial neurodegenerative conditions Inclusion Body Myopathy Paget's disease of the bone and/or Frontotemporal Dementia and Amyotrophic Lateral Sclerosis are defective at interacting with UBXD1, indicating that functions regulated by a p97-UBXD1 complex are altered in these diseases. We have performed liquid chromatography-mass spectrometric analysis of UBXD1-interacting proteins to identify pathways in which UBXD1 functions. UBXD1 displays prominent association with ERGIC-53, a hexameric type I integral membrane protein that functions in protein trafficking. The UBXD1-ERGIC-53 interaction requires the N-terminal 10 residues of UBXD1 and the C-terminal cytoplasmic 12 amino acid tail of ERGIC-53. Use of p97 and E1 enzyme inhibitors indicate that complex formation between UBXD1 and ERGIC-53 requires the ATPase activity of p97, but not ubiquitin modification. We also performed SILAC-based quantitative proteomic profiling to identify ERGIC-53 interacting proteins. This analysis identified known (e.g. COPI subunits) and novel (Rab3GAP1/2 complex involved in the fusion of vesicles at the cell membrane) interactions that are also mediated through the C terminus of the protein. Immunoprecipitation and Western blotting analysis confirmed the proteomic interaction data and it also revealed that an UBXD1-Rab3GAP association requires the ERGIC-53 binding domain of UBXD1. Localization studies indicate that UBXD1 modules the sub-cellular trafficking of ERGIC-53, including promoting movement to the cell membrane. We propose that p97-UBXD1 modulates the trafficking of ERGIC-53-containing vesicles by controlling the interaction of transport factors with the cytoplasmic tail of ERGIC-53. UBXD1 is a member of the poorly understood subfamily of p97 adaptors that do not harbor a ubiquitin association domain or bind ubiquitin-modified proteins. Of clinical importance, p97 mutants found in familial neurodegenerative conditions Inclusion Body Myopathy Paget's disease of the bone and/or Frontotemporal Dementia and Amyotrophic Lateral Sclerosis are defective at interacting with UBXD1, indicating that functions regulated by a p97-UBXD1 complex are altered in these diseases. We have performed liquid chromatography-mass spectrometric analysis of UBXD1-interacting proteins to identify pathways in which UBXD1 functions. UBXD1 displays prominent association with ERGIC-53, a hexameric type I integral membrane protein that functions in protein trafficking. The UBXD1-ERGIC-53 interaction requires the N-terminal 10 residues of UBXD1 and the C-terminal cytoplasmic 12 amino acid tail of ERGIC-53. Use of p97 and E1 enzyme inhibitors indicate that complex formation between UBXD1 and ERGIC-53 requires the ATPase activity of p97, but not ubiquitin modification. We also performed SILAC-based quantitative proteomic profiling to identify ERGIC-53 interacting proteins. This analysis identified known (e.g. COPI subunits) and novel (Rab3GAP1/2 complex involved in the fusion of vesicles at the cell membrane) interactions that are also mediated through the C terminus of the protein. Immunoprecipitation and Western blotting analysis confirmed the proteomic interaction data and it also revealed that an UBXD1-Rab3GAP association requires the ERGIC-53 binding domain of UBXD1. Localization studies indicate that UBXD1 modules the sub-cellular trafficking of ERGIC-53, including promoting movement to the cell membrane. We propose that p97-UBXD1 modulates the trafficking of ERGIC-53-containing vesicles by controlling the interaction of transport factors with the cytoplasmic tail of ERGIC-53. P97 (also called VCP for valosin-containing protein or Cdc48 in yeast) is a highly conserved and abundant protein and is a member of the AAA (ATPases Associated with diverse cellular Activities) family of ATPases. The ATPase is mutated in two familial diseases, Inclusion Body Myopathy Paget's disease of the bone and/or Frontotemporal Dementia (IBMPFD) 1The abbreviations used are:IBMPFDInclusion Body Myopathy Paget's disease of the bone and/or Frontotemporal DementiaALSAmyotrophic Lateral SclerosisUBAubiquitin associated. 