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

Abstract

Eukaryotic ribosomes are assembled by a complex pathway that extends from the nucleolus to the cytoplasm and is powered by many energy-consuming enzymes. Nuclear export is a key, irreversible step in pre-ribosome maturation, 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 human) 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 adaptor. 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.

Figure 1. Nug2 binds to inter-subunit face of the pre-60S subunit clashing with export factor Nmd3

(a) CRAC analyses of Nug2 and Nmd3 (performed twice; only sites were considered that were reproducibly found in both datasets). Total number of hits was plotted against the relative location along the rDNA. (b) Yeast 3-hybrid revealing interaction between Nug2 and identified 25S rRNA fragments. Negative control, empty vector and H25. (c) Nug2 (yellow) and Nmd3 binding sites (green) identified by CRAC and highlighted in the indicated 25S rRNA. (d, e) Mapping of CRAC Nug2 (yellow) and Nmd3 (green) binding sites on the 60S structure (PDB: 3O5H). (f) Overlapping binding sites (red) of Nug2 (yellow) and Nmd3 (green).

Figure 2. K⁺-dependent GTPase activity of Nug2

(a) Domain organization of Nug2. (b). Complementation of nug2Δ cells by NUG2, nug2K328R and nug2G369A on YPD plates. (c) Repression (+doxycycline) and overexpression (−doxycycline) of NUG2, nug2K328R and nug2G369A in NUG2 cells. (d) Polysomal (10mM MgCl₂; upper panel) and ribosomal profiles (1.5mM MgCl₂; lower panel) of NUG2, nug2K328R and nug2G369A cells analyzed by sucrose gradient centrifugation. Western analysis of gradient fractions using antibodies against Nug2 and RpL35 (upper panel) (e) Subcellular distribution of RpL25-GFP and RpS3-GFP in NUG2 and nug2 mutant cells analyzed by fluorescence microscopy. (f) GTPase activity of purified ctNug2 (SDS-PAGE; left panel) analyzed by thin-layer chromatography/autoradiography (middle panel). Ratio of hydrolyzed phosphate/total GTP plotted against time (right panel). (g) Binding of MANT-GTP (left panel) and MANT-GDP (right panel) to purified wild-type and mutant ctNug2. GTPase and binding assays were performed twice yielding highly reproducible datasets.

Figure 3. Nug2 release from pre-60S particles requires intrinsic K⁺-dependent GTPase and Rea1 ATPase activity

(a) Synthetic lethality (sl) between alleles rsa4-1 or rea1-DTS and nug2K328R revealed by growth on 5-FOA. (b-e) ATP-dependent release of Rsa4 and Nug2 from purified pre-60S particles. Scheme of the release assay (b) and experimental analyses (c-e). Affinity-purified Rix1-particles carrying wild-type or mutant Nug2 were incubated with ATP or GTP in NaCl or KCl buffer, before matured pre-60S particles were re-isolated via RpL3-Flag affinity-purification. Final eluates were analyzed by SDS-PAGE and Coomassie staining (upper panel; indicated bands were identified by mass spectrometry) and Western blotting using the indicated antibodies (lower panel). All in vitro assays were performed at least twice with highly reproducible datasets.

Figure 4. Nug2 release from the pre-60S subunit is linked to Nmd3 recruitment

(a) Affinity-purification of the indicated TAP-tagged pre-60S factors from NUG2 or nug2K328R mutant cells. (*) position of Rei1 identified by mass spectrometry. (b) Affinity-purification of Arx1-TAP from NUG2, nug2K328R and nug2G369A cells. (c) Affinity-purification of Rix1-TAP from sAid-Nug2-sAid degron strain after time-dependent auxin treatment. (d) Affinity-purification of Nug2-TAP particles with (lane 1) or without (lane 2) subsequent Rix1-Flag immunodepletion. (a-d) SDS-PAGE and Coomassie staining (upper panel) and Western blotting using the indicated antibodies (lower panel). Protein bands indicated were identified by mass spectrometry. All affinity-purifications were performed at least twice, yielding highly reproducible datasets. (e) Model of pre-60S subunit maturation starting from the Rix1-particle with final Nmd3-Crm1-RanGTP recruitment.