Visualizing maturation factor extraction from the nascent ribosome by the AAA-ATPase Drg1

Abstract

The AAA-ATPase Drg1 is a key factor in eukaryotic ribosome biogenesis that initiates cytoplasmic maturation of the large ribosomal subunit. Drg1 releases the shuttling maturation factor Rlp24 from pre-60S particles shortly after nuclear export, a strict requirement for downstream maturation. The molecular mechanism of release remained elusive. Here, we report a series of cryo-EM structures that captured the extraction of Rlp24 from pre-60S particles by Saccharomyces cerevisiae Drg1. These structures reveal that Arx1 and the eukaryote-specific rRNA expansion segment ES27 form a joint docking platform that positions Drg1 for efficient extraction of Rlp24 from the pre-ribosome. The tips of the Drg1 N domains thereby guide the Rlp24 C terminus into the central pore of the Drg1 hexamer, enabling extraction by a hand-over-hand translocation mechanism. Our results uncover substrate recognition and processing by Drg1 step by step and provide a comprehensive mechanistic picture of the conserved modus operandi of AAA-ATPases.

Structural dynamics of AAA + ATPase Drg1 and mechanism of benzo-diazaborine inhibition

“…Furthermore, Drg1 also contains a conserved D1-D2 linker, which again exhibits two translocating and non-translocating conformations (helix vs. loop), similar as p97 14 , 61 . Most recently, Prattes et al assembled a complex of pre-60S particle and Drg1 in vitro and reported a structure for the extraction of Rlp24 from pre-60S particle by Drg1 64 . They found that the C-terminal tail of Rlp24 is threaded into the central pore of Drg1, and translocated by the PLs of Drg1 via a hand-over-hand translocation mechanism.…”

“…Previously, it was reported that several nucleoporins of nuclear pore complex, including Nup116, could interact with Drg1, and Nup116 could facilitate the release of Rlp24 from pre-60S particles in vitro 32 . Most recently, it was found that Arx1 and ES27 on the pre-60S particle forms a docking platform to interact with the NTD of Drg1 to recruited it to the pre-60S particle 64 . Whether any of them is a bona fide adapter merits further investigation.…”

Targeting of client proteins to the VCP/p97/Cdc48 unfolding machine

“…The SDS22-PP1-I3 complex is very stable and needs to be dissociated by p97 to allow assembly of PP1 holoenzymes with activating subunits ( Weith et al, 2018 ; Cao et al, 2021 ). Conceptually, this process is reminiscent of ribosome biogenesis that involves the binding of a transient maturation factor, Rlp24, which needs to be extracted by the p97-related AAA protein Drg1 ( Prattes et al, 2022 ).…”

Principles of human pre-60Sbiogenesis

“…Traditionally, eukaryotic 60S assembly has been studied in the yeast system ((4) and references therein), (5)(6)(7)(8)(9)(10), which has offered several molecular snapshots of early nucleolar assembly intermediates from Saccharomyces cerevisiae (11)(12)(13). However, the mechanisms that drive early assembly forward remain unknown.…”