To identify components involved in the nuclear export of ribosomes in yeast, we developed an in vivo assay exploiting a green fluorescent protein (GFP)-tagged version of ribosomal protein L25. After its import into the nucleolus, L25-GFP assembles with 60S ribosomal subunits that are subsequently exported into the cytoplasm. In wild-type cells, GFP-labeled ribosomes are only detected by fluorescence in the cytoplasm. However, thermosensitive rna1-1 (Ran-GAP), prp20-1 (Ran-GEF), and nucleoporin nup49 and nsp1 mutants are impaired in ribosomal export as revealed by nuclear accumulation of L25-GFP. Furthermore, overexpression of dominant-negative RanGTP (Gsp1-G21V) and the tRNA exportin Los1p inhibits ribosomal export. The pattern of subnuclear accumulation of L25-GFP observed in different mutants is not identical, suggesting that transport can be blocked at different steps. Thus, nuclear export of ribosomes requires the nuclear/cytoplasmic Ran-cycle and distinct nucleoporins. This assay can be used to identify soluble transport factors required for nuclear exit of ribosomes.
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8 February 1999
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February 08 1999
A Novel In Vivo Assay Reveals Inhibition of Ribosomal Nuclear Export in Ran-Cycle and Nucleoporin Mutants
Ed Hurt,
Ed Hurt
*Biochemie-Zentrum Heidelberg, D-69120 Heidelberg, Germany; and ‡Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh EH9 37R, United Kingdom
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Stefan Hannus,
Stefan Hannus
*Biochemie-Zentrum Heidelberg, D-69120 Heidelberg, Germany; and ‡Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh EH9 37R, United Kingdom
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Birgit Schmelzl,
Birgit Schmelzl
*Biochemie-Zentrum Heidelberg, D-69120 Heidelberg, Germany; and ‡Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh EH9 37R, United Kingdom
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Denise Lau,
Denise Lau
*Biochemie-Zentrum Heidelberg, D-69120 Heidelberg, Germany; and ‡Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh EH9 37R, United Kingdom
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David Tollervey,
David Tollervey
*Biochemie-Zentrum Heidelberg, D-69120 Heidelberg, Germany; and ‡Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh EH9 37R, United Kingdom
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George Simos
George Simos
*Biochemie-Zentrum Heidelberg, D-69120 Heidelberg, Germany; and ‡Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh EH9 37R, United Kingdom
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Ed Hurt
*Biochemie-Zentrum Heidelberg, D-69120 Heidelberg, Germany; and ‡Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh EH9 37R, United Kingdom
Stefan Hannus
*Biochemie-Zentrum Heidelberg, D-69120 Heidelberg, Germany; and ‡Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh EH9 37R, United Kingdom
Birgit Schmelzl
*Biochemie-Zentrum Heidelberg, D-69120 Heidelberg, Germany; and ‡Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh EH9 37R, United Kingdom
Denise Lau
*Biochemie-Zentrum Heidelberg, D-69120 Heidelberg, Germany; and ‡Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh EH9 37R, United Kingdom
David Tollervey
*Biochemie-Zentrum Heidelberg, D-69120 Heidelberg, Germany; and ‡Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh EH9 37R, United Kingdom
George Simos
*Biochemie-Zentrum Heidelberg, D-69120 Heidelberg, Germany; and ‡Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh EH9 37R, United Kingdom
Address correspondence to Ed Hurt, Biochemie-Zentrum Heidelberg, Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany. Tel.: 49 6221 54 41 73. Fax: 49 6221 54 43 69. E-mail: [email protected]
E. Hurt was recipient of a grant from the Deutsche Forschungsgemeinschaft (Schwerpunktprogramm “Funktionelle Architektur des Zellkerns”).
Received:
August 31 1998
Revision Received:
November 12 1998
Online ISSN: 1540-8140
Print ISSN: 0021-9525
1999
J Cell Biol (1999) 144 (3): 389–401.
Article history
Received:
August 31 1998
Revision Received:
November 12 1998
Citation
Ed Hurt, Stefan Hannus, Birgit Schmelzl, Denise Lau, David Tollervey, George Simos; A Novel In Vivo Assay Reveals Inhibition of Ribosomal Nuclear Export in Ran-Cycle and Nucleoporin Mutants . J Cell Biol 8 February 1999; 144 (3): 389–401. doi: https://doi.org/10.1083/jcb.144.3.389
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