Coupled GTPase and remodeling 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 1 - 3 . Nuclear export is a key, irreversible step in pre-ribosome maturation 4 - 8 , but mechanisms underlying the timely acquisition of export competence remain poorly understood. Here we show that a conserved GTPase Nug2/Nog2 (called NGP-1, Gnl2 or nucleostemin 2 in human 9 ) plays 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 10 . Nug2 and Nmd3 are not present on the same pre-60S particles, with Nug2 binding prior to 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 remodeling 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 remodeling 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.

...0410462 6011 Nature Nature Nature 0028-0836 1476-4687 24240281 3880858 10.1038/nature12731 EMS55090 Article Coupled GTPase and remodeling ATPase activities form a checkpoint for ribosome export Matsuo Yoshitaka 1 Granneman Sander 2 3 Thoms Matthias 1 Manikas Rizos-Georgios 1 Tollervey David 2 Hurt Ed 1 1 Biochemie-Zentrum der Universitt Heidelberg, Im Neuenheimer Feld 328, Heidelberg D-69120, Germany 2 Wellcome Trust Centre for Cell Biology, The University of Edinburgh, Edinburgh UK 3 Centre for Synthetic and Systems Biology (SynthSys), University of Edinburgh, Edinburgh, UK Correspondence and requests for materials should be addressed to E.H. ( ed.hurt@bzh.uni.heidelberg.de ). Author Contributions Experiments were designed and the data were interpreted by Y.M. and E.H.; all experiments excepting 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, ct Nug2 complementation assay and immunodepletion assay; G.M. developed the methods of 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. 17 10 2013 17 11 2013 2 1 2014 02 7 2014 505 7481 112 116 Users may view, print, copy, download and text and data- mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms Eukaryotic ribosomes are assembled by a complex pathway that extends from the nucleolus to the cytoplasm and is powered by many energy-consuming enzymes 1 - 3 . Nuclear export is a key, irreversible step in pre-ribosome maturation 4 - 8 , but mechanisms underlying the timely acquisition of export competence remain poorly understood. Here we show that a conserved GTPase Nug2/Nog2 (called NGP-1, Gnl2 or nucleostemin 2 in human 9 ) plays 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 10 . Nug2 and Nmd3 are not present on the same pre-60S particles, with Nug2 binding prior to 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 remodeling 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 remodeling 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. The

... for Nmd3 becomes accessible on the pre-60S subunit, which further triggers Crm1 and RanGTP recruitment to generate nuclear export competence. Thus, our data indicate coordination between a remodeling AAA-ATPase and a conformation-sensing GTPase. The human Nug2 orthologue Gnl2 is highly expressed in proliferating cells including cancer cells and involved in the control of cell cycle progression 30 . The discovery of the role of Nug2 during surveillance of ribosome biogenesis may help reveal the

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Gene: GNL2 - guanine nucleotide binding protein-like 2 (nucleolar) - (NCBI)

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