In eukaryotes, nuclear export of the large (60S) ribosomal subunit requires the adapter protein Nmd3p to provide the nuclear export signal. Here, we show that in yeast release of Nmd3p from 60S subunits in the cytoplasm requires the ribosomal protein Rpl10p and the G-protein, Lsg1p. Mutations in LSG1 or RPL10 blocked Nmd3-GFP shuttling into the nucleus and export of pre-60S subunits from the nucleus. Overexpression of NMD3 alleviated the export defect, indicating that the block in 60S export in lsg1 and rpl10 mutants results indirectly from failing to recycle Nmd3p. The defect in Nmd3p recycling and the block in 60S export in both lsg1 and rpl10 mutants was also suppressed by mutant Nmd3 proteins that showed reduced binding to 60S subunits in vitro. We propose that the correct loading of Rpl10p into 60S subunits is required for the release of Nmd3p from subunits by Lsg1p. These results suggest a coupling between recycling the 60S export adapter and activation of 60S subunits for translation.

We characterized two essential putative GTPases, Nog1p and Lsg1p, that are found associated with free 60S ribosomal subunits affinity purified with the nuclear export adapter Nmd3p. Nog1p and Lsg1p are nucleolar and cytoplasmic, respectively, and are not simultaneously on the same particle, reflecting the path of Nmd3p shuttling in and out of the nucleus. Conditional mutants of both NOG1 and LSG1 are defective in 60S subunit biogenesis and display diminished levels of 60S subunits at restrictive temperature. Mutants of both genes also accumulate the 60S ribosomal reporter Rpl25-eGFP in the nucleolus, suggesting that both proteins are needed for subunit export from the nucleolus. Since Lsg1p is cytoplasmic, its role in nuclear export is likely to be indirect. We suggest that Lsg1p is needed to recycle an export factor(s) that shuttles from the nucleus associated with the nascent 60S subunit.In eukaryotic cells ribosomes are assembled in the nucleolus and must be transported through the nuclear pore complex to the cytoplasm, where they function in translation. The first discrete intermediate preribosomal species, the 90S particle (40), assembles around the 35S primary transcript in the nucleolus and contains integral ribosomal proteins, trans-acting rRNA processing factors, and subunit assembly factors. Recent evidence suggests that the 90S particle assembles cotranscriptionally on the primary transcript, is devoted to 40S biogenesis, and is almost entirely lacking in 60S proteins and assembly factors (5, 16). Cleavage of the transcript into 20S and 27S species in the nucleolus, the precursors of 18S and 25S, respectively, may release the pre-40S complex. The subsequent processing and assembly of the 66S particle, the nucleolar precursor of the 60S subunit (44), appears to be independent of 40S assembly (5, 16). Release of the large subunit from the nucleolus into the nucleoplasm likely coincides with the release of many of the 66S-associated biogenesis factors (9, 31), yielding a nucleoplasmic pre-60S subunit.

The large ribosomal subunit protein Rpl10p is required for subunit joining and 60S export in yeast. We have recently shown that Rpl10p as well as the cytoplasmic GTPase Lsg1p are required for releasing the 60S nuclear export adapter Nmd3p from subunits in the cytoplasm. Here, we more directly address the order of Nmd3p and Rpl10p recruitment to the subunit. We show that Nmd3p can bind subunits in the absence of Rpl10p. In addition, we examined the basis of the previously reported dominant negative growth phenotype caused by overexpression of C-terminally truncated Rpl10p and found that these Rpl10p fragments are not incorporated into subunits in the nucleus but instead sequester the WD-repeat protein Sqt1p. Sqt1p is an Rpl10p binding protein that is proposed to facilitate loading of Rpl10p into the 60S subunit. Although Sqt1p normally only transiently binds 60S subunits, the levels of Sqt1p that can be coimmunoprecipitated by the 60S-associated GTPase Lsg1p are significantly increased by a dominant mutation in the Walker A motif of Lsg1p. This mutant Lsg1 protein also leads to increased levels of Sqt1p in complexes that are coimmunoprecipitated with Nmd3p. Furthermore, the dominant LSG1 mutant also traps a mutant Rpl10 protein that does not normally bind stably to the subunit. These results support the idea that Sqt1p loads Rpl10p onto the Nmd3p-bound subunit after export to the cytoplasm and that Rpl10p loading involves the GTPase Lsg1p.In eukaryotic cells, ribosomes are assembled in the nucleolus and are transported across the nuclear membrane through nuclear pore complexes to the cytoplasm, where they are activated for translation. Many rRNA processing events must coincide with the assembly of proteins onto nascent ribosomal subunits during their biogenesis (25,34,35). The nascent 60S particle is preassembled in the nucleolus from locally transcribed and processed rRNAs and ribosomal proteins imported from the cytoplasm. The assembly events are highly organized and require over 150 trans-acting factors, many of which reside solely in the nucleolus. Much work has been done in elucidating the various intermediate forms of the pre-60S particle during its maturation in the nucleolus and nucleoplasm (references 7, 8, and 34 and references therein). Most of the assembly factors are removed from the preassembled complex before it leaves the nucleolus, and the complement of trans-acting factors is further reduced in the nucleoplasm prior to export (2, 29).Nuclear export of the large subunit requires the adapter protein Nmd3p, which provides the nuclear export signal (NES) for the subunit (9, 15). 60S export also depends on the export receptor Crm1p, which recognizes the leucine-rich NES of Nmd3p (9,15). This export pathway is conserved in humans, where it has also been suggested that Nmd3p binds to 60S subunits in the nucleolus and may be required for their release into the nucleoplasm (17,32,33).NMD3 functionally interacts with the ribosomal protein RPL10. The interaction of NMD3 and RPL10 was first observed in a ge...

BACKGROUND: Current guidelines for managing Philadelphia-positive chronic myeloid leukemia include monitoring the expression of the BCR-ABL1 (breakpoint cluster region/c-abl oncogene 1, nonreceptor tyrosine kinase) fusion gene by quantitative reverse-transcription PCR (RT-qPCR). Our goal was to establish and validate reference panels to mitigate the interlaboratory imprecision of quantitative BCR-ABL1 measurements and to facilitate global standardization on the international scale (IS).

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