title: Targeted proteomics reveals compositional dynamics of 60S pre-ribosomes after nuclear export.
abstract: 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.
authors.affiliation: Institute of Biochemistry, Department of Biology, ETH Zürich, Switzerland.
authors.forename: Martin
authors.initials: M
authors.lastname: Altvater
authors.forename: Yiming
authors.initials: Y
authors.lastname: Chang
authors.forename: Andre
authors.initials: A
authors.lastname: Melnik
authors.forename: Laura
authors.initials: L
authors.lastname: Occhipinti
authors.forename: Sabina
authors.initials: S
authors.lastname: Schütz
authors.forename: Ute
authors.initials: U
authors.lastname: Rothenbusch
authors.forename: Paola
authors.initials: P
authors.lastname: Picotti
authors.forename: Vikram Govind
authors.initials: VG
authors.lastname: Panse
mesh_terms: D021581:Active Transport, Cell Nucleus; D001692:Biological Transport; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence; D028861:Nuclear Pore Complex Proteins / Q000235:genetics / Q000378:metabolism; D001678:Organelle Biogenesis; D060066:Protein Interaction Maps; D020543:Proteome / Q000032:analysis / Q000235:genetics; D040901:Proteomics / Q000379:methods*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics; D054683:Ribosome Subunits, Large, Eukaryotic / Q000737:chemistry* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: C578305:BUD20 protein, S cerevisiae; D028861:Nuclear Pore Complex Proteins; D020543:Proteome; D016601:RNA-Binding Proteins; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins