Coupled GTPase and remodelling ATPase activities form a checkpoint for ribosome export


Eukaryotic ribosomes are assembled by a complex pathway that extends from the nucleolus to the cytoplasm and is powered by many energy-consuming enzymes1,2,3. Nuclear export is a key, irreversible step in pre-ribosome maturation4,5,6,7,8, but mechanisms underlying the timely acquisition of export competence remain poorly understood. Here we show that a conserved Saccharomyces cerevisiae GTPase Nug2 (also known as Nog2, and as NGP-1, GNL2 or nucleostemin 2 in human9) has a key role in the timing of export competence. Nug2 binds the inter-subunit face of maturing, nucleoplasmic pre-60S particles, and the location clashes with the position of Nmd3, a key pre-60S export adaptor10. Nug2 and Nmd3 are not present on the same pre-60S particles, with Nug2 binding before Nmd3. Depletion of Nug2 causes premature Nmd3 binding to the pre-60S particles, whereas mutations in the G-domain of Nug2 block Nmd3 recruitment, resulting in severe 60S export defects. Two pre-60S remodelling factors, the Rea1 ATPase and its co-substrate Rsa4, are present on Nug2-associated particles, and both show synthetic lethal interactions with nug2 mutants. Release of Nug2 from pre-60S particles requires both its K+-dependent GTPase activity and the remodelling ATPase activity of Rea1. We conclude that Nug2 is a regulatory GTPase that monitors pre-60S maturation, with release from its placeholder site linked to recruitment of the nuclear export machinery.

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Figure 1: Nug2 binds to inter-subunit face of the pre-60S subunit clashing with export factor Nmd3.
Figure 2: K+-dependent GTPase activity of Nug2.
Figure 3: Nug2 release from pre-60S particles requires intrinsic K+-dependent GTPase and Rea1 ATPase activity.
Figure 4: Nug2 release from the pre-60S subunit is linked to Nmd3 recruitment.


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We thank M. Remacha, M. Fromont- Racine, A. W. Johnson, C. Dargemont, M. Seedorf and J. Warner for antibodies. We thank the GenePool at the University of Edinburgh for performing the MiSeq sequencing, and E. Petfalski for performing the initial crosslinking experiments. We thank E. Thomson for careful reading the manuscript. This work was supported by a postdoctoral fellowship from Alexander von Humboldt Foundation to Y.M., and by the Wellcome Trust to S.G. and D.T. (077248), and by grants from the Deutsche Forschungsgemeinschaft to E.H. (DFG Hu363/10-4).

Author information




Experiments were designed and the data were interpreted by Y.M. and E.H.; all experiments except CRAC analysis were performed by Y.M.; CRAC experiments and data analyses were performed by S.G. in collaboration with D.T.; M.T. constructed rea1 mutants and performed the in vitro release assay of rea1 mutants, the ctNug2 complementation assay and the immunodepletion assay; R.-G.M. developed the methods of the in vitro assay for nucleotide binding and GTPase activity measurement; the manuscript was written by Y.M. and E.H.; all authors discussed the results and commented on the manuscript.

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Correspondence to Ed Hurt.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Multiple sequence alignment of various Nug2 orthologues.

Multiple sequence alignment of YlqF (bacterial homologue of Nug2; Bacillus cereus), ctNug2, DmNug2 (Drosophila melanogaster), DrNug2 (Danio rerio), HsNug2 (Homo sapiens), KlNug2 (Kluyveromyces lactis), MmNug2 (Mus musculus), ScNug2 (S. cerevisiae), SpNug2 (S. pombe), XlNug2 (Xenopus laevis) and YlNug2 (Yarrowia lipolytica), using T-Coffee multiple sequence alignment ( and Jalview. Indicated above the alignment are the different Nug2 domains including the N, G, and C domains and the C-terminal extension. Moreover, the DAR, G1, G3, G4 motifs, point mutation sites (in red) and truncated site of ctNUG2-510 amino acids (truncation of the non-conserved C-terminal extension; red line) are indicated.

Extended Data Figure 2 Nug2 and Nmd3 are not found on the same pre-60S particles.

Indicated different TAP-tagged bait proteins were affinity purified from yeast wild-type cells. The final eluates were analysed by SDS–PAGE and Coomassie staining (top), and by western blotting using the indicated antibody (bottom). Asterisks mark the position of each bait protein. Rea1 has been identified by mass spectrometry. All affinity purifications and western analyses were performed at least twice, yielding highly reproducible data sets.

Extended Data Figure 3 ctNug2 can complement the lethal phenotype of a nug2Δ null mutant.

Serial dilutions of the yeast Nug2 shuffle strain (MAT a , ade2, ade3, his3, ura3, leu2, trp1, nug2::kanMX4, pHT4467-NUG2) transformed with empty plasmid, yeast ScNUG2, ctNUG2 or ctNUG2-510 (truncation of the non-conserved C-terminal extension; see Extended Data Fig. 1) under the control of the constitutive ADH1 promoter in a single-copy-number (YCplac111) or multi-copy-number (pRS425) plasmid (see Supplementary Table 2) were spotted on SDC−Leu (loading control) and SDC plates containing 5-FOA at indicated temperatures for 6 days. Note that ctNug2 only partially complements the nug2 null mutant.

Extended Data Figure 4 Mutations in ATP-binding or MIDAS domain of Rea1 inhibit the release of Rsa4 and Nug2 from the pre-60S particle.

a, b, Wild-type REA1 and the rea1 mutants mapping in the ATP-binding site of the AAA2 domain (Lys659Ala) or in the MIDAS domain (DAA)21 were N-terminally tagged with eGFP and expressed in a REA1 shuffle strain (a) or overexpressed under the control of the inducible GAL1-10 promoter in REA1 wild-type strain DS1-2b (b). Transformants were spotted in tenfold serial dilution steps on the indicated plates and incubated at 30 °C for 3 days. Both of the rea1 mutant alleles do not complement the rea1 null strain (a, SDC + 5-FOA) and cause a dominant-negative phenotype after overexpression by replacing endogenous Rea1 (b, galactose). c, Overnight pre-cultures were grown in SRC−Leu to prevent plasmid loss, followed by shifting cells (A600 nm  = 0.75) to galactose medium (YPG) for 7 h. Rix1 particles, which were affinity purified from a Rix1–TAP, RpL3–Flag strain containing either endogenous wild-type or overexpressed wild-type eGFP–Rea1, eGFP–Rea1(DAA) and eGFP–Rea1(Lys659Ala), were incubated with or without 4 mM ATP in KCl buffer, before the different in vitro matured pre-60S particles were re-isolated by affinity-purification via the RpL3–Flag on Flag beads. Subsequently, the in vitro matured pre-60S particles (eluates) were analysed by SDS–PAGE and Coomassie staining. Relevant bands are indicated on the right. Note that in the case of the rea1 mutants, the release of Nug2, Rsa4 but also Rea1 and the Rix1 complex is significantly inhibited. All in vitro assays were performed at least twice, yielding highly reproducible data sets.

Extended Data Figure 5 Nug2 depletion assay using the auxin-inducible degron system.

a, Growth of Nug2 auxin degron strains (sAid–Nug2–sAid) in the PADH-OsTIR1 background on YPD plates with or without 500 μM auxin (IAA). The cell growth of sAid–Nug2–sAid strain was inhibited by the addition of auxin. b, Western blotting of sAid–Nug2–sAid after auxin treatment. The depletion of sAid–Nug2–sAid occurred within about 30 min of auxin addition.

Extended Data Table 1 Yeast strains used in this study
Extended Data Table 2 Plasmids used in this study
Extended Data Table 3 Adapters used for the CRAC experiments

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Matsuo, Y., Granneman, S., Thoms, M. et al. Coupled GTPase and remodelling ATPase activities form a checkpoint for ribosome export. Nature 505, 112–116 (2014).

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