Eukaryotic Ribosome assembly and Nucleocytoplasmic Transport

Abstract

The process of eukaryotic ribosome assembly stretches across the nucleolus, the nucleoplasm and the cytoplasm, and therefore relies on efficient nucleocytoplasmic transport. In yeast, the import machinery delivers ~140,000 ribosomal proteins every minute to the nucleus for ribosome assembly. At the same time, the export machinery facilitates translocation of ~2000 pre-ribosomal particles every minute through ~200 nuclear pore complexes (NPC) into the cytoplasm. Eukaryotic ribosome assembly also requires >200 conserved assembly factors, which transiently associate with pre-ribosomal particles. Their site(s) of action on maturing pre-ribosomes are beginning to be elucidated. In this chapter, we outline protocols that enable rapid biochemical isolation of pre-ribosomal particles for single particle cryo-electron microscopy (cryo-EM) and in vitro reconstitution of nuclear transport processes. We discuss cell-biological and genetic approaches to investigate how the ribosome assembly and the nucleocytoplasmic transport machineries collaborate to produce functional ribosomes.

1 Introduction

Eukaryotic ribosome assembly takes place across multiple cellular compartments : the nucleolus , the nucleoplasm and the cytoplasm (Fig. 1). This dynamic and energy consuming process requires the coordination of three RNA polymerases (I, II, and III), the RNA splicing, and nucleocytoplasmic transport machineries [1]. Despite this complexity, ribosome biogenesis is an incredibly efficient process, with yeast producing up to 60 ribosomes every second [2].

Fig. 1

Current model for eukaryotic ribosome assembly . Transcription of primary 35S rRNA transcript, the common precursor of 18S, 5.8S, and 25S rRNAs , by Pol I from rDNA repeats together with cotranscriptional joining of U3 snoRNP , r-proteins and 40S assembly factors form the 90S ribosomal precursor. 5S rRNA is transcribed separately by Pol III for assembly of 5S RNP before joining the 60S pre-ribosome. Cotranscriptional cleavage of 35S rRNA at A2 site separates the 60S and 40S r-subunit maturation pathways. Pre-ribosomal particles undergo a cascade of maturation steps in the nucleoplasm by transient association with assembly factors until reaching nuclear export competency. Once exported to the cytoplasm, last maturation events and quality control steps can occur resulting into functional r-subunits production. (Adapted from Peña et al. 2017)

The small subunit (SSU) processome is the first ribosome precursor assembled cotranscriptionally in the nucleolus through stepwise association with UTP-A, UTP-B, and UTP-C complexes and U3 snoRNP [1, 3]. Cryo-electron microscopy (cryo-EM) studies are revealing how UTP-A, UTP-B complexes, U3 snoRNP and additional biogenesis factors encapsulate and guide pre-rRNA folding in a hierarchical 5′-to-3′-oriented manner [4,5,6,7]. During nucleolar maturation, the 5′ domain of the 18S rRNA achieves a mature conformation, and the central domain is correctly positioned relative to the 5′ domain. In contrast, the 3′ major domain is buried within the SSU processome core, and its conformation is distinct as in the mature 40S subunit. Release of the 40S pre-ribosome requires endonucleolytic cleavages within the pre-rRNA [8,9,10,11], the release of U3 snoRNA and associated proteins catalyzed by the RNA helicase Dhr1 and its cofactor Utp14 [12, 13]. The RNA exosome mediates degradation of 5′-ETS (external transcribed spacer) rRNA within the 5′-ETS rRNA –UTP complex to disassemble and recycle the UTPs for a new round of 40S assembly [14].

Following 40S preribosome release the growing 27S pre-rRNA associates with 60S-specific r-proteins and maturation factors to initiate 60S pre-ribosome assembly . Cryo-EM structures of early states of the 60S pre-ribosome suggest a sequence of events during nucleolar 60S assembly. Early states the nucleolar 60S pre-ribosome show a characteristic arch-like morphology, whereas the later states adopt a compact shape closer to a mature 60S subunit. The 5′ domains (I and II) within 27S pre-rRNA adopt a near mature conformation, and connect with the 3′ terminal domain VI, thus forming the solvent-exposed backside of the 60S subunit. In all states, the pre-rRNA spacer ITS2, located in the 27S pre-rRNA between 5.8S and 25S rRNA , and the associated ITS2 factors form the “foot” structure. The central domains III, IV and V, which form the subunit interface, are not visible in the early states. Although cotranscriptional assembly of the 60S pre-ribosome occurs in a sequential manner, it undergoes nonlinear compaction of domains I, II, and VI, which then allows domains III, IV, and V to fall onto the arch-like structure, thus preparing the pre-ribosomal cargo for nuclear export [15,16,17].

