Identification of MARCH2 E3 ligase-substrates using proximity-dependent biotin labeling method
MARCH2 is known to be involved in intracellular vesicular trafficking. To identify its substrates, we have recently developed a method based on proximity-dependent biotin labelling. In this protocol, the MARCH2 ubiquitin ligase of interest is expressed as a fusion to Escherichia coli biotin ligase BirA together with a biotin acceptor peptide(AP)-tagged ubiquitin. The BirA-directed biotin labeling of AP depends on the proximity of the two fusion proteins in the cell, which leads to preferential labeling of ubiquitinated E3 substrates. In this study, we applied this procedure to MARCH2 and identified its substrates.
Sample Processing Protocol
Affinity purification of biotinylated proteins HeLa cells were transfected with plasmids encoding FLAG-BirA-MARCH2 and AP-HA-Ub; 24 h post-transfection, cells were lysed in lysis buffer (2% SDS, 250 mM NaCl, 50 mM Tris-Cl, pH 7.4), boiled at 95°C for 10 min and centrifuged at 12,000 × g for 10 min. The obtained supernatant was diluted 10-fold in dilution buffer (250 mM NaCl, 50 mM Tris-Cl, pH 7.4) and immunoprecipitated with anti-FLAG M2 agarose (Sigma) overnight at 4°C. The beads were separated from the lysate by centrifugation at 2000 × g for 3 min. The lysates containing biotinylated proteins were incubated with Dynabeads MyOne Streptavidin C1 (Invitrogen) overnight at room temperature. Dynabeads were sequentially washed with Buffer 1 (2% SDS, 250 mM NaCl, 50 mM Tris-Cl, pH 7.4), Buffer 2 (0.1% deoxycholic acid, 1% Triton X-100, 1 mM EDTA, 500 mM NaCl, 50 mM HEPES, pH 7.4), Buffer 3 (0.5% deoxycholic acid, 0.5% NP-40, 1 mM EDTA, 250 mM LiCl, 10 mM Tris-Cl, pH 8.0), and Buffer 4 (50 mM NaCl, 50 mM Tris-Cl, pH 7.4). Finally, the beads were washed with and resuspended in 200 μl of 100 mM ammonium bicarbonate (pH 7.8). Protein digestion and affinity purification of ubiquitinated peptides Purified proteins immobilized on Dynabeads were reduced with 5 mM dithiothreitol and alkylated with 25 mM iodoacetamide. Proteins were digested with trypsin (Promega) overnight at 37°C. The supernatant was separated from Dynabeads by centrifugation at 10,000 × g for 5 min. The collected supernatant was dried in a speed vacuum concentrator. The peptides were dissolved in immunoaffinity purification buffer (50 mM MOPS/NaOH, pH 7.2, 10 mM NaH2PO4, 50 mM NaCl) and incubated with Ubiquitin Branch Motif (K-ε-GG) Immunoaffinity Beads (Cell Signaling Technology) overnight at 4°C. The beads were washed twice with immunoaffinity purification buffer and three times with deionized water, and peptides were eluted with 0.15% trifluoroacetic acid and lyophilized. Protein identification and quantitation by mass spectrometry Lyophilized peptides were analyzed by nanoelectrospray LC–MS/MS on an LTQ Orbitrap Velos (Thermo Fisher Scientific Inc., San Jose, CA). The peptides were separated on a 75 μm × 15 cm reversed-phase column (EASY-Spray PepMap, C18, 3 μm, 100Å; Thermo Fisher Scientific Inc. San Jose, CA) with a gradient of 5% to 50% acetonitrile in 0.125% formic acid over 120 min.
Data Processing Protocol
The 20 most intense peaks from each full MS scan acquired in the Orbitrap were selected for MS/MS in the linear ion trap. The MS-MS spectra were used to search the National Center for Biotechnology Information nonredundant and expressed-sequence-tag databases using Mascot 2.2 (Matrix Science). The MS/MS scan range was m/z 200–2000.
Wonjin Yoo, Yonsei university
Jong-Bok Yoon, Department of Biochemistry, College of Life Science & Biotechnology, Yonsei University, 262 Seongsanno, Seodaemun-Gu, Seoul 120-749, Korea. Tel.: +82-2-2123-2704; Fax: 82-2-362-9897. ( lab head )
Yoo W, Cho EB, Kim S, Yoon JB. The E3 ubiquitin ligase MARCH2 regulates ERGIC3-dependent trafficking of secretory proteins. J Biol Chem. 2019 PubMed: 31142615