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.Most steps in ribosome synthesis take place in the nucleolus, a specialized subnuclear region. This process starts with the synthesis of two pre-rRNA transcripts, 35S and pre-5S rRNA, which are processed and base modified to yield the mature 25S/28S, 18S, 5.8S, and 5S rRNAs, respectively (18). During these processes about 80 ribosomal proteins assemble onto the rRNAs to yield preribosomal particles, which are exported into the cytoplasm (41). In contrast to pre-rRNA processing and modification, very little is known about the assembly pathway for eukaryotic ribosomal subunits or the features that make them competent for nuclear exit (for recent reviews, see references 18 and 40).The transport of macromolecules through the nuclear pores is thought to involve facilitated diffusion of soluble transport factors over the repeat sequences of the nuclear pore proteins (nucleoporins) that form and line the nuclear pore complex. Directionality of transport is provided by the small GTPase Ran, due to the presence of a step RanGTP/RanGDP gradient across the nuclear membrane (for a review, see reference 23). RanGTP binds with high affinity to nuclear import and export receptors (importins and exportins, respectively) of the karyopherin ␤ superfamily (10). For nuclear exit, export cargoes, which harbor nuclear export sequences (NESs) (e.g., leucinerich NESs), form an intranuclear complex with the NES receptor Xpo1p/Crm1 in the presence of RanGTP (8,33). This trimeric complex is then exported from the nucleus into the cytoplasm.Saccharomyces cerevisiae has been a useful system for the analysis of the nuclear pore complex as well as transport factors (6). We have reported an in vivo assay for ribosomal export in yeast that uses a fusion between green fluorescent protein (GFP) and ribosomal protein Rpl25p (15). Rpl25p is imported into the nucleus and assembles with ribosomes by direct binding to the rRNA inside the nucleolus (39). Passage of both the free Rpl25p-GFP and the preribosomal particles through the nucleoplasm appears to be rapid in wild-type cells, and GFP-labeled ribosomes were detected by fluorescence microscopy in the cytoplasm. Mutations causing defects in subunit e...

Mutations in the ribosomal protein gene RPL10 suggest a novel modulating disease mechanism for autism

“…34 Collectively, these observations indicate that the human mutant alleles RPL10[L206M] and RPL10[H213Q] found in autism patients may be functional hypomorphs in that they support growth of RPL10[G161D] cells at the restrictive temperature, possibly by supplying basic translation functions missing in these yeast ts mutant cells. 16 However, the distinct qualitative modifications in the RPL10[G161D] ribosomal profile induced by expression of human mutant alleles, but not by expression of the human WT ortholog (compare Figure 3b, e and f), is compatible with the hypothesis, that the human mutant RPL10 proteins may exhibit altered functions in the regulation and modulation of the actively transcribing ribosome complement. This might compromise the timely translation of mRNAs into polypeptide chains in general.…”

“…The latter is functionally linked to Nmd3 via the Xpo1/exportin export receptor pathway. 16,17 Studies in yeast show that in the cytoplasm, Rpl10 is necessary for joining the 60S and 40S subunits in a late step of translation initiation, 18 a functional reflection of its location at the interface between the two ribosomal subunits adjoining the A and P sites as well as the transpeptidyl transferase center. The high level of sequence conservation of RPL10 in all organisms and the location close to regulatory and catalytic sites of the ribosome indicate the importance of the structural and functional integrity of this protein in ribosome assembly and function, respectively.…”

Arx1 Is a Nuclear Export Receptor for the 60S Ribosomal Subunit in Yeast

“…Nmd3 is an essential nucleocytoplasmic shuttling protein that contains a leucine-rich NES and is required for Crm1-dependent export of the 60S subunit (Ho et al, 2000b;Gadal et al, 2001;Thomas and Kutay, 2003;Trotta et al, 2003). However, several questions arise regarding the export of ribosomal subunits.…”

“…In yeast the large and small ribosomal subunits are assembled in the nucleolus and exported to the cytoplasm separately but in a Crm1-dependent manner (Moy and Silver, 1999;Ho et al, 2000b;Stage-Zimmermann et al, 2000;Gadal et al, 2001). Nmd3 is an essential nucleocytoplasmic shuttling protein that contains a leucine-rich NES and is required for Crm1-dependent export of the 60S subunit (Ho et al, 2000b;Gadal et al, 2001;Thomas and Kutay, 2003;Trotta et al, 2003).…”

Evidence for a novel cytoplasmic processing event in ribosome maturation in the sea urchin Paracentrotus lividus

“…3). Mature particles are then exported to the cytoplasm, probably in the way previously described [37][38][39][40][41].…”