In yeast , before every cell division, ~200,000 pre-ribosomes are transported to the cytoplasm through ~200 NPCs (nuclear pore complexes) by the exportin Crm1 that recognizes nuclear export sequences (NESs) on cargos and interacts with the FG-rich (phenylalanine/glycine-rich) meshwork of the NPC transport channel [18,19,20,21,22]. Nmd3 is the only identified essential adaptor for Crm1-mediated 60S pre-ribosome export [23, 24]. In contrast, no essential NES-containing export adaptor has been identified for 40S pre-ribosome export. 40S pre-ribosome bound shuttling assembly factors Ltv1 and Rio2 can recruit Crm1 in the presence of RanGTP through a leucine-rich NES . Pre-ribosomes also employ multiple export factors (Mex67-Mtr2 , Arx1 , Ecm1 , Bud20 ) that directly interact with the FG-meshwork of the transport channel [1, 21, 22]. In addition, a non-FG pathway involving the mRNA export factor Gle2 also facilitates 60S pre-ribosome export [25].

Exported pre-ribosomes undergo final maturation and proofreading before initiating translation [26]. Cytoplasmic proofreading of 60S pre-ribosomes involves release of assembly factors that block binding of r-proteins , interactions with translation factors, or pairing with the 40S . Tif6 prevents binding of immature 60S pre-ribosomes to mature 40S subunits, ensuring that only properly assembled subunits engage in translation . Quality control of the 40S pre-ribosome relies on assembly factors that prevent the premature binding of initiation factors, mRNA , tRNA, and the 60S subunit. Ltv1 and Enp1 directly bind uS3 on its solvent side, thereby blocking the mRNA channel opening. Rio2 , Tsr1 , and Dim1 bind the subunit interface, thus preventing joining of the mature 60S subunit and translation initiation factor eIF1A. Nob1 and Pno1 block the binding of eIF3, thereby interfering with translation initiation [27]. After release of Rio2 , Tsr1 , and Dim1 initiated by the reorganization of the beak structure, the 40S pre-ribosome becomes competent to interact with a mature 60S subunit. This translation-like interaction is thought to test the ability of a 40S pre-ribosome to engage with a mature 60S subunit, and only then triggers Nob1 to cleave 20S pre-RNA to mature 18S rRNA in vitro [28, 29]. Cryo-EM studies are revealing how these late factors interact with the 40S pre-ribosome and have provided a structural framework for the ordering of cytoplasmic maturation events [30,31,32].

In addition to >200 assembly factors , ribosome assembly relies on efficient nucleocytoplasmic transport [1]. All r-proteins and assembly factors need to be imported into the nucleus, and correctly assembled pre-ribosomal particles need to be transported through nuclear pore complexes into the cytoplasm. How the ribosome assembly machinery collaborates with the cellular trafficking pathways is currently under intense investigation. Here, we outline biochemical approaches that permit rapid screening and analyses of pre-ribosomal particles for single particle cryo-EM studies, and reconstitution of nucleocytoplasmic transport processes. We discuss genetic and cell-biological approaches that will enable the unveiling of the functional interface between the ribosome assembly and nucleocytoplasmic machineries.

2 Materials, Reagents, and Yeast Media

2.1 Material (Listed in Alphabetical Order)

• 1. 15 mL Centrifuge tube (Greiner Bio-One, 188271)

• 2. 20 mL Luer Solo, Inject (Braun, 4606205V)

• 3. 50 mL Centrifuge tube (Greiner Bio-One, 227261)

• 4. 50 mL Magnetic Separation Rack (New BioLabs, S1507S)

• 5. Centrifuge 5804 R (Eppendorf, 5805 000.327).

