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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© The Rockefeller University Press, 0021-9525/1999//389 $5.00
The Journal of Cell Biology, Volume 144, Number 3, , 1999 389-401

Regular Articles

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 ribosomesin 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 assembleswith 60S ribosomal subunits that are subsequently exported into the cytoplasm. In wild-type cells, GFP-labeled ribosomesare only detected by fluorescence in the cytoplasm. However,thermosensitive rna1-1 (Ran-GAP), prp20-1 (Ran-GEF), and nucleoporinnup49 and nsp1 mutants are impaired in ribosomal export asrevealed by nuclear accumulation of L25-GFP. Furthermore, overexpression of dominant-negative RanGTP (Gsp1-G21V) and thetRNA exportin Los1p inhibits ribosomal export. The pattern ofsubnuclear accumulation of L25-GFP observed in different mutantsis not identical, suggesting that transport can be blockedat different steps. Thus, nuclear export of ribosomes requiresthe nuclear/cytoplasmic Ran-cycle and distinct nucleoporins.This assay can be used to identify soluble transport factorsrequired for nuclear exit of ribosomes.

Key Words: nuclear pore complex • ribosomal export • L25 • GFP • nucleocytoplasmic transport

Abbreviations used in this paper: GFP, green fluorescent protein; NES, nuclear export signal; NLS, nuclear localization sequence; NPC, nuclear pore complex; rRNA, ribosomal RNA.

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: cg5{at}ix.urz.uni-heidelberg.de

E. Hurt was recipient of a grant from the Deutsche Forschungsgemeinschaft(Schwerpunktprogramm "Funktionelle Architektur des Zellkerns").

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