Targeted proteomics reveals compositional dynamics of 60S pre‐ribosomes after nuclear export

Abstract

A combination of genetic trapping, affinity-capture and selected reaction monitoring mass spectrometry is used to characterize the dynamic proteome of pre-60S ribosomal particles after nuclear export. These results identify Bud20 as a novel shuttling factor for pre-60S export.

Mechanistic insight into eukaryotic 60S ribosomal subunit biogenesis by cryo-electron microscopy

“…the Yvh1-dependent exchange of the stalk protein uL10 for the placeholder Mrt4 leads to formation of the P-stalk (Kemmler et al 2009;Lo et al 2009;Rodríguez-Mateos et al 2009). Even though Yvh1 was initially found to be a shuttling protein (Kemmler et al 2009;Lo et al 2009), raising the possibility that it might associate with pre-60S subunits in the nucleus, more recent data obtained by innovative mass spectrometry techniques show that Yvh1 binding is cytoplasmic and occurs downstream from the Drg1-dependent release of Rlp24 (Altvater et al 2012). The final steps of maturation, which include the release of Nmd3 and eIF6 from the subunit interface side of the 60S particle, depend on the completion of both Arx1 release and P-stalk assembly (Lebreton et al 2006b;Lo et al 2010).…”

“…While small molecules can diffuse through the pore freely, large macromolecules need to recruit export factors that help them pass through a meshwork formed by FG-rich repeats of nucleoporins that fill the channel at the center of the NPC (Grossman et al 2012). The nascent 60S particle acquires export competence by recruiting its full set of nuclear export factors, which include Nmd3, Mex67-Mtr2, Arx1, Bud20, and Ecm1 in yeast (Ho et al 2000b;Gadal et al 2001;Bradatsch et al 2007;Yao et al 2007Yao et al , 2010Hung et al 2008;Altvater et al 2012;Bassler et al 2012). The requirement for a large number of export factors may originate from the large size of the pre-60S subunit, which at more than 2 MDa molecular weight is among the largest cargoes transported through the NPC (Knockenhauer and Schwartz 2016).…”

“…The mechanism by which Ecm1 and Bud20 contribute to nuclear export has not been fully clarified yet. Bud20 has been reported to bind to FG-rich nucleoporins directly (Altvater et al 2012) or to harbor a NES-like element (Bassler et al 2012). However, because Bud20 does not recruit Crm1 in vitro (Altvater et al 2012), this NES-like element may not be functionally active.…”

“…Bud20 has been reported to bind to FG-rich nucleoporins directly (Altvater et al 2012) or to harbor a NES-like element (Bassler et al 2012). However, because Bud20 does not recruit Crm1 in vitro (Altvater et al 2012), this NES-like element may not be functionally active. Besides these export factors, the protein Gle2 ensures efficient nuclear export of the pre-60S particle by engaging in non-FG interactions with the nucleoporin Nup116 (Occhipinti et al 2013).…”

Eukaryotic Ribosome Assembly and Nuclear Export

“…Although Nmd3 is the only known NES-containing adaptor, additional export adaptors are also required to shield the large and highly charged surface area of the pre-60S particles during translocation through the NPC. These additional factors include Ecm1, Bud20, and Arx1, which share affinity for the FG-repeats of nucleoporins as well as common genetic interactions with other export factors Bassler et al, 2012;Altvater et al, 2012). However, a mutant nmd3 lacking the NES is unable to complement the lethality caused by nmd3D (Ho et al, 2000;Gadal et al, 2001b).…”