Nuclear export of 60s ribosomal subunits depends on Xpo1p and requires a nuclear export sequence-containing factor, Nmd3p, that associates with the large subunit protein Rpl10p

Abstract

Nuclear export of ribosomes requires a subset of nucleoporins and the Ran system, but specific transport factors have not been identified. Using a large subunit reporter (Rpl25p-eGFP), we have isolated several temperature-sensitive ribosomal export (rix) mutants. One of these corresponds to the ribosomal protein Rpl10p, which interacts directly with Nmd3p, a conserved and essential protein associated with 60S subunits. We find that thermosensitive nmd3 mutants are impaired in large subunit export. Strikingly, Nmd3p shuttles between the nucleus and cytoplasm and is exported by the nuclear export receptor Xpo1p. Moreover, we show that export of 60S subunits is Xpo1p dependent. We conclude that nuclear export of 60S subunits requires the nuclear export sequence-containing nonribosomal protein Nmd3p, which directly binds to the large subunit protein Rpl10p.

FIG. 1

A genetic screen for rix mutants identifies the ribosomal protein Rpl10p. (A) Five rix mutants (rix1-1, rix2-1, rix3-1, rix5-1, and rix7-1) and the isogenic wild-type RIX⁺ strain expressing Rpl25p-eGFP were grown at 23°C before a shift for 5 h to 37°C and inspection by fluorescence microscopy. (B) rix5-1 is complemented by RPL10. Top, time course of nuclear accumulation of Rpl25p-eGFP in rix5-1 (=rpl10-1) and RIX5⁺ (=RPL10⁺) cells shifted for 1 or 2 h to 37°C; bottom, growth curve of rix5-1 cells and rix-5 strain complemented by the cloned RIX5 (=RPL10) at 37°C. (C) Pre-rRNA processing in an rpl10 ts mutant shifted to 37°C for 6 h. rRNAs were pulse-labeled for 2 min with [methyl-³H] methionine followed by a 5-min chase. rRNA was analyzed by autoradiography. (D) Rpl10p contains an NLS in the N domain. Full-length Rpl10p (residues 1 to 221), Rpl10p-NLS (residues 1 to 64), and Rpl25p-NLS (residues 1 to 52), all tagged with GFP, were analyzed in KAP123⁺ and kap123Δ strains by fluorescence microscopy. (E) Nuclear accumulation of Rpl10p-eGFP and Rpl25p-eGFP in rrp44-1 and wild-type (wt) cells grown at 23°C before transfer to 37°C for 5 h.

FIG. 1

A genetic screen for rix mutants identifies the ribosomal protein Rpl10p. (A) Five rix mutants (rix1-1, rix2-1, rix3-1, rix5-1, and rix7-1) and the isogenic wild-type RIX⁺ strain expressing Rpl25p-eGFP were grown at 23°C before a shift for 5 h to 37°C and inspection by fluorescence microscopy. (B) rix5-1 is complemented by RPL10. Top, time course of nuclear accumulation of Rpl25p-eGFP in rix5-1 (=rpl10-1) and RIX5⁺ (=RPL10⁺) cells shifted for 1 or 2 h to 37°C; bottom, growth curve of rix5-1 cells and rix-5 strain complemented by the cloned RIX5 (=RPL10) at 37°C. (C) Pre-rRNA processing in an rpl10 ts mutant shifted to 37°C for 6 h. rRNAs were pulse-labeled for 2 min with [methyl-³H] methionine followed by a 5-min chase. rRNA was analyzed by autoradiography. (D) Rpl10p contains an NLS in the N domain. Full-length Rpl10p (residues 1 to 221), Rpl10p-NLS (residues 1 to 64), and Rpl25p-NLS (residues 1 to 52), all tagged with GFP, were analyzed in KAP123⁺ and kap123Δ strains by fluorescence microscopy. (E) Nuclear accumulation of Rpl10p-eGFP and Rpl25p-eGFP in rrp44-1 and wild-type (wt) cells grown at 23°C before transfer to 37°C for 5 h.