• 6. Centrifuge Sorvall RC3BP with H-6000A Rotor (Thermo-Scientific, 75007530, 11250).

• 7. Cover glasses (VWR, 631-0137).

• 8. DynaMag™—Spin Magnet (Invitrogen, 12320D).

• 9. Fluorescence microscope Leica DM6000 B (Leica).

• 10. Incubator IFE 600 (Memmert).

• 11. Incubator shaker ISF1-X (Kuhner).

• 12. Inoculation loop (Greiner, 731101).

• 13. Membrane filters 0.45 mm (Millipore, HAWP04700).

• 14. Microcentrifuge 5415R (Eppendorf, 022621408).

• 15. Microscope slides (VWR, 631-1550).

• 16. Planetary mill Pulverisette 6 (Fritsch, 06.2000.00).

• 17. Spectrophotometer Ultrospec 2100 pro (GE Healthcare, 80-2112-21).

• 18. Stainless Steel Grinding Balls (Fritsch, 55.0200.10).

• 19. Stainless Steel Grinding Bowls, 80 mL (Fritsch, 50.4100.00).

• 20. Sterifil Aseptic System (Millipore, XX1104700).

• 21. Syringe filter, pore size: 2.7 μm (GE Healthcare, 6888-2527).

• 22. Syringe filter pore size: 1.6 μm (GE Healthcare, 6882-2516).

• 23. Thermomixer comfort (Eppendorf).

2.2 Reagents (Listed in Alphabetical Order)

• 1. Acetone (Merck, 1.00014.1000).

• 2. AcTEV protease (Invitrogen, 12575015).

• 3. Beta-mercaptoethanol (CalBioChem, 444203).

• 4. CaCl2(Sigma-Aldrich, 22,350-6).

• 5. cOmplete protease inhibitor cocktail tablets, EDTA free (Roche, 11873580001),

• 6. Coomassie Brilliant Blue R-250 (ThermoScientific, 20,278).

• 7. Difco Agar (BD, 214530).

• 8. Dithiothreitol (DTT) (AppliChem, A1101).

• 9. Dynabeads™ M-270 Epoxy (Invitrogen, 14301).

• 10. Glucose (Sigma-Aldrich, G8270).

• 11. Glutathione Sepharose 4 Fast Flow (GE Healthcare, 17-5132-01).

• 12. Glycerol (AppliChem, A1123).

• 13. HEPES (Sigma-Aldrich, H4034).

• 14. IgG from rabbit serum (Sigma-Aldrich, I5006).

• 15. KOAc (Sigma-Aldrich, P1147).

• 16. LDS sample buffer (Invitrogen, NP0008).

• 17. MgCl₂ (Fluka, 63072).

• 18. Mg(OAc)₂ (Sigma-Aldrich, M5661).

• 19. NaCl (Merck, 1064045000).

• 20. NH₄Cl (Sigma-Aldrich, A9434).

• 21. NuPAGE 4–12% Bis–Tris (Novex, NP0321BOX).

• 22. Phenylmethanesulfonyl fluoride (PMSF) (AppliChem, A0999).

• 23. Polyvinylpyrrolidone (K90) (AppliChem, A6393).

• 24. Sodium phosphate (Sigma-Aldrich, 342483).

• 25. TCA (Sigma-Aldrich, T6399).

• 26. Tris–HCl (Sigma-Aldrich, T3253).

• 27. Triton X-100 (Merck, 1086431000).

• 28. Tween 20 (Sigma-Aldrich, P9416).

2.3 Yeast Media

• 1. Synthetic complete (SC) media: 0.69% yeast nitrogen base without amino acids (ForMedium, CYN0410) 2% glucose (Sigma-Aldrich, G8270) 0.6–0.8% appropriate drop-out supplements complete mixtures (ForMedium).

• 2. SC plates (SC, 2% agar).

• 3. Yeast-extract peptone dextrose (YPD) media: 1% yeast extract, (ForMedium, YEM03), 2% peptone (ForMedium, PEP03), 2% glucose (Sigma-Aldrich, G8270).

• 4. YPD plates (YPD , 2% agar).

