Error-free translation of the genetic code by the ribosome is critical for proteome homeostasis and cellular function. This essential task necessitates that only correctly assembled ribosomal subunits are committed for translation. Eukaryotic ribosome assembly is initiated by production of pre-rRNA in the nucleolus that is driven by RNA polymerase I (Peña et al., 2017; Kressler et al., 2017). The emerging pre-rRNA associates with small subunit (40S)-specific r-proteins, snoRNAs, U3snoRNP, numerous U3 proteins (UTPs) and assembly factors to form a pre-40S. Endonucleolytic cleavage releases the earliest pre-40S, permitting the remaining growing pre-rRNA to recruit large subunit (60S)-specific r-proteins and assembly factors to form a pre-60S. On their way through the nucleoplasm, pre-ribosomal particles interact with >200 assembly factors, including >40 energy-consuming AAA-ATPases, ABC-ATPases, GTPases and ATP-dependent RNA helicases. Elucidating their order of action and the mechanisms that co-ordinate their activities during pre-ribosome maturation is an important challenge.

Export-competent pre-ribosomes are transported through nuclear pore complexes (NPCs) into the cytoplasm, where they undergo maturation and proofreading before acquiring translation competence (Nerurkar et al., 2015). These steps include the release of assembly factors, transport receptors, pre-rRNA processing steps and the incorporation of remaining r-proteins that are critical for ribosome function. Cytoplasmic maturation of an exported pre-60S is initiated by the AAA-ATPase Drg1, which directly binds to and evicts the placeholder ribosomal-like protein Rlp24 from the pre-60S (Pertschy et al., 2007; Kappel et al., 2012; Kressler et al., 2012). This step is a prerequisite for the release of two GTPases (Nog1 and Nug1), the assembly factor Nsa2, the subunit anti-association factor Tif6, the ribosomal-like protein Mrt4 and the export factors Mex67-Mtr2, Bud20, Arx1 and Nmd3 (Loibl et al., 2014; Zisser et al., 2018). Following Rlp24 release, the 60S maturation pathway, via a yet unknown mechanism, bifurcates to proofread the polypeptide exit tunnel (PET) and to assemble the ribosomal stalk (Lo et al., 2010). PET maturation is accomplished through the release of Arx1, an assembly factor that covers the tunnel and prevents the premature loading of the protein-folding chaperone machinery (Bradatsch et al., 2012; Greber et al., 2012).

Arx1 release from the pre-60S requires the cytoplasmic zinc-finger protein Rei1, the DnaJ domain-containing protein Jjj1 and Ssa1/Ssa2 (Hsp70) ATPase activity (Meyer et al., 2007; Meyer et al., 2010). Cryo-electron microscopy (cryo-EM) studies have revealed that Rei1 inserts its C-terminal tail (Rei1-CTT) into the PET (Greber et al., 2016). Failure to insert the Rei1-CTT into the tunnel impairs Arx1 release, and blocks subsequent pre-60S maturation (Greber et al., 2016). Cryo-EM analyses of a nuclear pre-60S revealed that the C-terminal tail of the GTPase Nog1 (Nog1-CTT) intertwines around Rlp24, makes contact with Arx1 and inserts its end into the PET (Wu et al., 2016). For Rei1-CTT to gain access to the PET, the Nog1-CTT end must be extracted. How these events are orchestrated during cytoplasmic maturation is unknown.

Another functional center whose assembly is completed in the cytoplasm is the 60S ribosomal stalk. The stalk is built from a single copy of ribosomal proteins uL10 (Rpp0) and two heterodimers of P1 (Rpp1) and P2 (Rpp2), and plays an essential role by recruiting and activating translation elongation factors. On a mature 60S subunit, the stalk is anchored through the interaction of uL10 with rRNA and uL11 (Rpl12) (Ben-Shem et al., 2011). During nuclear 60S assembly, the ribosomal-like protein Mrt4 functions as a placeholder for uL10 (Lo et al., 2009; Kemmler et al., 2009). Mrt4 removal from the pre-60S is triggered by the release factor Yvh1 (Kemmler et al., 2009; Lo et al., 2009). uL10 can be loaded onto the pre-60S to assemble the stalk only after Mrt4 is released. How stalk assembly is coordinated with other cytoplasmic maturation steps remains unclear.

Removal of the placeholders Rlp24, Arx1 and Mrt4 from the pre-60S is an essential prerequisite for PET maturation and quality control, polypeptidyl transferase center (PTC) maturation, stalk assembly and the incorporation of the remaining r-proteins. How these spatially distant events on the pre-60S are orchestrated remains unknown. By employing quantitative mass spectrometry, genetic and cell-biological approaches, we reveal that the ATPase Drg1 and the GTPase Nog1 license these events, and ensure that only correctly assembled 60S subunits enter translation.

The nuclear localized GTPase Nog1 co-enriches with a late pre-60S that contains the export factors Nmd3, Bud20, Mex67-Mtr2 and Arx1 (Jensen et al., 2003; Kallstrom et al., 2003). In wild-type (WT) cells, Nog1 is not detected on a cytoplasmic pre-60S purified through tandem-affinity purification (TAP) of Lsg1 (Kressler et al., 2008; Altvater et al., 2012). However, Nog1 mislocalizes to the cytoplasm, and accumulates on Lsg1-TAP particles upon impairment of the cytoplasmic AAA-ATPase Drg1 or upon treating yeast cells with the Drg1-inhibitor diazaborine (DIA) (Pertschy et al., 2007; Loibl et al., 2014). These data show that Nog1 travels to the cytoplasm, where it is released in a Drg1-dependent manner and rapidly recycled back to the nucleus. How and exactly when Nog1 is released from the pre-60S in the cytoplasm is unclear.

Nog1^DN accumulates on a cytoplasmic pre-60S particle

The Nog1 G-domain exhibits characteristic G1–G5 motifs (Figure 1A, upper panel) suggesting that GTP-binding or hydrolysis of Nog1, like that of other GTPases such as Nug2/Nog2 and Lsg1, might regulate interactions between Nog1 and the pre-60S particle (Hedges et al., 2005; Matsuo et al., 2014). Dominant-negative mutations have been described within the G1 motif of Lsg1(K349N/R/T) (Hedges et al., 2005) and the G3 motif of Nug2 (G369A) (Bourne et al., 1991; Hedges et al., 2005; Matsuo et al., 2014) that impair the release of these GTPases from a pre-60S. A G224A mutation in the G3 motif of human Nog1, which presumably blocks GTP hydrolysis, was shown to be dominant negative in mammalian cells, and induced pre-rRNA processing and assembly defects (Lapik et al., 2007).

Figure 1

Download asset Open asset

We investigated whether the G-domain contributes to Nog1 release from the pre-60S in the cytoplasm. To this end, the orthologous mutation in the G3 motif (G223A) of yeast Nog1 (Figure 1A), hereafter termed Nog1^DN, was transformed into a Nog1 shuffle strain, wherein the NOG1 gene was disrupted but the viability of the yeast cells was maintained through a centromeric plasmid containing a WT copy of NOG1. We did not obtain transformants for the Nog1^DN mutant. To demonstrate dominant-negative behavior, we placed the Nog1^DN mutant under the control of an inducible GAL1 promoter, and transformed this plasmid into WT yeast cells. On glucose-containing medium, where Nog1^DN expression is repressed, the resulting transformants grew similar to WT. By contrast, expression of Nog1^DN in galactose-containing medium was lethal to yeast cells (Figure 1B), confirming the dominant-negative behavior of the G223A mutation.