1The abbreviations used are:IBMPFDInclusion Body Myopathy Paget's disease of the bone and/or Frontotemporal DementiaALSAmyotrophic Lateral SclerosisUBAubiquitin associated. and Amyotrophic Lateral Sclerosis (ALS), both of which display accumulation of ubiquitin positive vacuoles in affected cell types (1Watts G.D. Wymer J. Kovach M.J. Mehta S.G. Mumm S. Darvish D. Pestronk A. Whyte M.P. Kimonis V.E. Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia is caused by mutant valosin-containing protein.Nat. Genet. 2004; 36: 377-381Crossref PubMed Scopus (1111) Google Scholar, 2Johnson J.O. Mandrioli J. Benatar M. Abramzon Y. Van Deerlin V.M. Trojanowski J.Q. Gibbs J.R. Brunetti M. Gronka S. Wuu J. Ding J. McCluskey L. Martinez-Lage M. Falcone D. Hernandez D.G. Arepalli S. Chong S. Schymick J.C. Rothstein J. Landi F. Wang Y.D. Calvo A. Mora G. Sabatelli M. Monsurrò M.R. Battistini S. Salvi F. Spataro R. Sola P. Borghero G. ITALSGEN Consortium Galassi G. Scholz S.W. Taylor J.P. Restagno G. Chiò A. Traynor B.J. Exome sequencing reveals VCP mutations as a cause of familial ALS.Neuron. 2010; 68: 857-864Abstract Full Text Full Text PDF PubMed Scopus (938) Google Scholar). The protein functions in numerous cellular pathways, including homotypic membrane fusion, ERAD (ER-Associated Degradation), mitotic spindle disassembly, degradation of protein aggregates by autophagy and endo-lysosomal sorting of ubiquitinated caveolins (reviewed in 3Uchiyama K. Kondo H. p97/p47-Mediated biogenesis of Golgi and ER.J. Biochem. 2005; 137: 115-119Crossref PubMed Scopus (90) Google Scholar, 4Wolf D.H. Stolz A. The Cdc48 machine in endoplasmic reticulum associated protein degradation.Biochim. Biophys. Acta. 2011; ([Epub ahead of print])Google Scholar, 5Dargemont C. Ossareh-Nazari B. Cdc48/p97, a key actor in the interplay between autophagy and ubiquitin/proteasome catabolic pathways.Biochim. Biophys. Acta. 2011; ([Epub ahead of print])PubMed Google Scholar, 6Yamanaka K. Sasagawa Y. Ogura T. Recent advances in p97/VCP/Cdc48 cellular functions.Biochim. Biophys. Acta. 2011; ([Epub ahead of print])PubMed Google Scholar, 7Meyer H. Popp O. 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Endolysosomal sorting of ubiquitylated caveolin-1 is regulated by VCP and UBXD1 and impaired by VCP disease mutations.Nat. Cell Biol. 2011; 13: 1116-1123Crossref PubMed Scopus (158) Google Scholar). Inclusion Body Myopathy Paget's disease of the bone and/or Frontotemporal Dementia Amyotrophic Lateral Sclerosis ubiquitin associated. Inclusion Body Myopathy Paget's disease of the bone and/or Frontotemporal Dementia Amyotrophic Lateral Sclerosis ubiquitin associated. P97 exists as a hexamer, with two centrally localized ATPase domains (reviewed in 3Uchiyama K. Kondo H. p97/p47-Mediated biogenesis of Golgi and ER.J. Biochem. 2005; 137: 115-119Crossref PubMed Scopus (90) Google Scholar, 4Wolf D.H. Stolz A. The Cdc48 machine in endoplasmic reticulum associated protein degradation.Biochim. Biophys. Acta. 2011; ([Epub ahead of print])Google Scholar, 5Dargemont C. Ossareh-Nazari B. Cdc48/p97, a key actor in the interplay between autophagy and ubiquitin/proteasome catabolic pathways.Biochim. 