3 Pre-ribosome Isolation for Single Particle Cryo-Electron Microscopy

During the last decade, affinity-purification protocols combined with sensitive mass spectrometry have dramatically altered our understanding of eukaryotic ribosome assembly [33]. By employing the powerful “tandem affinity-purification ” (TAP ), several groups have unravelled the compositions of the 90S, 60S , and 40S pre-ribosomes and thus expanded the inventory of the assembly machinery and ordered the 40S and 60S maturation pathways [34,35,36,37,38,39,40]. Recently, improvement in these biochemical approaches together with advances of cryo-EM have driven structural studies of pre-ribosomal particles, thus providing high-resolution snapshots of the process of eukaryotic ribosome assembly [4,5,6, 15,16,17, 30,31,32, 41, 42] (Fig. 2a, b).

Fig. 2

Purification and structure of a late 40S pre-ribosome. (a) Protein composition of 80S ribosomes Nob1-D15N particles, purified via ProteinA (pA)-tag. Proteins were separated by SDS-PAGE and visualized by silver staining. Labelled protein bands were characterized by mass spectrometry . (b) Front and back view of cryo-EM structure of a cytoplasmic 40S preribosome (PDB: 6FAI). The 20S rRNA is shown in light gray, r-proteins in dark gray—except of uS3 in yellow. Assembly factors are shown in color: Tsr1 in orange, Rio2 in blue, Pno1 in green, Ltv1 in purple, and Enp1 in red. (Adapted from Scaiola et al. 2018)

3.1 Preparing Yeast Cells for Cryogenic Lysis (Modified from Rout Lab Protocol: Harvesting Cells and Making Yeast Noodles [43])

3.1.1 Buffers and Solutions

Resuspension Buffer: 1.2% PVP-40 (polyvinylpyrrolidone), 20 mM HEPES pH 7.4, Supplemented with 1:100 complete protease inhibitor cocktail tablet (Roche), 2 mM PMSF, 1 mM DTT (see Note 1 ).

3.1.2 Yeast Cell Preparation

• 1. Grow cell culture to OD₆₀₀ = 2.5–3.5 in complete media/ 1.5–2.0 in synthetic media.

• 2. Spin cultures down (4500 × g, 15 min, 4 °C).

• 3. Resuspend pellet in 50 mL ddH₂O on ice. Put resuspended solution into 50 mL Falcon tube(s) and spin down (4500 × g, 5 min, 4 °C). Repeat this step.

• 4. Resuspend pellet on ice in a volume of resuspension buffer equal to the volume of the pellet. Spin down (4500 × g, 15 min, 4 °C). Aspirate all liquid from the pellet.

• 5. Spin just the pellet down again (4500 × g, 2 min, 4 °C) to remove the residual buffer (repeat if needed).

• 6. Pellet should be fairly dry and resemble a thick paste.

• 7. Fill up a Styrofoam box with a liquid nitrogen and prepare a metal holder next to it.

• 8. Use a metal clamp to precool and fill up completely a 50 mL Falcon tube with liquid nitrogen using and fix the tube to a metal holder.

• 9. With a spatula, scoop out cell paste and place into a 20 mL syringe. Press out the cell paste into the liquid nitrogen in the Falcon tube. Avoid touching the liquid nitrogen directly with the tip of the syringe to prevent syringe clogging.

• 10. When all cell paste is gone, remove liquid nitrogen from the tube (poke holes into cap of Falcon tube, screw on the cap and turn tube upside down to pour out the liquid nitrogen).

• 11. Do not tighten the tube completely, in order to allow liquid nitrogen vapor to escape. Store tubes at −80 °C.

3.1.3 Cryogenic Lysis of Yeast Cells (See Also Note 2 ) (Modified from Rout Lab Protocol: Cryogenic Lyses of yeast Cells [43])

• 1. Fill a rectangular ice bucket with liquid nitrogen.

• 2. Pre-chill everything. Immerse the 80 mL stainless steel grinding jars, the stainless steel lid, the grinding balls, steel spoon and the storage tube, with the frozen yeast noodles, in the liquid nitrogen.

• 3. Pre-cooling is finished when nitrogen bath is no longer bubbling vigorously.

