Proximity labeling provides a powerful in vivo tool to characterize the proteome of subcellular structures and the interactome of specific proteins. The nematode Caenorhabditis elegans is one of the most intensely studied organisms in biology, offering many advantages for biochemistry. Using the highly active biotin ligase TurboID, we optimize here a proximity labeling protocol for C. elegans. An advantage of TurboID is that biotin's high affinity for streptavidin means biotin-labeled proteins can be affinity-purified under harsh denaturing conditions. By combining extensive sonication with aggressive denaturation using SDS and urea, we achieved near-complete solubilization of worm proteins. We then used this protocol to characterize the proteomes of the worm gut, muscle, skin, and nervous system. Neurons are among the smallest C. elegans cells. To probe the method's sensitivity, we expressed TurboID exclusively in the two AFD neurons and showed that the protocol could identify known and previously unknown proteins expressed selectively in AFD. The active zones of synapses are composed of a protein matrix that is difficult to solubilize and purify. To test if our protocol could solubilize active zone proteins, we knocked TurboID into the endogenous elks-1 gene, which encodes a presynaptic active zone protein. We identified many known ELKS-1-interacting active zone proteins, as well as previously uncharacterized synaptic proteins. Versatile vectors and the inherent advantages of using C. elegans, including fast growth and the ability to rapidly make and functionally test knock-ins, make proximity labeling a valuable addition to the armory of this model organism.
Journal of Biological Chemistry
We thank de Bono lab members for helpful comments on the manuscript, IST Austria and University of Vienna Mass Spec Facilities for invaluable discussions and comments for the optimization of mass spec analyses of worm samples. The biotin auxotropic E. coli strain MG1655bioB:kan was gift from John Cronan (University of Illinois) and was kindly sent to us by Jessica Feldman and Ariana Sanchez (Stanford University). dg398 pEntryslot2_mNeongreen::3XFLAG::stop and dg397 pEntryslot3_mNeongreen::3XFLAG::stop::unc-54 3′UTR entry vector were kindly shared by Dr Dominique Glauser (University of Fribourg). Codon-optimized mScarlet vector was a generous gift from Dr Manuel Zimmer (University of Vienna).
Artan M, Barratt S, Flynn SM, et al. Interactome analysis of Caenorhabditis elegans synapses by TurboID-based proximity labeling. Journal of Biological Chemistry. 2021;297(3). doi:10.1016/J.JBC.2021.101094
Artan, M., Barratt, S., Flynn, S. M., Begum, F., Skehel, M., Nicolas, A., & de Bono, M. (2021). Interactome analysis of Caenorhabditis elegans synapses by TurboID-based proximity labeling. Journal of Biological Chemistry. Elsevier. https://doi.org/10.1016/J.JBC.2021.101094
Artan, Murat, Stephen Barratt, Sean M. Flynn, Farida Begum, Mark Skehel, Armel Nicolas, and Mario de Bono. “Interactome Analysis of Caenorhabditis Elegans Synapses by TurboID-Based Proximity Labeling.” Journal of Biological Chemistry. Elsevier, 2021. https://doi.org/10.1016/J.JBC.2021.101094.
M. Artan et al., “Interactome analysis of Caenorhabditis elegans synapses by TurboID-based proximity labeling,” Journal of Biological Chemistry, vol. 297, no. 3. Elsevier, 2021.
Artan M, Barratt S, Flynn SM, Begum F, Skehel M, Nicolas A, de Bono M. 2021. Interactome analysis of Caenorhabditis elegans synapses by TurboID-based proximity labeling. Journal of Biological Chemistry. 297(3), 101094.
Artan, Murat, et al. “Interactome Analysis of Caenorhabditis Elegans Synapses by TurboID-Based Proximity Labeling.” Journal of Biological Chemistry, vol. 297, no. 3, 101094, Elsevier, 2021, doi:10.1016/J.JBC.2021.101094.
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