Abstract
Subcellular localization critically influences protein function, and cells control protein localization to regulate biological processes. We have developed and applied Dynamic Organellar Maps, a proteomic method that allows global mapping of protein translocation events. We initially used maps statically to generate a database with localization and absolute copy number information for over 8700 proteins from HeLa cells, approaching comprehensive coverage. All major organelles were resolved, with exceptional prediction accuracy (estimated at >92%). Combining spatial and abundance information yielded an unprecedented quantitative view of HeLa cell anatomy and organellar composition, at the protein level. We subsequently demonstrated the dynamic capabilities of the approach by capturing translocation events following EGF stimulation, which we integrated into a quantitative model. Dynamic Organellar Maps enable the proteome-wide analysis of physiological protein movements, without requiring any reagents specific to the investigated process, and will thus be widely applicable in cell biology.
Highlights
The hallmark of eukaryotic cells is their compartmentalization into distinct membrane-bound organelles
We investigated the well-characterized process of epidermal growth factor receptor (EGFR) uptake
We have developed and applied a powerful new method for making quantitative organellar maps, to generate an extensive database of human protein subcellular localizations and organellar composition
Summary
The hallmark of eukaryotic cells is their compartmentalization into distinct membrane-bound organelles. In order to regulate protein activity, many biological processes involve changes in protein subcellular localization. Prominent examples include the endocytic uptake of activated plasma membrane signalling receptors, to terminate the signalling process (Jones and Rappoport, 2014), and the nucleo-cytoplasmic shuttling of many transcription factors, to regulate their access to DNA (Plotnikov et al, 2011). The ability to monitor changes in organellar composition would provide a powerful tool to investigate cell biological processes at the systems level. Microscopy-based approaches can provide spatial information on individual proteins (Uhlen et al, 2015), but are limited by the availability of specific antibodies, and are very labour-intensive for analysing complete proteomes (Marx, 2015). Genome-wide GFP-tagging in yeast circumvents the need for antibodies (Huh et al, 2003), but tags may inadvertently alter protein subcellular localisation, which is difficult to control for; in addition, serial imaging of cells for comparative purposes remains experimentally challenging (Breker et al, 2013)
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