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Direct binding of ubiquitin conjugates by the mammalian p97 adaptor complexes, p47 and Ufd1-Npl4.EMBO J. 2002; 21: 5645-5652Crossref PubMed Scopus (295) Google Scholar). This activity is mediated by adaptors that harbor an ubiquitin association domain (UBA) and a p97-docking module. Numerous adaptors have been identified, including those having PUB, SHP, UBD, UBX, VBM, and VIM p97 interaction motifs (reviewed in 16Madsen L. Seeger M. Semple C.A. Hartmann-Petersen R. New ATPase regulators–p97 goes to the PUB.Int. J. Biochem. Cell Biol. 2009; 41: 2380-2388Crossref PubMed Scopus (56) Google Scholar, 17Yeung H.O. Kloppsteck P. Niwa H. Isaacson R.L. Matthews S. Zhang X. Freemont P.S. Insights into adaptor binding to the AAA protein p97.Biochem. Soc. Trans. 2008; 36: 62-67Crossref PubMed Scopus (103) Google Scholar, 18Schuberth C. Buchberger A. UBX domain proteins: major regulators of the AAA ATPase Cdc48/p97.Cell Mol. Life Sci. 2008; 65: 2360-2371Crossref PubMed Scopus (209) Google Scholar). The majority of these adaptors interact with the N-terminal domain of p97. Interestingly, over half of the mammalian UBX-domain containing proteins (the largest family of adaptors) do not harbor an UBA domain, nor bind ubiquitinated proteins (19Alexandru G. Graumann J. Smith G.T. Kolawa N.J. Fang R. Deshaies R.J. UBXD7 binds multiple ubiquitin ligases and implicates p97 in the H1F1alpha turnover.Cell. 2008; 134: 804-816Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). There is currently very little information pertaining to the activities of proteins that comprise this sub-family of p97 adaptors. The biochemical mechanism by which disease-relevant P97 mutations alter the function of the ATPase is not well understood. Some of the mutations that cause IBMPFD stimulate the ATPase activity of p97 (20Halawani D. LeBlanc A.C. Rouiller I. Michnick S.W. Servant M.J. Latterich M. Hereditary inclusion body myopathy-linked p97/VCP mutations in the NH2 domain and the D1 ring modulate p97/VCP ATPase activity and D2 ring conformation.Mol. Cell. Biol. 2009; 29: 4484-4494Crossref PubMed Scopus (91) Google Scholar). Other studies indicate that they alter the binding of specific adaptors to the N-terminal domain of p97, where most of the IBMPFD mutations are found (21Fernández-Sáiz V. Buchberger A. Imbalances in p97 co-factor interactions in human proteinopathy.EMBO Rep. 2010; 11: 479-485Crossref PubMed Scopus (77) Google Scholar). Intriguingly, these alterations can both promote the binding of certain adaptors and suppress the interaction with others (21Fernández-Sáiz V. Buchberger A. Imbalances in p97 co-factor interactions in human proteinopathy.EMBO Rep. 2010; 11: 479-485Crossref PubMed Scopus (77) Google Scholar). UBXD1, a member of the non-UBA family of p97 adaptors, has recently been shown to be deficient at interacting with several p97 mutants, including those commonly found in familial IBMPFD and ALS (10Ritz D. Vuk M. Kirchner P. Bug M. Schütz S. Hayer A. Bremer S. Lusk C. Baloh R.H. Lee H. Glatter T. Gstaiger M. Aebersold R. Weihl C.C. Meyer H. Endolysosomal sorting of ubiquitylated caveolin-1 is regulated by VCP and UBXD1 and impaired by VCP disease mutations.Nat. Cell Biol. 2011; 13: 1116-1123Crossref PubMed Scopus (158) Google Scholar). This study also demonstrated that UBXD1 collaborates with p97 in the endo-lysosomal sorting of ubiquitinated caveolins and this process is altered in cells containing mutant p97 (10Ritz D. Vuk M. Kirchner P. Bug M. Schütz S. Hayer A. Bremer S. Lusk C. Baloh R.H. Lee H. Glatter T. Gstaiger M. Aebersold R. Weihl C.C. Meyer H. Endolysosomal sorting of ubiquitylated caveolin-1 is regulated by VCP and UBXD1 and impaired by VCP disease mutations.Nat. Cell