Nog1 is recruited to the pre-60S in the nucleolus (Kressler et al., 2008; Altvater et al., 2012), and is released from the particle in the cytoplasm (Pertschy et al., 2007; Lo et al., 2010; Altvater et al., 2012). We investigated whether the Nog1^DN mutant was released from the pre-60S in the cytoplasm. For this, we isolated the Lsg1-TAP particle after inducing expression of either Nog1 or the Nog1^DN mutant allele for 2.5 hr (Figure 1C). Western analyses revealed that Nog1^DN mutant protein, but not Nog1, accumulated on the Lsg1-TAP particle (Figure 1C). Whole cell extracts (WCE) revealed similar Nog1 and Nog1^DN protein levels (Figure 1C), suggesting that Nog1^DN co-enrichment with Lsg1-TAP is not due to altered expression of the mutant protein. Moreover, the Nog1^DN-GFP fusion showed an increase in cytoplasmic signal, supporting the notion that Nog1^DN release from the pre-60S in the cytoplasm is impaired (Figure 1D). Although a nuclear signal of Nog1^DN-GFP is observed in these cells, this mutant did not efficiently co-enrich with Ssf1-TAP under the same conditions (Figure 1C), possibly owing to blockage of downstream cytoplasmic maturation steps that indirectly impair early assembly steps (see later). We conclude that a functional G-domain is essential to evict Nog1 from the pre-60S in the cytoplasm.

Nog1^DN impairs cytoplasmic maturation of the pre-60S particle

We investigated the consequences of impaired Nog1^DN release on the composition of the cytoplasmic Lsg1-TAP particle by Sequential Window Acquisition of all THeoretical fragment ion spectra mass spectrometry, also termed SWATH-MS. SWATH-MS is a mass spectrometry approach that combines data-independent acquisition with a peptide-centric data query strategy (Gillet et al., 2012). In contrast to selected reaction monitoring mass spectrometry (SRM-MS) (Picotti and Aebersold, 2012), SWATH-MS can be extended to the analysis of any peptide and protein of interest post-acquisition, while maintaining optimal consistency of quantification in pull-down samples (Collins et al., 2013; Lambert et al., 2013).

We interrogated quantitatively the protein composition of four well-characterized pre-60S particles representing different maturation stages (Nissan et al., 2002): Ssf1-TAP, an early nucleolar particle; Rix1-TAP, a nucleoplasmic particle; Arx1-TAP, a particle loaded with nuclear export factors; and Lsg1-TAP, an exclusively cytoplasmic pre-60S. The data were analyzed using OpenSWATH software (Röst et al., 2014), and accuracy was compared with that of SRM-MS based analyses (Altvater et al., 2012). We found that the proteomic heat map obtained using SWATH-MS was in agreement with that generated through SRM-MS (Altvater et al., 2012) and Western analyses (Figure 2—figure supplement 1). In contrast to SRM-MS, SWATH-MS permitted the quantitation of the approximate residence time of nearly all assembly factors along the 60S maturation pathway (Figure 2).

Figure 2 with 1 supplement see all

Download asset Open asset

We correlated the protein heat map with reported pre-ribosome cryo-EM structures (Sanghai et al., 2018; Kater et al., 2017; Wu et al., 2016; Barrio-Garcia et al., 2016; Ma et al., 2017; Malyutin et al., 2017; Greber et al., 2016). These analyses allowed organization of assembly factors into different clusters on a maturing pre-60S. For example, the early nucleolar Ssf1-Rrp14-Rrp15-Mak11 cluster, the nuclear ITS2 factors Nop15-Rlp7-Cic1-Nop7, the 5S RNP-associated Rrs1-Rpf2, the Rix1-Rea1 machinery and the Drg1-dependent factors Rlp24-Nog1-Nsa2-Bud20 appear to undergo coordinated and grouped release. Our analyses also revealed temporal association of assembly factors for which structural information in the context of the pre-60S is lacking. These factors include nucleolar proteins and the uncharacterized nuclear-localized Tma16 (Figure 2). We conclude that SWATH-MS is a reliable tool to quantify the protein contents of pre-ribosomal particles.

Next, we quantified and compared the protein contents of genetically trapped cytoplasmic pre-60S particles isolated from yeast cells expressing either the dominant-negative Drg1-E617Q (Lsg1-TAP:Drg1^DN) (Altvater et al., 2012) or the Nog1^DN (Lsg1-TAP:Nog1^DN) mutant. We focused on those assembly factors that are known to participate in pre-60S cytoplasmic maturation (Figure 3A). In agreement with previous studies (Pertschy et al., 2007; Lo et al., 2010; Altvater et al., 2012), a Drg1^DN:Lsg1-TAP particle accumulated Rlp24, Bud20, Nog1, Mrt4, Nmd3 and Tif6 (Figure 3A and Figure 3B). In addition, the cytoplasmic chaperone Sqt1, which loads uL16 onto the pre-60S (Pausch et al., 2015), and the uncharacterized ribosome-associated factor Tma16 (Fleischer et al., 2006) accumulated on the Drg1^DN:Lsg1-TAP particle (Figure 3B). Like the Drg1^DN:Lsg1-TAP particle, the Lsg1-TAP:Nog1^DN particle accumulated Mrt4, Sqt1, Tma16, Nmd3 and Tif6 (Figure 3A and Figure 3B). However, unlike the Drg1^DN:Lsg1-TAP particle, Rlp24 and its interaction partner Bud20 did not accumulate on the Lsg1-TAP:Nog1^DN particle (Figure 3A and Figure 3B). Both Drg1^DN and Nog1^DN-trapped Lsg1-TAP particles failed to recruit Yvh1. Although Rei1 recruitment to the Lsg1-TAP:Drg1^DN particle was impaired, Nog1^DN-trapped particles recruited Rei1 (Figure 3C). By contrast, Reh1, which functionally overlaps with Rei1 (Parnell and Bass, 2009), accumulated on both Drg1^DN and Nog1^DN-trapped Lsg1-TAP particles (Figure 3B). The relative co-enrichments of assembly factors on the Lsg1-TAP:Nog1^DN particle monitored by SWATH-MS were in agreement with Western analyses (Figure 3C). These data suggest that Nog1^DN-expressing cells are impaired in a subset of events along the 60S cytoplasmic maturation pathway.

Figure 3

Download asset Open asset

Nog1^DN does not hinder initiation of cytoplasmic maturation

Cryo-EM studies have revealed that different domains of the assembly factor Nog1 (NTD, G-domain and CTT) meander from the peptidyl transferase center (PTC) to the PET, and contact assembly factors on the pre-60S (Wu et al., 2016). The Nog1-NTD interacts with the assembly factor Nsa2, whereas the Nog1-G domain contacts the ribosomal-like protein Mrt4, and the Nog1-CTT intertwines around Rlp24 and contacts Arx1 (Figure 1A). Although Nog1, Nsa2, Arx1, Bud20, Mrt4 and Rlp24 are already recruited to the pre-60S during nucleolar/nucleoplasmic maturation, this cluster of assembly factors (Figure 1A) is evicted only in the cytoplasm (Figure 3A) (Pertschy et al., 2007; Lo et al., 2010; Altvater et al., 2012).

