Puromycin reactivity does not accurately localize translation at the subcellular level
Abstract
Puromycin is a tyrosyl-tRNA mimic that blocks translation by labeling and releasing elongating polypeptide chains from translating ribosomes. Puromycin has been used in molecular biology research for decades as a translation inhibitor. The development of puromycin antibodies and derivatized puromycin analogs has enabled the quantification of active translation in bulk and single-cell assays. More recently, in vivo puromycylation assays have become popular tools for localizing translating ribosomes in cells. These assays often use elongation inhibitors to purportedly inhibit the release of puromycin-labeled nascent peptides from ribosomes. Using in vitro and in vivo experiments in various eukaryotic systems, we demonstrate that, even in the presence of elongation inhibitors, puromycylated peptides are released and diffuse away from ribosomes. Puromycylation assays reveal subcellular sites, such as nuclei, where puromycylated peptides accumulate post-release and which do not necessarily coincide with sites of active translation. Our findings urge caution when interpreting puromycylation assays in vivo.
Data availability
Raw data for all plots have been deposited in accompanying excel files.
Article and author information
Author details
Funding
National Institutes of Health (2R37GM059425-14)
- Rachel Green
National Institutes of Health (5R37HD037047-20)
- Geraldine Seydoux
National Institutes of Health (5K99GM135450-02)
- Boris Zinshteyn
Howard Hughes Medical Institute
- Rachel Green
Howard Hughes Medical Institute
- Geraldine Seydoux
Damon Runyon Cancer Research Foundation (DRG-2280-16)
- Daniel H Goldman
Damon Runyon Cancer Research Foundation (DRG-2250-16)
- Boris Zinshteyn
National Institutes of Health (T32 GM007445)
- Madeline Cassani
- Nathan M Livingston
National Science Foundation (DGE-1746891)
- Madeline Cassani
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2020, Enam et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 17,840
- views
-
- 1,301
- downloads
-
- 61
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Biochemistry and Chemical Biology
- Cancer Biology
Many cells in high glucose repress mitochondrial respiration, as observed in the Crabtree and Warburg effects. Our understanding of biochemical constraints for mitochondrial activation is limited. Using a Saccharomyces cerevisiae screen, we identified the conserved deubiquitinase Ubp3 (Usp10), as necessary for mitochondrial repression. Ubp3 mutants have increased mitochondrial activity despite abundant glucose, along with decreased glycolytic enzymes, and a rewired glucose metabolic network with increased trehalose production. Utilizing ∆ubp3 cells, along with orthogonal approaches, we establish that the high glycolytic flux in glucose continuously consumes free Pi. This restricts mitochondrial access to inorganic phosphate (Pi), and prevents mitochondrial activation. Contrastingly, rewired glucose metabolism with enhanced trehalose production and reduced GAPDH (as in ∆ubp3 cells) restores Pi. This collectively results in increased mitochondrial Pi and derepression, while restricting mitochondrial Pi transport prevents activation. We therefore suggest that glycolytic flux-dependent intracellular Pi budgeting is a key constraint for mitochondrial repression.
-
- Biochemistry and Chemical Biology
Glycans play an important role in modulating the interactions between natural killer cells and antibodies to fight pathogens and harmful cells.