Proximity Protein Labeling In Dictyostelium With Engineered Ascorbic Acid Peroxidase 2
To fully understand any cellular process, we not only need to identify the proteins implicated, but also how the protein network is structurally and spatially organized and changes over time. However, the dynamic nature of many protein interactions involved in cellular signaling pathways continues to be the bottleneck in mapping and studying protein networks. Fortunately, a recently developed proximity labeling method using engineered ascorbic acid peroxidase 2 (APEX2) in mammalian cells allows the identification of weak and/or transient protein interactions with spatial and temporal resolution. Here, we describe a protocol for successfully using the APEX2-proximity labeling method in Dictyostelium, using the cAMP receptor cAR1 as example. Coupled to the identification of the labeled proteins by mass spectrometry, this method expands Dictyostelium’s proteomics toolbox and should be widely useful for identifying interacting partners involved in a variety of biological processes in Dictyostelium.
1. Chen C-L, Perrimon N. Proximity-dependent labeling methods for proteomic profiling in living cells. Wiley Interdiscip Rev Dev Biol. 2017 Jul;6(4):10.1002/wdev.272. https://doi.org/10.1002/wdev.272 PMID:28387482
2. Roux KJ, Kim DI, Raida M, Burke B. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. J Cell Biol. 2012 Mar;196(6):801–10. https://doi.org/10.1083/jcb.201112098 PMID:22412018
3. Sears RM, May DG, Roux KJ. BioID as a Tool for Protein-Proximity Labeling in Living Cells. Methods Mol Biol. 2019;2012:299–313. https://doi.org/10.1007/978-1-4939-9546-2_15 PMID:31161514
4. Kim DI, Jensen SC, Noble KA, Birendra KC, Roux KH, Motamedchaboki K, et al. An improved smaller biotin ligase for BioID proximity labeling. Mol Biol Cell. 2016 Apr;27(8):1188-96. https://doi.org/10.1091/mbc.E15-12-0844. PMID:269112792
5. Branon TC, Bosch JA, Sanchez AD, Udeshi ND, Svinkina T, Carr SA, et al. Efficient proximity labeling in living cells and organisms with TurboID. Nat Biotechnol. 2018 Oct;36(9):880–7. https://doi.org/10.1038/nbt.4201 PMID:30125270
6. Batsios P, Meyer I, Gräf R. Proximity-Dependent Biotin Identification (BioID) in Dictyostelium Amoebae. Methods Enzymol. 2016;569:23–42. https://doi.org/10.1016/bs.mie.2015.09.007 PMID:26778551
7. Martell JD, Deerinck TJ, Sancak Y, Poulos TL, Mootha VK, Sosinsky GE, et al. Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy. Nat Biotechnol. 2012 Nov;30(11):1143–8. https://doi.org/10.1038/nbt.2375 PMID:23086203
8. Rhee HW, Zou P, Udeshi ND, Martell JD, Mootha VK, Carr SA, et al. Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging. Science. 2013 Mar;339(6125):1328–31. https://doi.org/10.1126/science.1230593 PMID:23371551
9. Udeshi ND, Pedram K, Svinkina T, Fereshetian S, Myers SA, Aygun O, et al. Antibodies to biotin enable large-scale detection of biotinylation sites on proteins. Nat Methods. 2017 Dec;14(12):1167–70. https://doi.org/10.1038/nmeth.4465 PMID:29039416
10. Lam SS, Martell JD, Kamer KJ, Deerinck TJ, Ellisman MH, Mootha VK, et al. Directed evolution of APEX2 for electron microscopy and proximity labeling. Nat Methods. 2015 Jan;12(1):51–4. https://doi.org/10.1038/nmeth.3179 PMID:25419960
11. Hung V, Udeshi ND, Lam SS, Loh KH, Cox KJ, Pedram K, et al. Spatially resolved proteomic mapping in living cells with the engineered peroxidase APEX2. Nat Protoc. 2016 Feb;11(3):456–75. https://doi.org/10.1038/nprot.2016.018 PMID:26866790
12. Lobingier BT, Hüttenhain R, Eichel K, Miller KB, Ting AY, von Zastrow M, et al. An Approach to Spatiotemporally Resolve Protein Interaction Networks in Living Cells. Cell. 2017 Apr 6;169(2):350-360.e12. https://doi.org/10.1016/j.cell.2017.03.022 PMID:28388416
13. Fey P, Dodson RJ, Basu S, Chisholm RL. One stop shop for everything Dictyostelium: DictyBase and the Dicty Stock Center in 2012. Methods Mol Biol. 2013;983:59–92. https://doi.org/10.1007/978-1-62703-302-2_4 PMID:23494302
14. Fey P, Kowal AS, Gaudet P, Pilcher KE, Chisholm RL. Protocols for growth and development of Dictyostelium discoideum. 2007 May;2(6):1307–16. https://doi.org/10.1038/nprot.2007.178 PMID:17545967
15. Sekine R, Kawata T, Muramoto T. CRISPR/Cas9 mediated targeting of multiple genes in Dictyostelium. Sci Rep. 2018 May;8(1):8471. https://doi.org/10.1038/s41598-018-26756-z PMID:29855514
16. Singer-Krüger B, Fröhlich T, Franz-Wachtel M, Nalpas N, Macek B, Jansen R-P. APEX2-mediated proximity labeling resolves protein networks in Saccharomyces cerevisiae cells. FEBS J. 2020 Jan;287(2):325–44. https://doi.org/10.1111/febs.15007 PMID:31323700
17. Scavello M, Petlick AR, Ramesh R, Thompson VF, Lotfi P, Charest PG. Protein kinase A regulates the Ras, Rap1 and TORC2 pathways in response to the chemoattractant cAMP in Dictyostelium. J Cell Sci. 2017 May;130(9):1545–58. https://doi.org/10.1242/jcs.177170 PMID:28302905
18. Huang M, Lin W, Chang J, Cheng C, Wang HY, Mou KY. The cysteine‐free single mutant C32S of APEX2 is a highly expressed and active fusion tag for proximity labeling applications. Protein Sci. 2019 Sep;28(9):1703-1712. https://doi.org/10.1002/pro.3685 PMID:31306516