To dissect the impact of impaired Nog1^DN release from the pre-60S on the cytoplasmic maturation pathway, we employed a cell-biological approach. Release of the ribosomal-like-protein Rlp24 from the pre-60S initiates the cytoplasmic maturation cascade (Figure 3A) (Pertschy et al., 2007; Lo et al., 2010). On the pre-60S, Rlp24 directly contacts Bud20, and then intertwines around the Nog1-CTT, forming a Nog1-CTT:Rlp24 complex (Figure 4A). The last 40 residues of the Rlp24 C-terminal tail (155–190), which remain unresolved in cryo-EM structures (Wu et al., 2016; Zhou et al., 2019), recruit and activate the AAA-ATPase Drg1 to extract Rlp24 from the pre-60S (Pertschy et al., 2007; Lo et al., 2010; Kappel et al., 2012; Altvater et al., 2012). Given the intimate interactions between Nog1-CTT and Rlp24 on the pre-60S (Wu et al., 2016) (Figure 4A), we wondered whether Nog1^DN hinders Drg1-mediated Rlp24 release. As judged by immunofluorescence, Rlp24-TAP did not mislocalize to the cytoplasm upon Nog1^DN expression (Figure 4B), suggesting that Nog1^DN accumulation on a cytoplasmic pre-60S did not interfere with the release and recycling of Rlp24 back to the nucleus. These data are consistent with those from SWATH-MS analyses, which show that Rlp24 does not accumulate on the Lsg1-TAP:Nog1^DN particle (Figure 3B). This finding is in agreement with previous studies which showed that Drg1-ATPase activity is necessary and sufficient to release Rlp24 from the pre-60S (Kappel et al., 2012). The presence of Nog1^DN on the pre-60S did not interfere with recruitment of r-protein eL24 (yeast Rpl24), as judged by Western analyses (Figure 3C). Cell-biological studies indicate that the release of the Rlp24-interacting assembly factor Bud20 from the pre-60S is also not impaired in Nog1^DN-expressing cells (Figure 4C). We conclude that Nog1^DN does not disturb initiation of the pre-60S cytoplasmic maturation cascade.

Figure 4

Download asset Open asset

While the Lsg1-TAP:Drg1^DN particle accumulated Rlp24, Drg1^DN, Nog1 and another GTPase Nug1 (Altvater et al., 2012), the Lsg1-TAP:Nog1^DN particle accumulated only Nog1^DN, and not Rlp24, Drg1 or Nug1 (Figure 3B and Figure 4C). On the basis of these data, we conclude that Nog1^DN eviction from the pre-60S occurs after Drg1-mediated Rlp24 release.

Nog1^DN does not impair PET maturation

We investigated the impact of Nog1^DN expression on PET maturation and quality control (Figure 3A). During nuclear assembly of the pre-60S, the aminopeptidase fold of Arx1 covers the PET (Bradatsch et al., 2012; Greber et al., 2012). Arx1 eviction from the pre-60S is triggered in the cytoplasm by Rei1 (Meyer et al., 2010). Rei1 is recruited to the pre-60S through its N-terminal domain (Lebreton et al., 2006), whereas its C-terminal tail-end (Rei-CTT) is inserted into the PET (Greber et al., 2016). Rei1 recruitment to the pre-60S requires Drg1-mediated Rlp24 release (Figure 5A) (Lo et al., 2010; Altvater et al., 2012). We found that Nog1^DN expression did not interfere with Rei1 recruitment (Figure 3B and Figure 3C). However, co-expression of Nog1^DN and Drg1^DN impaired Rei1 recruitment to the pre-60S (Figure 5A). Accordingly, Nog1^DN-expressing cells when treated with the Drg1-inhibitor diazaborine (DIA) mislocalized Arx1-GFP to the cytoplasm in these cells (Figure 5B). Cryo-EM studies indicate that the Rei1-binding site on the pre-60S clashes with the Nog1-CTT:Rlp24 complex (Figure 5C) (Zhou et al., 2019), providing an explanation as to why PET maturation is inhibited in Drg1^DN-expressing (or DIA-treated) cells. All of these data show that Rlp24 extraction from the Nog1-CTT is critical to recruit Rei1 to the pre-60S in order to initiate PET maturation.

Figure 5

Download asset Open asset

Nog1^DN-expressing cells did not mislocalize Arx1-GFP into the cytoplasm (Figure 5D), suggesting that Nog1^DN presence on the pre-60S does not interfere with the ability of Rei1-CTT to probe the PET. Failure to insert Rei1-CTT into the PET by attaching a bulky domain (rei1-TAP) blocks Arx1 release and progression of cytoplasmic maturation, suggesting that PET proofreading represents a quality control check point (Greber et al., 2016). Consistent with this, Nog1^DN expression in a rei1-TAP mutant (in which Rei1-CTT function in probing the PET is compromised) mislocalized Arx1-GFP to the cytoplasm (Figure 5D). Before Rei1-CTT can probe PET integrity, the terminal end of Nog1-CTT needs to be extracted from the PET. Given that Rei1 recruitment and PET probing is not impaired in Nog1^DN-expressing cells, we suggest that Nog1^DN-CTT has been removed from the PET, and this does not require a functional Nog1-G3 domain. We conclude that the presence of Nog1^DN on the pre-60S does not hinder Rei1 recruitment or the insertion of the Rei1-CTT into the PET.

The Nog1-CTT:Rlp24 complex negatively regulates PET maturation

Nog1-CTT intertwines around the helical segments of Rlp24, then contacts Arx1 and finally inserts its terminal end into the PET (Figure 1A). We investigated which regions of Nog1-CTT contribute to pre-60S cytoplasmic maturation (Figure 6A and Figure 6B). We found that a Nog1 mutant lacking the entire CTT, including the Rlp24-interacting region, Nog1^1–426, was lethal in yeast (Figure 6B). Overexpression of a Nog1 mutant lacking the Rlp24-interacting region, Nog1^∆427–536, severely impaired the growth of yeast in a dominant-negative manner (Figure 6C). However, unlike the Nog1^DN mutant, Nog1^∆427–536-expressing cells did not mislocalize Mrt4-GFP and Tif6-GFP to the cytoplasm (Figure 6C). Likewise, a Rlp24 mutant lacking Nog1-interacting helices, Rlp24^∆91–105, also impaired yeast growth in a dominant-negative manner (Figure 6A and Figure 6C). Expression of the Rlp24^1–146 mutant (Figure 6A) that lacks the Drg1-binding platform, and therefore cannot be extracted from pre-60S, mislocalized Arx1-GFP, Mrt4-GFP and Tif6-GFP to the cytoplasm (Figure 6C) (Lo et al., 2010). However, Rlp24^∆91–105-expressing cells did not mislocalize these factors to the cytoplasm (Figure 6C). Given that Nog1 and Rlp24 rely on each other for their recruitment to the pre-60S (Saveanu et al., 2003), the toxicity of Nog1^∆427–536 and Rlp24^∆91–105 mutants probably reflects a failure to form a Nog1-CTT:Rlp24 complex on the pre-60S during early nuclear maturation. In contrast to the lethal Nog1^1–426 mutant, yeast strains expressing Nog1-CTT truncation mutants containing the Rlp24-interacting region but lacking the rest of the CTT complemented the lethality of the nog1∆ strain, but induced impaired growth at 20°C and 25°C (Figure 6A and Figure 6B). In agreement with these observations, growth of the Nog1-GFP strain was severely impaired at these temperatures (Figure 6B). We monitored PET maturation (Arx1-GFP), stalk assembly (Mrt4-GFP) and Tif6-GFP localization after treating the Nog1^1–479 truncation mutant (which retains the Rlp24-interaction platform) with DIA, which impairs Drg1-mediated Rlp24 release. We found that Arx1-GFP mislocalized to the cytoplasm in these cells (Figure 6D), emphasizing the importance of Rlp24 release and of the disassembly of the Nog1-CTT:Rlp24 complex for PET maturation. As expected, Mrt4-GFP and Tif6-GFP also mislocalized to the cytoplasm (Figure 6D), consistent with the idea that Nog1 release from the pre-60S requires Rlp24 removal. In conjunction with cryo-EM studies (Figure 5C), these data support the idea that the Nog1-CTT:Rlp24 complex negatively regulates PET maturation.

