A Novel In Vivo Assay Reveals Inhibition of Ribosomal Nuclear Export in Ran-Cycle and Nucleoporin Mutants

doi:10.1083/jcb.144.3.389

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J. Cell Biol., Volume 144, Number 3, February 8, 1999 389-401

A Novel In Vivo Assay Reveals Inhibition of Ribosomal Nuclear Export in Ran-Cycle and Nucleoporin Mutants

Ed Hurt,^* Stefan Hannus,^* Birgit Schmelzl,^* Denise Lau,^* David Tollervey, and George Simos^*

^* Biochemie-Zentrum Heidelberg, D-69120 Heidelberg, Germany; and Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh EH9 37R, United Kingdom

To identify components involved in the nuclear export of ribosomes in yeast, we developed an in vivo assay exploiting a greenfluorescent protein (GFP)-tagged version of ribosomal proteinL25. After its import into the nucleolus, L25-GFP assembles with60S ribosomal subunits that are subsequently exported into thecytoplasm. In wild-type cells, GFP-labeled ribosomes are onlydetected by fluorescence in the cytoplasm. However, thermosensitiverna1-1 (Ran-GAP), prp20-1 (Ran-GEF), and nucleoporin nup49 andnsp1 mutants are impaired in ribosomal export as revealed by nuclearaccumulation of L25-GFP. Furthermore, overexpression of dominant-negativeRanGTP (Gsp1-G21V) and the tRNA exportin Los1p inhibits ribosomalexport. The pattern of subnuclear accumulation of L25-GFP observedin different mutants is not identical, suggesting that transportcan be blocked at different steps. Thus, nuclear export of ribosomesrequires the nuclear/cytoplasmic Ran-cycle and distinct nucleoporins.This assay can be used to identify soluble transport factors requiredfor nuclear exit ofribosomes.

Key words: nuclear pore complex; ribosomal export; L25; GFP; nucleocytoplasmic transport

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