• 4. Once everything is chilled pour the noodles into the grinding jar.

• 5. Weigh the grinding jar with noodles and then adjust the counterbalance weight.

• 6. Use 7–8 of the 20 mm stainless steel balls based on the amount of noodles.

• 7. Be sure no liquid nitrogen is in the grinding jar prior to grinding to avoid an explosion.

• 8. Grinding is done in 4 cycles, each cycle composed of 500 rpm, 3 min and in reverse rotation 500 rpm, 2 min (see also Note 3 ).

• 9. Between each cycle the jars are removed and cooled in liquid nitrogen. Do not remove the lid (removal of lid may result in cell loss). To ensure lid is chilled use an empty Falcon tube to pour liquid nitrogen over the top of the grinding jar while the bowl of the grinding jar cools in the liquid nitrogen bath. Do not submerge the jar completely as this will allow liquid nitrogen into the grinding bowl and may also result in cell loss.

• 10. When 4 cycles are complete, carefully remove powder from the balls with a pre-cooled spatula and transfer the powder into a new prechilled 50 mL Falcon tube (if there is powder stuck to the side of the jar repeat 1 grinding cycle) (see Note 4 ).

• 11. Jars and balls are cleaned with warm water.

• 12. Frozen ground cells are stored at −80 °C.

3.1.4 Isolating Pre-ribosomes Using Magnetic Beads (Modified from Oeffinger et al. 2007 [43])

3.1.4.1 Buffers and Solutions

M-IgG Buffer: 20 mM HEPES pH 7.4, KOAc, 40 mM NaCl, 0.5% Triton-X, 0,1% Tween 20.

Supplemented with (see Note 1 ) 2 mM PMSF, 1 mM DTT, 2–10 mM MgCl₂ (optional).

3.1.4.2 Method (See Also Notes 5–8)

• 1. Transfer 2–5 g of ground yeast cells, to a pre-cooled 50 mL falcon, and put it into liquid nitrogen.

• 2. Pour 40 mL M-IgG Buffer + Detergents into cooled sterile glass beaker with sterile magnet fish and start mixing.

• 3. Cool down and sterilize metal sieve in liquid nitrogen.

• 4. Add yeast powder slowly through the sieve into mixing buffer (avoid freezing of clumps).

• 5. Allow powder to resuspend completely in buffer (5–10 min).

• 6. Transfer yeast lysate to 50 mL Falcon tube. Spin down (4500 × g, 5 min, 4 °C) to get rid of big chunks.

• 7. Meanwhile take 100 μL bead slurry and wash 3× with 1 mL M-IgG Buffer + Detergents using Magnet rack to wash away buffer and resuspend in 1 mL M-IgG Buffer + Detergents.

• 8. Filter lysate with 60 mL syringe through 2.7 μM Whatman filter slowly into new 50 mL Falcon (do not push too hard, rather exchange the filter for new one).

• 9. Repeat with 60 mL syringe through 1.6 μM Whatman filter into new 50 mL Falcon (stop as soon as white foam comes out).

• 10. Add beads to clarified yeast lysate, seal with parafilm and incubate 30 min at 4 °C on rotating wheel.

• 11. Collect beads with magnetic rack, wait for complete bead binding (solution clears up).

• 12. Collect flow-through in a new Falcon tube and repeat bead collection to avoid loss of the beads.

• 13. Resuspend all collected beads in 1 mL M-IgG Buffer + Detergents and transfer it into 1.5 mL tube.

• 14. Wash the beads 3× with 1 mL M-IgG Buffer + Detergents using magnetic rack.

• 15. Wash the beads further 3–5× with 1 mL M-IgG Buffer without Detergents (to get rid of foaming).

• 16. Resuspend beads in 50–75 μL M-IgG buffer without Detergents.

• 17. Add 0.5 μL (10 U) TEV protease (Sigma, 20 U/L), seal with parafilm and incubate overnight at 4 °C on rotating wheel.

• 18. Collect flow-through (purified sample) in new tube using magnetic rack.

• 19. Use 5 μL sample per EM Copper Grid.

• 20. To analyze samples, proceed with TCA precipitation, RNA extraction , Silver gel, or RNA gel.