Figure 6

Download asset Open asset

Nog1^DN blocks the terminal cytoplasmic maturation steps

The terminal steps during cytoplasmic maturation of a pre-60S involve sequential release of the nuclear export signal (NES)-containing Crm1 adaptor Nmd3 and the 60S anti-association factor Tif6 (Weis et al., 2015; Ma et al., 2017; Kargas et al., 2019). Nog1^DN expression accumulated Nmd3 and Tif6 on the pre-60S as determined by SWATH-MS and Western analyses (Figure 3B and Figure 3C). To monitor Nmd3 accumulation on a cytoplasmic pre-60S by cell-biological means, we employed the nuclear-localized Nmd3^3A-GFP mutant fusion, which harbors mutations in its NES that decrease its nuclear export rate (Hedges et al., 2005). As expected, at steady state, Nmd3^3A-GFP and Tif6-GFP localize to the nucleus in cells expressing WT Nog1 (Figure 4C). However, in Nog1^DN-expressing cells, Nmd3^3A-GFP and Tif6-GFP mislocalized to the cytoplasm (Figure 4C). We suggest that the presence of Nog1^DN on the pre-60S impairs Nmd3 and Tif6 release.

Like Nog1^DN-expressing cells, yvh1Δ cells mislocalize both Mrt4-GFP and Tif6-GFP to the cytoplasm, impairing stalk assembly and progression of the cytoplasmic maturation pathway (Kemmler et al., 2009; Lo et al., 2010). Given that Yvh1 recruitment to the pre-60S is impaired in Nog1^DN-expressing cells, we investigated whether the dominant-negative behavior of Nog1^DN is due to the failure to release Mrt4. To test this, we employed a dominant gain-of-function allele MRT4^G68E that bypasses Yvh1-mediated Mrt4 release from the pre-60S (Figure 7A and Figure 7B) and relocalizes Tif6-GFP to the nucleus (Figure 7A) (Kemmler et al., 2009). We found that Nog1^DN expression is toxic in yvh1∆MRT4^G68E cells (Figure 7B). Although Mrt4^G68E-GFP relocalized to the nucleus, Tif6-GFP still mislocalized to the cytoplasm in these cells (Figure 7A), showing that the presence of Mrt4 on the pre-60S is not the cause of the dominant-negative behavior of Nog1^DN. Consistent with the sequential release of Nmd3 and Tif6 from the pre-60S, Western analyses show that Nmd3 accumulates on the Lsg1-TAP particle isolated from yvh1∆MRT4^G68E cells expressing Nog1^DN (Figure 7C). Notably, the r-protein uL10 is recruited to this Lsg1-TAP particle (Figure 7C), indicating that Nog1^DN does not interfere with stalk assembly in yvh1∆MRT4^G68E cells. In light of a recent cryo-EM study, these data suggest that Nog1^DN presence on the pre-60S prevents the rearrangement of rRNA helix H89, and sterically hinders the incorporation of uL16 into its RNA-binding site (Figure 7D), a critical step that triggers downstream events that drive a pre-60S toward translation competence (Kargas et al., 2019; Zhou et al., 2019).

Figure 7

Download asset Open asset

During its journey from the nucleolus to the cytoplasm, and concomitant progression towards the mature subunit, a pre-60S interacts with diverse energy-consuming enzymes. However, the precise order of recruitment and eviction, as well as the mechanisms that co-ordinate the different energy-consuming steps remain unknown. Here, we reveal that sequential Drg1-ATPase and Nog1-GTPase activities license early cytoplasmic maturation events that eventually drive a pre-60S towards translation competence.

Nog1 co-ordinates the assembly, maturation and quality control of distant ribosomal centers

Previously, the Johnson laboratory had ordered cytoplasmic maturation events on the pre-60S into a coherent pathway (Lo et al., 2010). In that pioneering study, AAA-ATPase Drg1 activity was proposed to initiate two parallel branches of the pathway, namely PET maturation with quality control and ribosome stalk assembly (Figure 3A). The two branches converge to initiate Nmd3 and Tif6 release prior to acquiring translational competence. Our data suggest that Nog1 plays a pivotal role in co-ordinating bifurcation and convergence of the 60S cytoplasmic maturation pathway. Cryo-EM structures and the data presented here permit the refinement of early events that drive the cytoplasmic pre-60S maturation (Figure 8). Upon arrival in the cytoplasm, the pre-60S recruits Drg1 via the C-terminal region of Rlp24. This interaction stimulates Drg1 ATPase activity resulting in Rlp24 removal, and as a consequence, the terminal end of Nog1-CTT is extracted from the PET (State I to State II). Removal of Rlp24 permits Rei1 recruitment, and allows Rei1-CTT to probe PET integrity in order to trigger Arx1 release through the J-domain protein Jjj1 and ATPases Ssa1/2 (State III) (Meyer et al., 2007; Greber et al., 2016). Release of Rei1 then permits the assembly factor Reh1 to probe the PET (State IV) (Ma et al., 2017; Kargas et al., 2019). Nog1 eviction from the pre-60S requires the extraction of Rlp24 from Nog1-CTT by the ATPase Drg1 and GTP hydrolysis within the Nog1-G domain. This event unleashes a sequence of events in different regions of the pre-60S: (1) it permits recruitment of the release factor Yvh1 to the pre-60S in order to mediate Mrt4 release and initiate stalk assembly (State IV to State V); (2) it permits loading of r-proteins eL40 and uL16 into their rRNA-binding sites (State V), (Zhou et al., 2019; Kargas et al., 2019); and (3) it re-orientates rRNA helices H89 and H38 to promote a conformational rearrangement of the Nmd3 C-terminal domain (Malyutin et al., 2017; Zhou et al., 2019; Kargas et al., 2019), leading to Nmd3 eviction and thus completing PTC maturation (State V to State VI). We therefore propose that Nog1 serves as a hub that coordinates spatially distant maturation events to ensure only a correctly assembled 60S subunit enters translation.

Figure 8

Download asset Open asset

Finally, Nsa2 and Reh1 accumulate on Lsg1-TAP in Drg1^DN- as well as Nog1^DN- expressing cells (Altvater et al., 2012; Figure 3C). These data are consistent with the idea that Nog1 eviction from the pre-60S precedes Nsa2 and Reh1 release. However, the precise mechanisms that drive Nsa2 and Reh1 release remain unknown.

Nmd3 release licenses Efl1/Sdo1 to mediate Tif6 release (State VI) (Kargas et al., 2019). Although Lsg1 appears to be required only during a specific step of the cytoplasmic maturation pathway, it seems that it can be recruited to a newly exported pre-60S. Lsg1 recruitment to the pre-60S does not seem to depend on the initial cytoplasmic maturation events, such as Rlp24 extraction by Drg1 or Nog1 release, as we efficiently isolated Drg1^DN- and Nog1^DN-trapped particles via Lsg1-TAP (Altvater et al., 2012) (Figure 3B). These purifications suggest that the GTPases Nog1 and Lsg1 can bind simultaneously to cytoplasmic pre-60S particles.

The mass spectrometry data reported in this study has been deposited into the ProteomeXchange Consortium via the PRIDE partner repository with dataset identifier PXD011382.

