Targeted proteomics reveals compositional dynamics of 60S pre-ribosomes after nuclear export.

Construction and intracellular targeting of eukaryotic pre-ribosomal particles involve a multitude of diverse transiently associating trans-acting assembly factors, energy-consuming enzymes, and transport factors. The ability to rapidly and reliably measure co-enrichment of multiple factors with maturing pre-ribosomal particles presents a major biochemical bottleneck towards revealing their function and the precise ...
contribution of >50 energy-consuming steps that drive ribosome assembly. Here, we devised a workflow that combines genetic trapping, affinity-capture, and selected reaction monitoring mass spectrometry (SRM-MS), to overcome this deficiency. We exploited this approach to interrogate the dynamic proteome of pre-60S particles after nuclear export. We uncovered assembly factors that travel with pre-60S particles to the cytoplasm, where they are released before initiating translation. Notably, we identified a novel shuttling factor that facilitates nuclear export of pre-60S particles. Capturing and quantitating protein interaction networks of trapped intermediates of macromolecular complexes by our workflow is a reliable discovery tool to unveil dynamic processes that contribute to their in vivo assembly and transport.
Mesh Terms:
Active Transport, Cell Nucleus, Biological Transport, Mass Spectrometry, Microscopy, Fluorescence, Nuclear Pore Complex Proteins, Organelle Biogenesis, Protein Interaction Maps, Proteome, Proteomics, RNA-Binding Proteins, Ribosomal Proteins, Ribosome Subunits, Large, Eukaryotic, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins
Mol. Syst. Biol.
Date: Dec. 06, 2012
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