• 21. Boil beads in 25–50 μL 2× LDS and collect LDS in new tube (using magnetic rack) for TEV cleavage control.

3.2 Nuclear Import Assays for Ribosomal Proteins

It is assumed that like a typical import cargo, RanGTP dissociates the r-protein from the importin after arriving in the nuclear compartment. However, r-proteins contain disordered regions, which make them prone to non-specific interactions with other nucleic acids, aggregation and degradation in their non-assembled state. During a single 90 min generation time ~14 million r-proteins synthesized in the cytoplasm, need to be targeted to the yeast nucleus and handed over to the ribosome assembly machinery [1, 44]. Thus, safe and efficient transport of r-proteins to their rRNA binding site is a logistical challenge.

A number of groups have uncovered dedicated chaperones that interact with newly synthesized r-proteins in the cytoplasm [44,45,46,47,48,49,50,51,52,53,54,55]. The dedicated chaperone–r-protein complexes recruit the import machinery and are transported to the nucleus where they are released by the action of RanGTP .

A different mechanism is employed by the r-protein eS26 to reach the 90S pre-ribosome. Like a typical import cargo protein, eS26 uses importins for targeting to the nucleus. However, after reaching the nuclear compartment, eS26 is removed from the importin by an unloading factor, the escortin Tsr2 , without the aid of RanGTP . Tsr2 shields eS26 from proteolysis and enables its safe transfer to the 90S pre-ribosome [56]. Below, we outline a step-by step protocol to investigate the canonical RanGTP dependent disassembly of a Pse1–Slx9 complex (Fig. 3a), and non-canonical Tsr2 dependent disassembly of a Kap123–eS26 complex (Fig. 3b). Slx9 is a predominantly nuclear localized protein, yet it shuttles between the nucleus and the cytoplasm [57, 58]. The import receptor transports Slx9 to the nuclear compartment, where Slx9 facilitates assembly of a Crm1-export complex and efficient export of 40S pre-ribosomes from the nucleus [57,58,59,60].

Fig. 3

In vitro binding assays for nuclear import . (a) RanGTP-dependent release of Slx9 from Pse1 importin. Left panel: Workflow overview of the importin binding assay. POI = protein of interest. Right panel: GST–Pse1 complex formation with Slx9 dissociated in the RanGFP-dependent manner. GST–Pse1 (lanes 1–6) or GST alone (lanes 7–8) was immobilized on Glutathione Sepharose, washed with PBS-KMT and incubated with 4 μM Slx9 for 1 h at 4 °C (lanes 2–4 and 6). Pse1-Slx9 complex was subsequently incubated with 1.5 μM RanGTP under same conditions (lane 5). After washing with PBS-KMT, bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining and Western analyses using α-Slx9 antibodies. L = input. (b) RanGTP-independent release of eS26 from Kap123 importin. Immobilized GST-Kap123 on Glutathione Sepharose was washed and incubated with 4 μM eS26^FLAG for 1 h at 4 °C. GST-Kap123:eS26^FLAG complex was washed and incubated with either buffer alone (lane 2 and 8), 0.375 μM His₆-Ran^QLGTP (left panel lanes 3–6) or 0.375 μM His₆-Tsr2 (right panel lanes 9–12). Samples were withdrawn at the indicated time points and washed with PBS-KMT. Bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining and Western analyses using α-eS26 and α-RanGTP antibodies. L = input. GST-Kap123 is indicated by asterisk. (Adapted from Schütz et al. 2014)

3.2.1 A. RanGTP Mediated Disassembly of a Pse1–Slx9 Complex

3.2.1.1 Buffers and Solutions

PBS-KMT buffer (pH 7.3): 150 mM NaCl, 25 mM Sodium phosphate, 3 mM KCl, 1 mM MgCl₂, 0,1% Tween 20.

3.2.1.2 Method

• 1. Pipet 50 μL of GSH-Sepharose slurry (per reaction) into a 1.5 mL reaction tube and wash 3 times with 1 mL PBS-KMT buffer, spin down in between for 30 s at 850 x g . Wash GSH-Sepharose beads for all reactions together.

• 2. Meanwhile thaw recombinant GST-Pse1 and GST-alone sample on ice.