1. 1. Purnima Klingauf-Nerurkar
   2. Ludovic C Gillet
   3. Daniela Portugal-Calisto
   4. Michaela Oborská-Oplová
   5. Martin Jäger
   6. Olga T Schubert
   7. Agnese Pisano
   8. Cohue Peña
   9. Sanjana Rao
   10. Martin Altvater
   11. Yiming Chang
   12. Ruedi Aebersold
   13. Vikram G Panse

   (2018) ProteomeXchange

   ID PXD011382. The GTPase Nog1 co-ordinates assembly, maturation and quality control of distant ribosomal functional centers.

   http://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD011382

1. 1. Altvater M
   2. Chang Y
   3. Melnik A
   4. Occhipinti L
   5. Schütz S
   6. Rothenbusch U
   7. Picotti P
   8. Panse VG

   (2012) Targeted proteomics reveals compositional dynamics of 60S pre-ribosomes after nuclear export

   Molecular Systems Biology 8:628.

   https://doi.org/10.1038/msb.2012.63

   • PubMed
   • Google Scholar
2. 1. Altvater M
   2. Schütz S
   3. Chang Y
   4. Panse VG

   (2014) Dissecting ribosome assembly and transport in budding yeast

   Methods in Cell Biology 122:437–461.

   https://doi.org/10.1016/B978-0-12-417160-2.00020-5

   • PubMed
   • Google Scholar
3. 1. Ash MR
   2. Maher MJ
   3. Mitchell Guss J
   4. Jormakka M

   (2012) The cation-dependent G-proteins: in a class of their own

   FEBS Letters 586:2218–2224.

   https://doi.org/10.1016/j.febslet.2012.06.030

   • PubMed
   • Google Scholar
4. 1. Barrio-Garcia C
   2. Thoms M
   3. Flemming D
   4. Kater L
   5. Berninghausen O
   6. Baßler J
   7. Beckmann R
   8. Hurt E

   (2016) Architecture of the Rix1–Rea1 checkpoint machinery during pre-60S-ribosome remodeling

   Nature Structural & Molecular Biology 23:37–44.

   https://doi.org/10.1038/nsmb.3132

   • Google Scholar
5. 1. Bassler J
   2. Klein I
   3. Schmidt C
   4. Kallas M
   5. Thomson E
   6. Wagner MA
   7. Bradatsch B
   8. Rechberger G
   9. Strohmaier H
   10. Hurt E
   11. Bergler H

   (2012) The conserved Bud20 zinc finger protein is a new component of the ribosomal 60S subunit export machinery

   Molecular and Cellular Biology 32:4898–4912.

   https://doi.org/10.1128/MCB.00910-12

   • PubMed
   • Google Scholar
6. 1. Ben-Shem A
   2. Garreau de Loubresse N
   3. Melnikov S
   4. Jenner L
   5. Yusupova G
   6. Yusupov M

   (2011) The structure of the eukaryotic ribosome at 3.0 å resolution

   Science 334:1524–1529.

   https://doi.org/10.1126/science.1212642

   • PubMed
   • Google Scholar
7. 1. Bourne HR
   2. Sanders DA
   3. McCormick F

   (1991) The GTPase superfamily: conserved structure and molecular mechanism

   Nature 349:117–127.

   https://doi.org/10.1038/349117a0

   • PubMed
   • Google Scholar
8. 1. Bradatsch B
   2. Leidig C
   3. Granneman S
   4. Gnädig M
   5. Tollervey D
   6. Böttcher B
   7. Beckmann R
   8. Hurt E

   (2012) Structure of the pre-60S ribosomal subunit with nuclear export factor Arx1 bound at the exit tunnel

   Nature Structural & Molecular Biology 19:1234–1241.

   https://doi.org/10.1038/nsmb.2438

   • PubMed
   • Google Scholar
9. 1. Collins BC
   2. Gillet LC
   3. Rosenberger G
   4. Röst HL
   5. Vichalkovski A
   6. Gstaiger M
   7. Aebersold R

   (2013) Quantifying protein interaction dynamics by SWATH mass spectrometry: application to the 14-3-3 system

   Nature Methods 10:1246–1253.

   https://doi.org/10.1038/nmeth.2703

   • PubMed
   • Google Scholar
10. 1. Dong J
    2. Lai R
    3. Jennings JL
    4. Link AJ
    5. Hinnebusch AG

    (2005) The novel ATP-binding cassette protein ARB1 is a shuttling factor that stimulates 40S and 60S ribosome biogenesis

    Molecular and Cellular Biology 25:9859–9873.

    https://doi.org/10.1128/MCB.25.22.9859-9873.2005

    • PubMed
    • Google Scholar
11. 1. Escher C
    2. Reiter L
    3. MacLean B
    4. Ossola R
    5. Herzog F
    6. Chilton J
    7. MacCoss MJ
    8. Rinner O

    (2012) Using iRT, a normalized retention time for more targeted measurement of peptides

    Proteomics 12:1111–1121.

    https://doi.org/10.1002/pmic.201100463

    • PubMed
    • Google Scholar
12. 1. Faza MB
    2. Chang Y
    3. Occhipinti L
    4. Kemmler S
    5. Panse VG

    (2012) Role of Mex67-Mtr2 in the nuclear export of 40S pre-ribosomes

    PLOS Genetics 8:e1002915.

    https://doi.org/10.1371/journal.pgen.1002915

    • PubMed
    • Google Scholar
13. 1. Feng B
    2. Mandava CS
    3. Guo Q
    4. Wang J
    5. Cao W
    6. Li N
    7. Zhang Y
    8. Zhang Y
    9. Wang Z
    10. Wu J
    11. Sanyal S
    12. Lei J
    13. Gao N

    (2014) Structural and functional insights into the mode of action of a universally conserved obg GTPase

    PLOS Biology 12:e1001866.

    https://doi.org/10.1371/journal.pbio.1001866

    • PubMed
    • Google Scholar
14. 1. Fischer U
    2. Schäuble N
    3. Schütz S
    4. Altvater M
    5. Chang Y
    6. Boulos Faza M
    7. Panse VG

    (2015) A non-canonical mechanism for Crm1-export cargo complex assembly

    eLife 4:e05745.

    https://doi.org/10.7554/eLife.05745

    • Google Scholar
15. 1. Fleischer TC
    2. Weaver CM
    3. McAfee KJ
    4. Jennings JL
    5. Link AJ

    (2006) Systematic identification and functional screens of uncharacterized proteins associated with eukaryotic ribosomal complexes

    Genes & Development 20:1294–1307.

    https://doi.org/10.1101/gad.1422006

    • PubMed
    • Google Scholar
16. 1. Ford B
    2. Skowronek K
    3. Boykevisch S
    4. Bar-Sagi D
    5. Nassar N

    (2005) Structure of the G60A mutant of ras: implications for the dominant negative effect

    The Journal of Biological Chemistry 280:25697–25705.

    https://doi.org/10.1074/jbc.M502240200

    • PubMed
    • Google Scholar
17. 1. Gillet LC
    2. Navarro P
    3. Tate S
    4. Röst H
    5. Selevsek N
    6. Reiter L
    7. Bonner R
    8. Aebersold R

    (2012) Targeted data extraction of the MS/MS spectra generated by data-independent acquisition: a new concept for consistent and accurate proteome analysis

    Molecular & Cellular Proteomics 11:O111.016717.

    https://doi.org/10.1074/mcp.O111.016717

    • PubMed
    • Google Scholar
18. 1. Greber BJ
    2. Boehringer D
    3. Montellese C
    4. Ban N

    (2012) Cryo-EM structures of Arx1 and maturation factors Rei1 and Jjj1 bound to the 60S ribosomal subunit

    Nature Structural & Molecular Biology 19:1228–1233.

    https://doi.org/10.1038/nsmb.2425

    • PubMed
    • Google Scholar
19. 1. Greber BJ
    2. Gerhardy S
    3. Leitner A
    4. Leibundgut M
    5. Salem M
    6. Boehringer D
    7. Leulliot N
    8. Aebersold R
    9. Panse VG
    10. Ban N