Biol. 2011; 13: 1116-1123Crossref PubMed Scopus (158) Google Scholar). To gain further insights into the pathways in which p97-UBXD1 complex functions, we used immunopurification and mass spectrometric methods to identify proteins that associate with UBXD1. The results obtained with these methods as well as follow-up protein interaction and localization studies indicate that p97-UBXD1 modulates the subcellular localization of ERGIC-53 containing vesicles. Supplementary Table S1 describes plasmids used in this study and how they were generated. Constructs encoding amino-terminal FLAG tagged adaptors have been described previously (19Alexandru G. Graumann J. Smith G.T. Kolawa N.J. Fang R. Deshaies R.J. UBXD7 binds multiple ubiquitin ligases and implicates p97 in the H1F1alpha turnover.Cell. 2008; 134: 804-816Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). Antibodies used in experiments presented here are anti-FLAG mouse monoclonal antibody M2 (SIGMA), anti-UBXD1 mouse monoclonal antibody 5C3–1 (22Ramkumar P. Smith B.A. Akinbamidele A.C. Kapcia J. Beauparlant S.L. Haines D.S. Generation and characterization of novel monoclonal antibodies recognizing UBXD1.Hybridoma. 2009; 28: 459-462Crossref PubMed Scopus (1) Google Scholar), anti-ERGIC-53 H-245 rabbit polyclonal (Santa Cruz, Santa Cruz, CA), anti-p97 H-120 rabbit polyclonal (Santa Cruz), anti-Rab3GAP1 rabbit polyclonal (Novus, Littleton, CO), and anti-Rab3-GAP2 rabbit polyclonal (GeneTex, San Antonio, TX). The human lung adenocarcinoma cell line H1299 was first transfected by the calcium phosphate method with the pVgRXR plasmid (Invitrogen, Carlsbad, CA). Cells were cultured in 400 μg/ml zeocin until visible colonies were evident. Fourteen colonies were isolated, expanded, and tested for ponasterone inducibility using a transiently transfected pIND-LacZ reporter (Invitrogen) and liquid β-galactosidase assay. The clone (#9) giving the best inducibility and the lowest background in the absence of ponasterone was transfected by calcium phosphate with a second pVgRXR construct that harbors an introduced puromycin resistance gene. Cells were cultured in the presence of zeomycin (400 μg/ml) and puromycin (0.5 μg/ml) until visible colonies were present. Twenty-two colonies were isolated, expanded, and tested for ponasterone inducibility as described above. The clone (#9–8) giving the best inducibility with the lowest background was transfected by FuGENE6 (Roche) with pIND (Invitrogen) and pIND-UBXD1FLAG expression constructs. Cells were cultured in the presence of zeomycin (400 μg/ml), puromycin (0.5 μg/ml), and hygromycin (200 μg/ml) for 2 weeks to generate a pool of stably transfected cells. To induce UBXD1FLAG expression, cells were exposed to the noted concentration of ponasterone for 24–48 h. For experiments using transiently transfected cells, H1299 or Hek 293Ts (293T) were transfected with the indicated amount of DNA using FuGENE6 (Roche) according to the manufacturer's instructions. Cells were harvested or treated with inhibitors 48 h after exposure to plasmid DNA-FuGENE6 mixtures. Mass spectrometry was performed as described previously (23Lee J.E. Sweredoski M.J. Graham R. Ll Kolawa N.J. Smith G.T. Hess S. Deshaies R.J. The steady-state repertoire of human SCF ubiquitin ligase complexes does not require ongoing Nedd8 conjugation.Mol. Cell. Proteomics. 