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

Abstract

Construction and intracellular targeting of eukaryotic pre-ribosomal particles involve a multitude of diverse transiently associating trans-acting assembly factors, energy-consuming enzymes, and transport factors. The ability to rapidly and reliably measure co-enrichment of multiple factors with maturing pre-ribosomal particles presents a major biochemical bottleneck towards revealing their function and the precise contribution of >50 energy-consuming steps that drive ribosome assembly. Here, we devised a workflow that combines genetic trapping, affinity-capture, and selected reaction monitoring mass spectrometry (SRM-MS), to overcome this deficiency. We exploited this approach to interrogate the dynamic proteome of pre-60S particles after nuclear export. We uncovered assembly factors that travel with pre-60S particles to the cytoplasm, where they are released before initiating translation. Notably, we identified a novel shuttling factor that facilitates nuclear export of pre-60S particles. Capturing and quantitating protein interaction networks of trapped intermediates of macromolecular complexes by our workflow is a reliable discovery tool to unveil dynamic processes that contribute to their in vivo assembly and transport.

Figure 1

Dynamic association of trans-acting factors with maturing pre-60S subunits revealed by SRM. (A) A representative analysis (three independent biological replicates) of relative enrichment of 40 trans-acting factors with maturing pre-60S particles are depicted using purple to gold color-scale. Maximum enrichment is depicted as purple and minimum enrichment as gold. Each measurement represents the average of different SRM transitions per peptide and different peptides per protein. Akin to western analysis, the acquired intensity of each factor was normalized based on the average intensities of 10 depicted large-subunit r-proteins. (B) The enrichment of selected factors (labeled as red dots in (A)) is represented using histograms with error bars (±s.d.) to assess the precision of our analyses for the relative co-enrichment of individual factors. The color of each column corresponds to the relative enrichment as shown in (A). (C) Western analyses of selected pre-60S trans-acting factors (labeled as green dots in (A)) using TAP purified pre-60S and Bud20 particles. The purifications were separated on 4–12% gradient gels, and subjected to silver staining and western analyses. The large-subunit r-proteins Rpl1, Rpl3, and Rpl35 served as loading controls.

Figure 2

Proteome of genetically trapped cytoplasmic pre-60S particles revealed by SRM. (A) Proposed pathway of 60S cytoplasmic maturation initiated by Drg1. Maturation events indicated on the pathway only represent the order of action but not necessarily the association with the pre-60S particle. (B) A representative analysis (three independent biological replicates) of the relative enrichment of the depicted factors on the indicated pre-60S particles isolated at different stages of maturation, and genetically trapped cytoplasmic pre-60S particles are shown using purple to gold color-scale. Maximum enrichment is depicted as purple and minimum enrichment as gold. Each measurement represents the average of different SRM transitions per peptide and different peptides per protein. Akin to western analyses, the acquired intensities of each protein were normalized based on a set of 10 large-subunit r-proteins. (C) The relative enrichment of selected proteins (labeled in (B) with red dots) in Kre35-TAP WT (DRG1) and DRG1^DN samples is represented using histograms showing the fold change with error bars (±s.d.). The color of each bar corresponds to the enrichment shown in (B). (D) Western analyses of selected pre-60S trans-acting factors (labeled in (B) with green dots) using affinity-purified pre-60S particles. Samples were analyzed on NuPAGE 4–12% gradient gels followed by western analyses. The large-subunit r-protein Rpl1, Rpl3, and Rpl35 served as loading controls.

Figure 3

Overexpression of DRG1^DN results in mislocalization of Bud20–GFP and Nug1–GFP. Cells carrying a plasmid containing DRG1^DN under the control of CUP1 promoter were grown to early log phase. Expression of DRG1^DN was induced by 0.5 mM copper sulfate for 5–7 h. Cells were analyzed by fluorescence microscopy. Empty vector (pYEX4-T1) was used as controls. Scale bar=5 μm.

Figure 4

Bud20–GFP and Nug1–GFP shuttle between the nucleus and cytoplasm. Cells expressing Arx1–GFP, Gar1–GFP, Bud20–GFP, Nug1–GFP were mated with the kar1-1 overexpressing strain containing Nup82–mCherry. Heterokaryons were analyzed by fluorescence microscopy. Arx1–GFP and Gar1–GFP served as positive and negative controls, respectively. Scale bar=5 μm.

Figure 5

Bud20 is required for proper pre-60S subunit export. (A) The bud20Δ mutant is impaired in growth at different temperatures. BUD20 and bud20Δ cells were spotted in 10-fold dilutions on YPD plates and grown at indicated temperatures for 3–7 days. (B) The bud20Δ mutant is impaired in nuclear export of pre-60S subunits. BUD20 and bud20Δ cells expressing the indicated GFP fusion proteins were grown at 30°C until mid log phase. Cells were analyzed by fluorescence microscopy. The yrb2Δ mutant served as positive control for cytoplasmic S2–GFP localization. Scale bar=5 μm.

Figure 6

Proteome of late pre-60S particles in the bud20Δ mutant revealed by SRM. (A) A representative analysis (of three independent biological replicates) of relative enrichment of the trans-acting factors in the indicated pre-60S particles and in the bud20Δ mutant is shown using a purple to gold color-scale. Maximum enrichment is depicted as purple and minimum enrichment as gold. Each measurement represents the average of different SRM transitions per peptide and different peptides per protein. The acquired intensities for each protein were normalized based on a set of 10 large-subunit r-proteins. (B) The relative enrichment of selected proteins (labeled in (A) with red dots) in Arx1-TAP WT (BUD20) and bud20Δ samples is represented using histograms showing the fold change with error bars (±s.d.). The color of each bar corresponds to the enrichment shown in (A). (C) Western analyses of selected pre-60S trans-acting factors (labeled in (A) as green dots) using affinity-purified pre-60S particles. Samples were analyzed on NuPAGE 4–12% gradient gels followed by western analyses. The large-subunit r-protein Rpl1, Rpl3, and Rpl35 served as loading controls.

Figure 7

Bud20 functionally overlaps with pre-60S export factors and interacts with FG repeat containing nucleoporins. (A) Overexpression of NMD3 can partially rescue slow growth and pre-60S export defect of bud20Δ mutants. The bud20Δ strain was transformed with indicated plasmids and grown on SD plates at 30°C. Pictures of single colonies were taken after 4 days. Localization of the L5–GFP reporter in these cells was determined by fluorescence microscopy. Cells were grown in SD medium to mid-log phase before picture acquisition. Scale bar=5 μm. (B) Bud20 genetically interacts with factors required for proper pre-60S subunit export. Synthetic lethality (sl) or synthetic enhancement (se) of the bud20Δ mutant combined with indicated mutants strains. Strains carrying the WT and mutant alleles were spotted in 10-fold dilutions on 5-FOA (SD/SG) plates (when sl) or YPG/YPD plates (when se) and grown at 20–30°C for 3–9 days. Solid line indicates sl, dashed line indicates se. (C) Recombinant Bud20 interacts with FG repeat sequences of different nucleoporins. Different GST-nucleoporin fusion proteins were expressed in E. coli cells and immobilized on glutathione sepharose before incubation with recombinant Bud20, Bud20-ZnF, Bud20ΔZnF, or Mex67-Mtr2 (positive control). Bound proteins were eluted by SDS sample buffer and analyzed by SDS–PAGE followed by Coomassie staining or western analyses. L=Load.