• 3. Centrifuge the GST-Pse1 and GST-alone or lysates for 10 min at 4 °C at 16,000 x g to pellet aggregates and store the tubes on ice until use.

• 4. Add 12 μg GST-Pse1 or GST-alone (per reaction) to washed GSH-Sepharose beads suspended in 500 μL of PBS-KMT buffer.

• 5. Incubate on a rotating platform for 1 h at 4 °C to immobilize GST-Pse1 or GST-alone on GSH-Sepharose beads.

• 6. Wash samples as indicated in step 1 to remove unbound protein. Keep samples on ice between each washing step.

• 7. To assess the amount of GST-Pse1 or GST-alone bound to the GST-Sepharose beads remove supernatant completely. Add 30 μL 2× LDS sample buffer, mix and heat up at 70 °C for 10 min to elute the bound proteins. Separate the eluted denatured proteins on an 12–15% SDS-PAGE and analyze by Coomassie Blue staining.

• 8. To form an importin–protein complex, add 3–6 μM Slx9 to the immobilized GST-Pse1 or GST-alone on GSH-Sepharose beads suspended in 500 μL PBS-KMT buffer (from step 6).

• 9. Incubate on a rotating platform for 1 h at 4 °C.

• 10. Wash samples as indicated in step 1 to remove unbound protein. Samples are kept on ice between each washing step.

• 11. To assess the amount of GST-Pse1–Slx9 complex bound to the GST-Sepharose beads remove supernatant completely. Add 30 μL 2× LDS sample buffer, mix and heat up at 70 °C for 10 min to elute the bound proteins. Separate the eluted denatured proteins on a 12–15% SDS-PAGE and analyze by Western blotting using antibodies directed against Slx9 .

• 12. To investigate RanGTP sensitivity, incubate the GST-Pse1–Slx9 complex with 500 μL PBS-KMT containing 1.5 μM Ran^QLGTP for 1 h at 4 °C on a rotating wheel. In parallel incubate GST-Pse1–Slx9 in 500 μL PBS-KMT buffer without Ran^QLGTP to assess whether Slx9 is released by buffer alone.

• 13. Wash samples as indicated in step 1 to remove released protein. Keep samples on ice between each washing step.

• 14. Remove supernatant completely, and add 30 μL 2× LDS sample buffer, mix and heat up at 70 °C for 10 min to elute the bound proteins. Separate the eluted denatured proteins on a 12–15% SDS-PAGE and analyze by Coomassie Blue staining and Western blotting using antibodies directed against Slx9 .

3.2.2 Tsr2 Mediated Disassembly of a Kap123–eS26^FLAG Complex (See Also Notes 9–14)

• 1. Pipet 50 μL of GSH-Sepharose slurry (per reaction) into a 1.5 mL reaction tube and wash 3 times with 1 mL PBS-KMT buffer, spin down in between for 30 s at 850 × g. Wash GSH-Sepharose beads for all reactions together.

• 2. Meanwhile thaw recombinant GST-Kap123 and GST-alone sample on ice.

• 3. Centrifuge the GST-Kap123 and GST-alone or lysates for 10 min at 4 °C at 16,000  × g to pellet aggregates and store the tubes on ice until use.

• 4. Add 12 μg GST-Kap123 or GST-alone to washed GSH-Sepharose beads suspended in 500 μL of PBS-KMT buffer.

• 5. Incubate on a rotating platform for 1 h at 4 °C to immobilize GST-Kap123 or GST-alone on GSH-Sepharose beads.

• 6. Wash samples as indicated in step 1 to remove unbound protein. Keep samples on ice between each washing step.

• 7. The amount of GST-Kap123 or GST-alone bound to the GST-Sepharose beads can be assessed at this stage. For this, remove supernatant completely. Add 30 μL 2× LDS sample buffer, mix and heat up at 70 °C for 10 min to elute the bound proteins. Separate the eluted denatured proteins on a 12–15% SDS-PAGE and analyze by Coomassie Blue staining.

• 8. To form importin–protein incubate the immobilized GST-Pse1 or GST-alone on GSH-Sepharose beads with 3–6 μM eS26^FLAG in 500 μL PBS-KMT buffer from step 6.

