Abstract
In order to understand the mechanisms through which apical and basolateral membrane proteins achieve their subcellular distributions in polarized epithelial cells, it is critical to develop techniques that permit the selective observation of newly synthesized populations of these proteins. The SNAP tag system permits the detection and visualization of distinct spatially and temporally defined cohorts of tagged proteins. Thus, this technique is especially well suited to studying the trafficking routes pursued by newly synthesized proteins. The SNAP tag can be applied in the setting of fixed or live cell fluorescence microscopic analysis and can also be used in the context of various biochemical approaches. Here, we describe the use of the SNAP tag in association with confocal microscopy and SDS-PAGE to follow the biosynthetic pool of a membrane protein as it exits from the trans-Golgi network and makes its way to the plasma membrane.
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References
Rodriguez-Boulan E, Kreitzer G, Müsch A (2005) Organization of vesicular trafficking in epithelia. Nat Rev Mol Cell Biol 6:233–247
Fölsch H, Mattila PE, Weisz OA (2009) Taking the scenic route: biosynthetic traffic to the plasma membrane in polarized epithelial cells. Traffic 10:972–981
Weisz OA, Rodriguez-Boulan E (2009) Apical trafficking in epithelial cells: signals, clusters and motors. J Cell Sci 122:4253–4266
Juillerat A, Gronemeyer T, Keppler A et al (2003) Directed evolution of O6-alkylguanine-DNA alkyltransferase for efficient labeling of fusion proteins with small molecules in vivo. Chem Biol 10:313–317
Keppler A, Kindermann M, Gendreizig S et al (2004) Labeling of fusion proteins of O6-alkylguanine-DNA alkyltransferase with small molecules in vivo and in vitro. Methods 32:437–444
Griffin BA, Adams SR, Tsien RY (1998) Specific covalent labeling of recombinant protein molecules inside live cells. Science 281:269–272
Machleidt T, Robers M, Hanson GT (2007) Protein labeling with FlAsH and ReAsH. Methods Mol Biol 356:209–220
Los GV, Encell LP, McDougall MG et al (2008) HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem Biol 3:373–382
Maurel D, Banala S, Laroche T, Johnsson K (2010) Photoactivatable and photoconvertible fluorescent probes for protein labeling. ACS Chem Biol 5:507–516
Kamiya M, Johnsson K (2010) Localizable and highly sensitive calcium indicator based on a BODIPY fluorophore. Anal Chem 82:6472–6479
Sun X, Zhang A, Baker B et al (2011) Development of SNAP-Tag fluorogenic probes for wash-free fluorescence imaging. Chembiochem 12:2217–2226
Lukinavičius G, Umezawa K, Olivier N et al (2013) A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins. Nat Chem 5:132–139
Farr GA, Hull M, Mellman I, Caplan MJ (2009) Membrane proteins follow multiple pathways to the basolateral cell surface in polarized epithelial cells. J Cell Biol 186:269–282
Gautier A, Juillerat A, Heinis C et al (2008) An engineered protein tag for multiprotein labeling in living cells. Chem Biol 15:128–136
Harder JL, Whiteman EL, Pieczynski JN, Liu C-J, Margolis B (2012) Snail destabilizes cell surface Crumbs3a. Traffic 13:1170–1185
Milenkovic L, Scott MP, Rohatgi R (2009) Lateral transport of smoothened from the plasma membrane to the membrane of the cilium. J Cell Biol 187:365–374
Morton MJ, Farr GA, Hull M, Capendeguy O, Horisberger J-D, Caplan MJ (2010) Association with beta-COP regulates the trafficking of the newly synthesized Na, K-ATPase. J Biol Chem 285:33737–33746
Acknowledgements
The authors would like to thank Dr. Ivan Correa for helpful discussions and support. This material is based upon work supported by NIH training grant 5T32GM007223-35 and National Science Foundation Graduate Research Fellowship Grant No. DGE-1122492 (E.H. Stoops) and NIH grants DK17433 and DK072612 (M.J. Caplan).
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Stoops, E.H., Farr, G.A., Hull, M., Caplan, M.J. (2014). SNAP-Tag to Monitor Trafficking of Membrane Proteins in Polarized Epithelial Cells. In: Ivanov, A. (eds) Exocytosis and Endocytosis. Methods in Molecular Biology, vol 1174. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0944-5_11
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DOI: https://doi.org/10.1007/978-1-4939-0944-5_11
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