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Nucleotide independant as well as ATP-hydrolysis driven steps of Rea1 Linker remodelling drive the removal of assembly factors from pre-ribosomal particles: PhoX cross-linking of Rea1 in presence of ATPgS, AMP-PNP or Apo state.

ABSTRACT: The ribosome maturation factor Rea1 (or Midasin) catalyses the removal of assembly factors from large ribosomal subunit precursors and to promotes their export from the nucleus to the cytosol. Rea1 consists of nearly 5000 amino-acid residues and belongs to the AAA+ protein family. It consists of a ring of six AAA+ domains from which the ≈ 1700 amino-acid residue linker emerges that is subdivided into stem, middle and top domains. A flexible and unstructured D/E rich region connects the linker top to a MIDAS (metal ion dependent adhesion site) domain, which is able to bind the assembly factor substrates. Despite its key importance for ribosome maturation, the Rea1 mechanism driving assembly factor removal by Rea1 is still poorly understood. Here we demonstrate that the Rea1 linker 30 is essential for assembly factor removal. It rotates and swings towards the AAA+ ring following a complex remodelling scheme involving nucleotide independent as well as nucleotide dependent steps. ATP-hydrolysis is required to engage the linker with the AAA+ ring and ultimately with the AAA+ ring docked MIDAS domain. The interaction between the linker top and the MIDAS domain allows direct force transmission for assembly factor removal. To evaluate the conformational changes and spatial proximities in presence or absence of nucleotides we carried out XL-MS experiments using PhoX on purified Rea1 in presence of ATPgS, AMP-PNP or in Apo state (in XL triplicates each).

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Hugo Gizardin-Fredon

LAB HEAD: Sarah Cianferani

PROVIDER: PXD053636 | Pride | 2024-07-29

REPOSITORIES: Pride

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