    (2016) Insertion of the biogenesis factor Rei1 probes the ribosomal tunnel during 60S maturation

    Cell 164:91–102.

    https://doi.org/10.1016/j.cell.2015.11.027

    • PubMed
    • Google Scholar
20. 1. Hedges J
    2. West M
    3. Johnson AW

    (2005) Release of the export adapter, Nmd3p, from the 60S ribosomal subunit requires Rpl10p and the cytoplasmic GTPase Lsg1p

    The EMBO Journal 24:567–579.

    https://doi.org/10.1038/sj.emboj.7600547

    • PubMed
    • Google Scholar
21. 1. Janke C
    2. Magiera MM
    3. Rathfelder N
    4. Taxis C
    5. Reber S
    6. Maekawa H
    7. Moreno-Borchart A
    8. Doenges G
    9. Schwob E
    10. Schiebel E
    11. Knop M

    (2004) A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes

    Yeast 21:947–962.

    https://doi.org/10.1002/yea.1142

    • PubMed
    • Google Scholar
22. 1. Jensen BC
    2. Wang Q
    3. Kifer CT
    4. Parsons M

    (2003) The NOG1 GTP-binding protein is required for biogenesis of the 60 S ribosomal subunit

    Journal of Biological Chemistry 278:32204–32211.

    https://doi.org/10.1074/jbc.M304198200

    • PubMed
    • Google Scholar
23. 1. Kallstrom G
    2. Hedges J
    3. Johnson A

    (2003) The putative GTPases Nog1p and Lsg1p are required for 60S ribosomal subunit biogenesis and are localized to the nucleus and cytoplasm, respectively

    Molecular and Cellular Biology 23:4344–4355.

    https://doi.org/10.1128/MCB.23.12.4344-4355.2003

    • PubMed
    • Google Scholar
24. 1. Kappel L
    2. Loibl M
    3. Zisser G
    4. Klein I
    5. Fruhmann G
    6. Gruber C
    7. Unterweger S
    8. Rechberger G
    9. Pertschy B
    10. Bergler H

    (2012) Rlp24 activates the AAA-ATPase Drg1 to initiate cytoplasmic pre-60S maturation

    The Journal of Cell Biology 199:771–782.

    https://doi.org/10.1083/jcb.201205021

    • PubMed
    • Google Scholar
25. 1. Kargas V
    2. Castro-Hartmann P
    3. Escudero-Urquijo N
    4. Dent K
    5. Hilcenko C
    6. Sailer C
    7. Zisser G
    8. Marques-Carvalho MJ
    9. Pellegrino S
    10. Wawiórka L
    11. Freund SM
    12. Wagstaff JL
    13. Andreeva A
    14. Faille A
    15. Chen E
    16. Stengel F
    17. Bergler H
    18. Warren AJ

    (2019) Mechanism of completion of peptidyltransferase centre assembly in eukaryotes

    eLife 8:e44904.

    https://doi.org/10.7554/eLife.44904

    • PubMed
    • Google Scholar
26. 1. Kater L
    2. Thoms M
    3. Barrio-Garcia C
    4. Cheng J
    5. Ismail S
    6. Ahmed YL
    7. Bange G
    8. Kressler D
    9. Berninghausen O
    10. Sinning I
    11. Hurt E
    12. Beckmann R

    (2017) Visualizing the assembly pathway of nucleolar Pre-60S ribosomes

    Cell 171:1599–1610.

    https://doi.org/10.1016/j.cell.2017.11.039

    • PubMed
    • Google Scholar
27. 1. Keller A
    2. Nesvizhskii AI
    3. Kolker E
    4. Aebersold R

    (2002) Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search

    Analytical Chemistry 74:5383–5392.

    https://doi.org/10.1021/ac025747h

    • PubMed
    • Google Scholar
28. 1. Kemmler S
    2. Occhipinti L
    3. Veisu M
    4. Panse VG

    (2009) Yvh1 is required for a late maturation step in the 60S biogenesis pathway

    The Journal of Cell Biology 186:863–880.

    https://doi.org/10.1083/jcb.200904111

    • PubMed
    • Google Scholar
29. 1. Kressler D
    2. Roser D
    3. Pertschy B
    4. Hurt E

    (2008) The AAA ATPase Rix7 powers progression of ribosome biogenesis by stripping Nsa1 from pre-60S particles

    The Journal of Cell Biology 181:935–944.

    https://doi.org/10.1083/jcb.200801181

    • PubMed
    • Google Scholar
30. 1. Kressler D
    2. Hurt E
    3. Bergler H
    4. Baßler J

    (2012) The power of AAA-ATPases on the road of pre-60S ribosome maturation — Molecular machines that strip pre-ribosomal particles

    Biochimica Et Biophysica Acta (BBA) - Molecular Cell Research 1823:92–100.

    https://doi.org/10.1016/j.bbamcr.2011.06.017

    • Google Scholar
31. 1. Kressler D
    2. Hurt E
    3. Baßler J

    (2017) A puzzle of life: crafting ribosomal subunits

    Trends in Biochemical Sciences 42:640–654.

    https://doi.org/10.1016/j.tibs.2017.05.005

    • PubMed
    • Google Scholar
32. 1. Lam H
    2. Deutsch EW
    3. Eddes JS
    4. Eng JK
    5. Stein SE
    6. Aebersold R

    (2008) Building consensus spectral libraries for peptide identification in proteomics

    Nature Methods 5:873–875.

    https://doi.org/10.1038/nmeth.1254

    • PubMed
    • Google Scholar
33. 1. Lambert JP
    2. Ivosev G
    3. Couzens AL
    4. Larsen B
    5. Taipale M
    6. Lin ZY
    7. Zhong Q
    8. Lindquist S
    9. Vidal M
    10. Aebersold R
    11. Pawson T
    12. Bonner R
    13. Tate S
    14. Gingras AC

    (2013) Mapping differential interactomes by affinity purification coupled with data-independent mass spectrometry acquisition

    Nature Methods 10:1239–1245.

    https://doi.org/10.1038/nmeth.2702

    • PubMed
    • Google Scholar
34. 1. Lapik YR
    2. Misra JM
    3. Lau LF
    4. Pestov DG

    (2007) Restricting conformational flexibility of the switch II region creates a dominant-inhibitory phenotype in obg GTPase Nog1

    Molecular and Cellular Biology 27:7735–7744.

    https://doi.org/10.1128/MCB.01161-07

    • PubMed
    • Google Scholar
35. 1. Lebreton A
    2. Saveanu C
    3. Decourty L
    4. Rain JC
    5. Jacquier A
    6. Fromont-Racine M

    (2006) A functional network involved in the recycling of nucleocytoplasmic pre-60S factors

    The Journal of Cell Biology 173:349–360.

    https://doi.org/10.1083/jcb.200510080

    • PubMed
    • Google Scholar
36. 1. Lo KY
    2. Li Z
    3. Wang F
    4. Marcotte EM
    5. Johnson AW

    (2009) Ribosome stalk assembly requires the dual-specificity phosphatase Yvh1 for the exchange of Mrt4 with P0

    The Journal of Cell Biology 186:849–862.

    https://doi.org/10.1083/jcb.200904110

    • PubMed
    • Google Scholar
37. 1. Lo KY
    2. Li Z
    3. Bussiere C
    4. Bresson S
    5. Marcotte EM
    6. Johnson AW

    (2010) Defining the pathway of cytoplasmic maturation of the 60S ribosomal subunit

    Molecular Cell 39:196–208.

    https://doi.org/10.1016/j.molcel.2010.06.018

    • PubMed
    • Google Scholar
38. 1. Loibl M
    2. Klein I
    3. Prattes M
    4. Schmidt C
    5. Kappel L
    6. Zisser G
    7. Gungl A
    8. Krieger E
    9. Pertschy B
    10. Bergler H