FIG. 2

The rsa1 disruption mutant and an nmd3 ts mutant accumulate Rpl25p-eGFP in the nucleus. (A) Left, growth of the rsa1Δ disruption and its isogenic wild-type strain on a YPD plate and in liquid medium. Equivalent amounts of cells (dilution in 10⁻¹ steps) were grown at the indicated temperatures for 3 days. Right, nuclear accumulation of Rpl25p-eGFP in the rsa1Δ mutant. rsa1Δ and RSA1⁺ strains were transformed with RPL25-eGFP and grown at 23°C before incubation at 37°C for 8 h. Rpl25p-eGFP localization was analyzed by fluorescence microscopy. (B) Growth of the nmd3-2 ts mutant at 23 and 37°C. (C) Rpl25p-eGFP accumulates in the nucleus of the nmd3-2 ts mutant. Cells were shifted for 0, 30, 90, and 180 min to 37°C, and then the Rpl25p-eGFP signal was analyzed by fluorescence microscopy.

FIG. 3

Nmd3p binds directly to Rpl10p and associates with 60S subunits in a salt-dependent manner. (A) Nmd3p binds to GST-tagged Rpl10p when expressed in E. coli. Expression and purification of recombinant Rpl10p-GST in the absence or presence of Nmd3p-His₆ is described in Materials and Methods. The supernatants of E. coli cell lysates (Sup) were incubated with glutathione-Sepharose 4B beads. After washing, bound proteins were eluted with sample buffer (Bound), and the fractions were analyzed by SDS-PAGE and Coomassie blue staining (top) or Western blotting using α-Rpl10p and α-His (bottom). Lanes: 1, protein standard (10-kDa ladder); 2 to 4, supernatants; 5 to 7, bound fractions; 2 and 5, Rpl10p-GST in the absence of Nmd3p-His₆; 3 and 6, Rpl10p-GST coexpressed with Nmd3p-His₆; 4 and 7, GST coexpressed with Nmd3p-His₆. Note that full-length Nmd3p-His₆ is sensitive to proteolysis in E. coli, yielding a more stable breakdown product Nmd3p-His₆*, which apparently lacks a short N-terminal part. Indicated is also an unknown E. coli contaminant (?) which coisolates with Rpl10p-GST. (B) Nmd3p, which was tagged with ProtA and is fully functional, is extracted from 60S ribosomes by high-salt (500 mM KCl) treatment. Ribosomes were analyzed by sucrose gradient centrifugation in low- and high-salt buffer, and ribosomal profiles were determined by OD₂₆₀ measurement of the gradient fractions (top). Western blot analysis of these gradient fractions reveals ProtA-tagged Nmd3p and Rpl10p (bottom).

FIG. 4

Two NESs in the Nmd3p carboxy-terminal domain. (A) Schematic drawing of the Nmd3p domains. Sequence comparison of classical NESs (PKI, mitogen-activated protein kinase kinase [MAPKK], human immunodeficiency virus Rev) and NES-like sequences present in the Nmd3p C domains from S. cerevisiae (S.C-1 and S.C-2), humans (HUM-1, HUM-2), Schizosaccharomyces pombe (S.P-1 and S.P-2) and Drosophila (DRO-1 and DRO-2). (B) The nmd3 null strain is complemented by full-length Nmd3p-eGFP, by an Nmd3p truncation construct lacking one of the two C-terminal NESs (ΔNES1) but not by one lacking both NESs (ΔNES1/2) after 5 days on 5-fluoroorotic acid-containing plates. (C) Intracellular location of the indicated Nmd3p constructs tagged with GFP in yeast cells. The NLS of Npl3p (NLS_RGG) tagged with GFP was used to test for NES activity of the Nmd3p C domain. (D) Nuclear accumulation of Rpl25p-eGFP expressed in the slow-growing nmd3ΔNES1 mutant grown at 30°C.

FIG. 5

Nmd3p and 60S large subunits are exported through the Xpo1p-dependent transport pathway. (A) Nmd3p-eGFP expressed in the LMB-sensitive xpo1 mutant and isogenic wild-type strain. (B) Rpl25p-eGFP expressed in the LMB-sensitive xpo1 mutant and isogenic wild-type strain. At time point 0 min, LMB (100 ng/ml) was added to the culture medium, and the fluorescence signals from GFP fusion proteins were observed by fluorescence microscopy.