2011; 10 (M110.006460)Abstract Full Text Full Text PDF Scopus (53) Google Scholar). Briefly, cell pellets were collected and lysed in lysis buffer (50 mm HEPES, pH 7.5; 70 mm KOAc; 5 mm Mg(OAc)2; 0.2% n-dodecyl-β-d-maltoside) containing 1 × protease inhibitor tablet (Roche) for 30 min on a nutator at 4 °C. The lysates were centrifuged at 16,600 × g for 15 min to remove cell debris, and the supernatant was incubated with anti-FLAG beads on a nutator for 1 h at 4 °C. Beads were washed with lysis buffer 5 times, followed by 2 washes with 100 mm Tris-HCl (pH 8.5). Proteins were eluted from beads in 10 m freshly prepared urea. Digestion was performed in 100 mm Tris-HCl (pH 8.5) containing 8 m urea at 37 °C first with Lys-C (35 ng/mg lysate) for 4 h, and then the urea concentration was reduced to 2 m for trypsin (30 ng/mg lysate) digestion overnight. Following digestion, the tryptic peptides were desalted on a reversed-phase Vivapure C18 micro spin column (Sartorius Stedim Biotech, Gottingen, Germany) and concentrated using a SpeedVac. Dried samples were acidified by 0.2% formic acid prior to liquid chromatography-mass spectrometric analysis. All liquid chromatography-mass spectrometry experiments were performed on an EASY-nLC (Thermo Scientific, West Palm Beach, FL) connected to a hybrid LTQ Orbitrap Classic or LTQ FT (Thermo Scientific) equipped with a nano-electrospray ion source (Thermo Scientific). Peptides were separated on a 15-cm reversed phase analytical column (75 μm internal diameter) in-house packed with 3 μm C18AQ beads (ReproSil-Pur C18AQ) using a 120-min gradient from 13% to 25% acetonitrile in 0.2% formic acid at a flow rate of 350 nL/minute. The mass spectrometer was operated in data-dependent mode to automatically switch between full-scan MS and tandem MS acquisition. Survey full scan mass spectra were acquired in Orbitrap or FT (300–1700 m/z), after accumulation of 500,000 ions, with a resolution of 60,000 at 400 m/z. The top ten most intense ions from the survey scan were isolated and, after the accumulation of 5000 ions, fragmented in the linear ion trap by collisionally induced dissociation (collisional energy 35% and isolation width 2 Da). Precursor ion charge state screening was enabled and all singly charged and unassigned charge states were rejected. The dynamic exclusion list was set with a maximum retention time of 90 s, a relative mass window of 10 ppm and early expiration was enabled. For data analysis, peaks were generated from raw data files using MaxQuant (version 1.2.2.5) with default parameters (24Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide quantification.Nat. Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (9150) Google Scholar) and searched using the built-in search engine Andromeda (25Cox J. Neuhauser N. Michalski A. Scheltema R.A. Olsen J.V. Mann M. Andromeda: A Peptide Search Engine Integrated into the MaxQuant Environment.J. Proteome Res. 2011; 10: 1794-1805Crossref PubMed Scopus (3448) Google Scholar). Peak lists were searched against the International Protein Index (IPI) human database (version 3.54, 75448 sequences) and a contaminant database (262 sequences). The search parameters were tryptic digestion, maximum of two missed cleavages, fixed carboxyamidomethyl modifications of cysteine, variable oxidation modifications of methionine, and variable protein N-terminal carbamylations. SILAC samples were searched with Arg6 and Lys8 as variable modifications as well. Mass tolerance for precursor ions were 7 ppm and that for fragment ions were 0.5 Da. Protein inference and quantitation were performed by MaxQuant with 1% false discovery rate thresholds for both peptides and proteins as calculated using a decoy search. Additionally, at least two different peptide sequences were required for protein identification. No threshold was employed for individual MS/MS spectra because we were primarily interested in protein identification and not specific peptide sequences or post-translational modifications. Peptides