Abstract
We previously described the ribopuromyclation method (RPM) to visualize and quantitate translating ribosomes in fixed and permeabilized cells by standard immunofluorescence. RPM is based on puromycylation of nascent chains bound to translating ribosomes followed by detection of puromycylated nascent chains with a puromycin-specific mAb. We now demonstrate that emetine optimally enhances nascent chain puromycylation, and describe a modified RPM protocol for identifying ribosome-bound nascent chains in metabolically inert permeabilized cells.
References
David A, Netzer N, Strader MB, Das SR, Chen CY et al (2011) RNA binding targets aminoacyl-tRNA synthetases to translating ribosomes. J Biol Chem 286:20688–20700
David A, Dolan BP, Hickman HD, Knowlton JJ, Clavarino G et al (2012) Nuclear translation visualized by ribosome-bound nascent chain puromycylation. J Cell Biol 197:45–57
Eggers DK, Welch WJ, Hansen WJ (1997) Complexes between nascent polypeptides and their molecular chaperones in the cytosol of mammalian cells. Mol Biol Cell 8:1559–1573
Fallon AM, Stollar V (1982) Isolation and characterization of puromycin-resistant clones from cultured mosquito cells. Somatic Cell Genet 8:521–532
Grollman AP (1968) Inhibitors of protein biosynthesis. V. Effects of emetine on protein and nucleic acid biosynthesis in HeLa cells. J Biol Chem 243:4089–4094
Hansen JL, Moore PB, Steitz TA (2003) Structures of five antibiotics bound at the peptidyl transferase center of the large ribosomal subunit. J Mol Biol 330:1061–1075
Jimenez A, Carrasco L, Vazquez D (1977) Enzymic and nonenzymic translocation by yeast polysomes. Site of action of a number of inhibitors. Biochemistry 16:4727–4730
Kappen LS, Suzuki H, Goldberg IH (1973) Inhibition of reticulocyte peptide-chain initiation by pactamycin: accumulation of inactive ribosomal initiation complexes. Proc Natl Acad Sci USA 70:22–26
Kedersha N, Chen S, Gilks N, Li W, Miller IJ et al (2002) Evidence that ternary complex (eIF2-GTP-tRNA(i)(Met))-deficient preinitiation complexes are core constituents of mammalian stress granules. Mol Biol Cell 13:195–210
Pestka S (1971) Inhibitors of ribosome functions. Annu Rev Microbiol 25:487–562
Schmidt EK, Clavarino G, Ceppi M, Pierre P (2009) SUnSET, a nonradioactive method to monitor protein synthesis. Nat Methods 6:275–277
Acknowledgments
Glennys Reynoso provided outstanding technical support. This work was generously supported by the Division of Intramural Research, NIAID.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
David, A., Bennink, J.R. & Yewdell, J.W. Emetine optimally facilitates nascent chain puromycylation and potentiates the ribopuromycylation method (RPM) applied to inert cells. Histochem Cell Biol 139, 501–504 (2013). https://doi.org/10.1007/s00418-012-1063-8
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00418-012-1063-8