    (2014) The drug diazaborine blocks ribosome biogenesis by inhibiting the AAA-ATPase Drg1

    Journal of Biological Chemistry 289:3913–3922.

    https://doi.org/10.1074/jbc.M113.536110

    • PubMed
    • Google Scholar
39. 1. Longtine MS
    2. McKenzie A
    3. Demarini DJ
    4. Shah NG
    5. Wach A
    6. Brachat A
    7. Philippsen P
    8. Pringle JR

    (1998) Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae

    Yeast 14:953–961.

    https://doi.org/10.1002/(SICI)1097-0061(199807)14:10<953::AID-YEA293>3.0.CO;2-U

    • PubMed
    • Google Scholar
40. 1. Ma C
    2. Wu S
    3. Li N
    4. Chen Y
    5. Yan K
    6. Li Z
    7. Zheng L
    8. Lei J
    9. Woolford JL
    10. Gao N

    (2017) Structural snapshot of cytoplasmic pre-60S ribosomal particles bound by Nmd3, Lsg1, Tif6 and Reh1

    Nature Structural & Molecular Biology 24:214–220.

    https://doi.org/10.1038/nsmb.3364

    • Google Scholar
41. 1. Malyutin AG
    2. Musalgaonkar S
    3. Patchett S
    4. Frank J
    5. Johnson AW

    (2017) Nmd3 is a structural mimic of eIF5A, and activates the cpGTPase Lsg1 during 60S ribosome biogenesis

    The EMBO Journal 36:854–868.

    https://doi.org/10.15252/embj.201696012

    • PubMed
    • Google Scholar
42. 1. Matsuo Y
    2. Granneman S
    3. Thoms M
    4. Manikas RG
    5. Tollervey D
    6. Hurt E

    (2014) Coupled GTPase and remodelling ATPase activities form a checkpoint for ribosome export

    Nature 505:112–116.

    https://doi.org/10.1038/nature12731

    • PubMed
    • Google Scholar
43. 1. Meyer AE
    2. Hung NJ
    3. Yang P
    4. Johnson AW
    5. Craig EA

    (2007) The specialized cytosolic J-protein, Jjj1, functions in 60S ribosomal subunit biogenesis

    PNAS 104:1558–1563.

    https://doi.org/10.1073/pnas.0610704104

    • PubMed
    • Google Scholar
44. 1. Meyer AE
    2. Hoover LA
    3. Craig EA

    (2010) The cytosolic J-protein, Jjj1, and Rei1 function in the removal of the pre-60 S subunit factor Arx1

    Journal of Biological Chemistry 285:961–968.

    https://doi.org/10.1074/jbc.M109.038349

    • PubMed
    • Google Scholar
45. 1. Navarro P
    2. Kuharev J
    3. Gillet LC
    4. Bernhardt OM
    5. MacLean B
    6. Röst HL
    7. Tate SA
    8. Tsou CC
    9. Reiter L
    10. Distler U
    11. Rosenberger G
    12. Perez-Riverol Y
    13. Nesvizhskii AI
    14. Aebersold R
    15. Tenzer S

    (2016) A multicenter study benchmarks software tools for label-free proteome quantification

    Nature Biotechnology 34:1130–1136.

    https://doi.org/10.1038/nbt.3685

    • PubMed
    • Google Scholar
46. 1. Nerurkar P
    2. Altvater M
    3. Gerhardy S
    4. Schütz S
    5. Fischer U
    6. Weirich C
    7. Panse VG

    (2015) Eukaryotic ribosome assembly and nuclear export

    International Review of Cell and Molecular Biology 319:107–140.

    https://doi.org/10.1016/bs.ircmb.2015.07.002

    • PubMed
    • Google Scholar
47. 1. Nissan TA
    2. Bassler J
    3. Petfalski E
    4. Tollervey D
    5. Hurt E

    (2002) 60s pre-ribosome formation viewed from assembly in the nucleolus until export to the cytoplasm

    The EMBO Journal 21:5539–5547.

    https://doi.org/10.1093/emboj/cdf547

    • PubMed
    • Google Scholar
48. 1. Parnell KM
    2. Bass BL

    (2009) Functional redundancy of yeast proteins Reh1 and Rei1 in cytoplasmic 60S subunit maturation

    Molecular and Cellular Biology 29:4014–4023.

    https://doi.org/10.1128/MCB.01582-08

    • PubMed
    • Google Scholar
49. 1. Pausch P
    2. Singh U
    3. Ahmed YL
    4. Pillet B
    5. Murat G
    6. Altegoer F
    7. Stier G
    8. Thoms M
    9. Hurt E
    10. Sinning I
    11. Bange G
    12. Kressler D

    (2015) Co-translational capturing of nascent ribosomal proteins by their dedicated chaperones

    Nature Communications 6:7494.

    https://doi.org/10.1038/ncomms8494

    • PubMed
    • Google Scholar
50. 1. Peña C
    2. Hurt E
    3. Panse VG

    (2017) Eukaryotic ribosome assembly, transport and quality control

    Nature Structural & Molecular Biology 24:689–699.

    https://doi.org/10.1038/nsmb.3454

    • PubMed
    • Google Scholar
51. 1. Pertschy B
    2. Saveanu C
    3. Zisser G
    4. Lebreton A
    5. Tengg M
    6. Jacquier A
    7. Liebminger E
    8. Nobis B
    9. Kappel L
    10. van der Klei I
    11. Högenauer G
    12. Fromont-Racine M
    13. Bergler H

    (2007) Cytoplasmic recycling of 60S preribosomal factors depends on the AAA protein Drg1

    Molecular and Cellular Biology 27:6581–6592.

    https://doi.org/10.1128/MCB.00668-07

    • PubMed
    • Google Scholar
52. 1. Picotti P
    2. Aebersold R

    (2012) Selected reaction monitoring-based proteomics: workflows, potential, pitfalls and future directions

    Nature Methods 9:555–566.

    https://doi.org/10.1038/nmeth.2015

    • PubMed
    • Google Scholar
53. 1. Puig O
    2. Caspary F
    3. Rigaut G
    4. Rutz B
    5. Bouveret E
    6. Bragado-Nilsson E
    7. Wilm M
    8. Séraphin B

    (2001) The Tandem Affinity Purification (TAP) Method: A General Procedure of Protein Complex Purification

    Methods 24:218–229.

    https://doi.org/10.1006/meth.2001.1183

    • Google Scholar
54. 1. Reiter L
    2. Claassen M
    3. Schrimpf SP
    4. Jovanovic M
    5. Schmidt A
    6. Buhmann JM
    7. Hengartner MO
    8. Aebersold R

    (2009) Protein identification false discovery rates for very large proteomics data sets generated by tandem mass spectrometry

    Molecular & Cellular Proteomics 8:2405–2417.

    https://doi.org/10.1074/mcp.M900317-MCP200

    • PubMed
    • Google Scholar
55. 1. Röst HL
    2. Rosenberger G
    3. Navarro P
    4. Gillet L
    5. Miladinović SM
    6. Schubert OT
    7. Wolski W
    8. Collins BC
    9. Malmström J
    10. Malmström L
    11. Aebersold R

    (2014) OpenSWATH enables automated, targeted analysis of data-independent acquisition MS data

    Nature Biotechnology 32:219–223.

    https://doi.org/10.1038/nbt.2841

    • PubMed
    • Google Scholar
56. 1. Röst HL
    2. Liu Y
    3. D'Agostino G
    4. Zanella M
    5. Navarro P
    6. Rosenberger G
    7. Collins BC
    8. Gillet L
    9. Testa G
    10. Malmström L
    11. Aebersold R