were assigned to proteins using the principle of maximum parsimony. Additionally, protein groups were formed where there was no evidence to disambiguate protein isoforms. Relative protein amounts were semiquantitatively measured using spectral counts using all peptides (distinct and shared) within a protein group. In the case of SILAC experiments, spectral counting was still employed because H/L ratios were often incalculable because of the binary nature of the experiments. To identify proteins with spectral counts significantly different, binomial tests were performed assuming equal probability of observation in either case (i.e. bait or empty, heavy or light). Proteins were determined to be significant if their p value was lower than the Bonferroni adjusted threshold of 0.05/n where n is the number of tests performed. Complete files of the proteomic analysis are presented in supplementary Tables S1 to S5. Cell pellets were lysed in EBC (50 mm Tris-HCl pH7.5, 120 mm NaCl, 1% Nonidet P-40) or n-dodecyl-β-d-maltoside-based buffer (see previous section) supplemented with protease inhibitors for 15 min at 4 °C. Extracts were subjected to centrifugation and supernatants transferred to a new tube. Protein concentration was determined by the Bradford method using a kit from Bio-Rad. For immunoprecipitations, samples containing 0.5–1 mg of lysate were incubated with anti-FLAG beads for 1–3 h on a nutator at 4 °C. Beads were then pelleted, washed three times in lysis buffer, resuspended in 1 × SDS-PAGE loading buffer and placed at 95 °C for 5 min. Proteins were resolved by SDS-PAGE in running buffer (250 mm Glycine, 25 mm Tris, 0.1% SDS) and transferred to nitrocellulose membranes in methanol-containing transfer buffer (200 mm Glycine, 25 mm Tris, 20% Methanol) for 1 h at 125 V. Membranes were then washed in phosphate-buffered saline (PBS)-Tween-20 (PBS-T) (63 mm Na2HPO4, 15.5 mm NaH2PO4, 7.5 mm NaCl, 0.1% Tween-20) and blocked in 5% milk in PBS-T for 1 h at room temperature. Primary antibodies were added at appropriate dilutions in 5% milk in PBS-T and rocked overnight at 4 °C. Following primary antibody, membranes were washed and incubated with secondary antibody (at appropriate dilution) in 5% milk in PBS-T for an hour. Membranes were washed and treated with Western Lightning Plus - ECL (PerkinElmer) as per manufacturer's instructions. Chemiluminescence was detected by exposure on X-ray film. Cells were grown to 40% confluency on glass cover slips that had been placed in six-well plates. Cells were washed twice in PBS, and then fixed in 4% paraformaldehyde PBS for 15 min. Samples were washed twice in PBS. Samples were blocked for 1 h at room temperature in blocking solution (10% FBS, 0.1% Triton X-100 in PBS). After two more washes in PBS, samples were incubated in primary antibodies (0.5 ml of 10% fetal bovine serum in PBS) for 1 h at 37°C with gentle rocking. After two more PBS washes, samples were incubated in secondary antibodies (0.5 ml of 10% FBS in PBS) for 45 min at 37°C. After the final 2 washes in PBS, cover slips were inverted onto glass slides with 1 drop mounting solution (SloFade Gold with DAPI, Invitrogen). Cells were visualized using a Leica TCS SP5 confocal microscope. For confocal microscopy, all scans were created using sequential capture to prevent bleed through or cascading fluorescence. Excitation lasers and detection ranges are as follows: DAPI: 405 nm, 415 nm–476 nm; AlexaFluor 568 (ERGIC-53): 561 nm, 571 nm–638 nm; AlexaFluor 647 (UBXD1): 633 nm, 644 nm–703 nm. Images were modified and analyzed with either Spot Advanced sofware or LAS AF software. Post-processing of images obtained with LAS AF software consisted