title: Dissecting ribosome assembly and transport in budding yeast.
abstract: Construction of the eukaryotic ribosome begins in the nucleolus and requires >300 evolutionarily conserved nonribosomal trans-acting factors, which transiently associate with preribosomal subunits at distinct assembly stages. A subset of trans-acting and transport factors passage assembled preribosomal subunits in a functionally inactive state through the nuclear pore complexes (NPC) into the cytoplasm, where they undergo final maturation before initiating translation. Here, we summarize the repertoire of tools developed in the model organism budding yeast that are spearheading the functional analyses of trans-acting factors involved in the assembly and intracellular transport of preribosomal subunits. We elaborate on different GFP-tagged ribosomal protein reporters and a pre-rRNA reporter that reliably monitors the movement of preribosomal particles from the nucleolus to cytoplasm. We discuss the powerful yeast heterokaryon assay, which can be employed to uncover shuttling trans-acting factors that need to accompany preribosomal subunits to the cytoplasm to be released prior to initiating translation. Moreover, we present two biochemical approaches, namely sucrose gradient analyses and tandem affinity purification, that are rapidly facilitating the uncovering of regulatory processes that control the compositional dynamics of trans-acting factors on maturing preribosomal particles. Altogether, these approaches when combined with traditional analytical biochemistry, targeted proteomics and structural methodologies, will contribute to the dissection of the assembly and intracellular transport of preribosomal subunits, as well as other macromolecular assemblies that influence diverse biological pathways.
authors.affiliation: Institute of Biochemistry (IBC), ETH Zürich, Otto-Stern-Weg 3, Zurich, Switzerland; MLS Program, Life Science Zurich Graduate School, Winterthurerstrasse 190, Zurich, Switzerland.
authors.forename: Martin
authors.initials: M
authors.lastname: Altvater
authors.affiliation: Institute of Biochemistry (IBC), ETH Zürich, Otto-Stern-Weg 3, Zurich, Switzerland; MLS Program, Life Science Zurich Graduate School, Winterthurerstrasse 190, Zurich, Switzerland.
authors.forename: Sabina
authors.initials: S
authors.lastname: Schütz
authors.affiliation: Institute of Biochemistry (IBC), ETH Zürich, Otto-Stern-Weg 3, Zurich, Switzerland.
authors.forename: Yiming
authors.initials: Y
authors.lastname: Chang
authors.affiliation: Institute of Biochemistry (IBC), ETH Zürich, Otto-Stern-Weg 3, Zurich, Switzerland.
authors.forename: Vikram Govind
authors.initials: VG
authors.lastname: Panse
mesh_terms: D001692:Biological Transport / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D028884:Karyopherins / Q000235:genetics; D013058:Mass Spectrometry / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D022022:Nuclear Pore / Q000235:genetics / Q000378:metabolism*; D009697:Nucleolus Organizer Region / Q000235:genetics*; D012335:RNA, Ribosomal / Q000096:biosynthesis / Q000235:genetics; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics; D054683:Ribosome Subunits, Large, Eukaryotic / Q000235:genetics / Q000378:metabolism*; D054682:Ribosome Subunits, Small, Eukaryotic / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D014461:Ultracentrifugation / Q000379:methods; D000097606:Exportin 1 Protein
chemical_list: D049452:Green Fluorescent Proteins; D028884:Karyopherins; D012335:RNA, Ribosomal; D018160:Receptors, Cytoplasmic and Nuclear; D000097606:Exportin 1 Protein
keywords: Budding yeast; Fluorescence in situ hybridization; Heterokaryon assay; Nuclear export; Ribosome biogenesis; Ribosome export; Sucrose sedimentation; TAP purification