    (2016) TRIC: an automated alignment strategy for reproducible protein quantification in targeted proteomics

    Nature Methods 13:777–783.

    https://doi.org/10.1038/nmeth.3954

    • PubMed
    • Google Scholar
57. 1. Sanghai ZA
    2. Miller L
    3. Molloy KR
    4. Barandun J
    5. Hunziker M
    6. Chaker-Margot M
    7. Wang J
    8. Chait BT
    9. Klinge S

    (2018) Modular assembly of the nucleolar pre-60S ribosomal subunit

    Nature 556:126–129.

    https://doi.org/10.1038/nature26156

    • PubMed
    • Google Scholar
58. 1. Saveanu C
    2. Namane A
    3. Gleizes PE
    4. Lebreton A
    5. Rousselle JC
    6. Noaillac-Depeyre J
    7. Gas N
    8. Jacquier A
    9. Fromont-Racine M

    (2003) Sequential protein association with nascent 60S ribosomal particles

    Molecular and Cellular Biology 23:4449–4460.

    https://doi.org/10.1128/MCB.23.13.4449-4460.2003

    • PubMed
    • Google Scholar
59. 1. Schubert OT
    2. Gillet LC
    3. Collins BC
    4. Navarro P
    5. Rosenberger G
    6. Wolski WE
    7. Lam H
    8. Amodei D
    9. Mallick P
    10. MacLean B
    11. Aebersold R

    (2015) Building high-quality assay libraries for targeted analysis of SWATH MS data

    Nature Protocols 10:426–441.

    https://doi.org/10.1038/nprot.2015.015

    • PubMed
    • Google Scholar
60. 1. Sengupta J
    2. Bussiere C
    3. Pallesen J
    4. West M
    5. Johnson AW
    6. Frank J

    (2010) Characterization of the nuclear export adaptor protein Nmd3 in association with the 60S ribosomal subunit

    The Journal of Cell Biology 189:1079–1086.

    https://doi.org/10.1083/jcb.201001124

    • PubMed
    • Google Scholar
61. 1. Shteynberg D
    2. Deutsch EW
    3. Lam H
    4. Eng JK
    5. Sun Z
    6. Tasman N
    7. Mendoza L
    8. Moritz RL
    9. Aebersold R
    10. Nesvizhskii AI

    (2011) iProphet: multi-level integrative analysis of shotgun proteomic data improves peptide and protein identification rates and error estimates

    Molecular & Cellular Proteomics 10:M111.007690.

    https://doi.org/10.1074/mcp.M111.007690

    • PubMed
    • Google Scholar
62. 1. Su T
    2. Izawa T
    3. Thoms M
    4. Yamashita Y
    5. Cheng J
    6. Berninghausen O
    7. Hartl FU
    8. Inada T
    9. Neupert W
    10. Beckmann R

    (2019) Structure and function of Vms1 and Arb1 in RQC and mitochondrial proteome homeostasis

    Nature 570:538–542.

    https://doi.org/10.1038/s41586-019-1307-z

    • PubMed
    • Google Scholar
63. 1. Teleman J
    2. Röst HL
    3. Rosenberger G
    4. Schmitt U
    5. Malmström L
    6. Malmström J
    7. Levander F

    (2015) DIANA--algorithmic improvements for analysis of data-independent acquisition MS data

    Bioinformatics 31:555–562.

    https://doi.org/10.1093/bioinformatics/btu686

    • PubMed
    • Google Scholar
64. 1. Weis F
    2. Giudice E
    3. Churcher M
    4. Jin L
    5. Hilcenko C
    6. Wong CC
    7. Traynor D
    8. Kay RR
    9. Warren AJ

    (2015) Mechanism of eIF6 release from the nascent 60S ribosomal subunit

    Nature Structural & Molecular Biology 22:914–919.

    https://doi.org/10.1038/nsmb.3112

    • PubMed
    • Google Scholar
65. 1. Wu S
    2. Tutuncuoglu B
    3. Yan K
    4. Brown H
    5. Zhang Y
    6. Tan D
    7. Gamalinda M
    8. Yuan Y
    9. Li Z
    10. Jakovljevic J
    11. Ma C
    12. Lei J
    13. Dong MQ
    14. Woolford JL
    15. Gao N

    (2016) Diverse roles of assembly factors revealed by structures of late nuclear pre-60S ribosomes

    Nature 534:133–137.

    https://doi.org/10.1038/nature17942

    • PubMed
    • Google Scholar
66. 1. Zhou Y
    2. Musalgaonkar S
    3. Johnson AW
    4. Taylor DW

    (2019) Tightly-orchestrated rearrangements govern catalytic center assembly of the ribosome

    Nature Communications 10:958.

    https://doi.org/10.1038/s41467-019-08880-0

    • PubMed
    • Google Scholar
67. 1. Zisser G
    2. Ohmayer U
    3. Mauerhofer C
    4. Mitterer V
    5. Klein I
    6. Rechberger GN
    7. Wolinski H
    8. Prattes M
    9. Pertschy B
    10. Milkereit P
    11. Bergler H

    (2018) Viewing pre-60S maturation at a minute's timescale

    Nucleic Acids Research 46:3140–3151.

    https://doi.org/10.1093/nar/gkx1293

    • PubMed
    • Google Scholar

Author details

1. Purnima Klingauf-Nerurkar

   Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland

   Contribution

   Conceptualization, Formal analysis, Investigation, Methodology

   Contributed equally with

   Ludovic C Gillet

   Competing interests

   No competing interests declared

2. Ludovic C Gillet

   Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland

   Contribution

   Conceptualization, Data curation, Formal analysis, Methodology

   Contributed equally with

   Purnima Klingauf-Nerurkar

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1001-3265

3. Daniela Portugal-Calisto

   Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland

   Contribution

   Formal analysis

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1591-5812

4. Michaela Oborská-Oplová

   1. Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland
   2. Institute of Biochemistry, ETH Zurich, Zurich, Switzerland

   Contribution

   Formal analysis, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0976-4341

5. Martin Jäger

   Institute of Biochemistry, ETH Zurich, Zurich, Switzerland

   Contribution

   Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

6. Olga T Schubert

   Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland

   Present address

   Department of Human Genetics, University of California, Los Angeles, Los Angeles, United States

   Contribution

   Conceptualization, Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2613-0714

7. Agnese Pisano

   Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland

   Contribution

   Formal analysis

   Competing interests

   No competing interests declared

8. Cohue Peña

   Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland

   Contribution

   Conceptualization, Formal analysis, Methodology

   Competing interests

   No competing interests declared

9. Sanjana Rao

   Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland

   Contribution

   Formal analysis, Methodology

   Competing interests

   No competing interests declared

10. Martin Altvater

    Institute of Biochemistry, ETH Zurich, Zurich, Switzerland

    Contribution

    Conceptualization, Formal analysis, Methodology

    Competing interests

    No competing interests declared

11. Yiming Chang

    Institute of Biochemistry, ETH Zurich, Zurich, Switzerland

    Contribution

    Conceptualization, Formal analysis, Investigation, Methodology

    Competing interests

    No competing interests declared

12. Ruedi Aebersold

    1. Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland
    2. Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland

    Present address

    Faculty of Science, University of Zurich, Zurich, Switzerland

    Contribution

    Conceptualization, Resources, Supervision, Methodology, Writing - review and editing

    Competing interests

    No competing interests declared

13. Vikram G Panse

    Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland

    Contribution

    Conceptualization, Formal analysis, Supervision, Investigation, Project administration

    For correspondence

    vpanse@imm.uzh.ch

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-7950-5746

• 2,679

  views

• 332

  downloads

• 34

  citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.