of mean baseline correction and medium noise reduction. Mass spectrometric analysis identifies ERGIC-53 as a high abundance UBXD1 interacting protein. We first generated a H1299 derived cell line that allows for regulated expression control of a C-terminally FLAG-tagged UBXD1 protein (UBXD1FLAG) using the Drosophila hormone ponasterone (Fig. 1A). After generation of this line, cells were exposed to two different concentrations (0.1 and 0.3 μm) of hormone, which results in modest overexpression of UBXD1FLAG (in the two- fivefold range over endogenous UBXD1) (Fig. 1A). UBXD1FLAG and interacting proteins were immunopurified from extracts using anti-FLAG antibody-conjugated beads and subjected to mass spectrometric analysis using an LTQ-FT instrument. Table I provides a list of proteins present in the UBXD1FLAG samples and not present in control anti-FLAG immunoprecipitations from mock-induced cells harboring the empty vector. As expected, UBXD1 and p97 were abundant constituents of both UBXD1FLAG immunoprecipitates. The protein that yielded the next highest number of spectra counts was ERGIC-53. Also present in both UBXD1FLAG immunoprecipitations was the actin binding protein LIMA1 (also called EPLIN1), SEPT9 and DST (Table I). CAV1, a recently identified UBXD1 interacting protein (10Ritz D. Vuk M. Kirchner P. Bug M. Schütz S. Hayer A. Bremer S. Lusk C. Baloh R.H. Lee H. Glatter T. Gstaiger M. Aebersold R. Weihl C.C. Meyer H. Endolysosomal sorting of ubiquitylated caveolin-1 is regulated by VCP and UBXD1 and impaired by VCP disease mutations.Nat. Cell Biol. 2011; 13: 1116-1123Crossref PubMed Scopus (158) Google Scholar), was detected but the number of spectral counts for CAV1 was low and did not reach statistical significance.Table ISpectral counts of proteins associated with UBXD1FLAG upon ponasterone induction in H1299 cells.aMass spectrometry was performed with LTQ-FT instrument. *Indicates significant enrichment in UBXD1FLAG immunoprecipitations (p < 0.05 Bonferroni corrected)ProteinsbPeptides and proteins were filtered at a 1% false discovery rate.# of assigned spectracInclusion limit set at a minimal of 10 spectra counts for 0.3 μm.0.1 μM0.3 μMTransitional endoplasmic reticulum ATPase (VCP; p97)183*512*UBX domain-containing protein 1 (UBXD1)74*89*ER-Golgi intermediate compartment 53kDa protein (ERGIC-53)33*34*Epithelial protein lost in neoplasm (EPLIN, LIMA1)622*SEPT9 protein (SEPT9)316*Dystonin (DST)813*a Mass spectrometry was performed with LTQ-FT instrument.b Peptides and proteins were filtered at a 1% false discovery rate.c Inclusion limit set at a minimal of 10 spectra counts for 0.3 μm. Open table in a new tab In addition to performing the analysis with the inducible H1299 system, we characterized FLAGUBXD1 interacting proteins using transiently transfected 293T cells. For these studies, we used SILAC (stable isotope labeling with amino acids in culture) and an LTQ-Orbitrap instrument. Samples were comprised of a 1:1 mixture of FLAG immunoprecipitates from "light" 293T cells (cultured in media supplemented with standard lysine and arginine) that had been transiently transfected with UBXD1FLAG plasmid versus "heavy" 293T cells (cultured in media containing Arg6 (U-13C6) and Lys8 (U-13C6, U-15N2)) that had been transfected with the empty vector control. Table II provides a list of proteins that were present in UBXD1FLAG immunoprecipitates. These proteins were not found in the control immunoprecipitations (i.e. no heavy peptides found). P97, UBXD1 and ERGIC-53 were present at high abundance. Numerous proteins were also identified in this second round of analysis, includin
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