• 9. Incubate on a rotating platform for 1 h at 4 °C.

• 10. Wash samples as indicated in step 1 to remove unbound protein. Samples are kept on ice between each washing step.

• 11. To assess the amount of GST-Kap123–eS26^FLAG complex bound to the GST-Sepharose beads, remove supernatant completely. Add 30 μL 2× LDS sample buffer, mix and heat up at 70 °C for 10 min to elute the bound proteins. Separate the eluted denatured proteins on a 12–15% SDS-PAGE and analyze by Western blotting using antibodies directed against FLAG tag.

• 12. To investigate Tsr2 dependent disassembly of the complex from step 10, incubate the GST-Kap123–eS26^FLAG complex with 500 μL PBS-KMT containing 1.5 μM Tsr2 . In parallel, incubate GST-Kap123–eS26^FLAG with 500 μL of PBS-KMT buffer as a control to assess whether eS26^FLAG, is released by buffer itself.

• 13. Wash samples as indicated in step 1 to remove released protein. Keep samples on ice between each washing step.

• 14. Remove supernatant completely, and add 30 μL 2× LDS sample buffer, mix and heat up at 70 °C for 10 min to elute and denature the bound proteins. Separate the eluted denatured proteins on a 12–15% SDS-PAGE and analyze by Coomassie Blue staining and Western blotting using antibodies directed against FLAG tag.

3.2.3 Cell Biological Assays for r-protein Nuclear Import

It is complicated to monitor the nuclear targeting of r-proteins in vivo since at steady state they reside in the cytoplasm. Therefore, one strategy is to uncouple r-protein nuclear import from their export by selectively impairing recruitment to the pre-ribosome. Fusing GFP to either the N- or C- terminus can prevent r-protein incorporation into the pre-ribosome. These non-functional proxy constructs can be employed to determine nuclear localization signals within r-proteins [56]. Further, by monitoring their mislocalization to the cytoplasm in different importin-deficient (Table 1) and/or mutant strains, these non-functional r-protein fusions (listed in Table 3) can pinpoint their nuclear pathway(s).

Table 1 Yeast strains used for ribosomal nuclear import studies

3.3 Nuclear Export Assays for Pre-ribosomes

3.3.1 Monitoring Nuclear Export of Pre-ribosomes (See Also Notes 15 and 16)

Transport of 60S and 40S pre-ribosomes from the nucleus into the cytoplasm can be investigated by monitoring the localization of r-protein fusions with GFP (uL23-GFP, uL5-GFP, uL18-GFP for the 60S subunit and uS5-GFP for the 40S subunit) in distinct mutant strains [72,73,74,75]. These r-protein fusions are stably incorporated into pre-ribosomes, and their steady-state localization in wild type (WT) cells is cytoplasmic. Nuclear accumulation of these reporters indicates either an early pre-ribosome assembly and/or export defect. Next to the r-protein GFP reporters, nuclear accumulation of GFP fused export adaptor factors serve as reporters for impairment in late assembly steps and/or of export of 60S (Nmd3-GFP) and 40S (Rrp12-GFP and Rio2-GFP) pre-ribosomes, respectively (Fig. 4).

Fig. 4

Monitoring ribosome export defect by fluorescence microscopy. GFP-reporters for large r-subunit (uL18-GFP or Nmd3-GFP) and small r-subunit (uS5-GFP or Rrp12-GFP) were transformed into WT or indicated mutant strains and they localization was analyzed by fluorescence microscopy. Scale bar = 5 μm. Output of the experiment is indicated on the left scheme: accumulation of the reporter in the nucleus in case of export defect as seen for bud20Δ and yrb2Δ

3.3.1.1 Method

• 1. Transform yeast strains with the reporter plasmids (uL18-GFP, uS5-GFP) and plate on appropriate media.

• 2. The transformants expressing reporters are grown in appropriate liquid media (for mutants at permissive temperature) until saturation (overnight).

• 3. The next day, dilute cells into 10 mL fresh media and grown until mid-log phase for at least 2–3 divisions. (In the case of cold or temperature sensitive mutants, shift the cultures to the appropriate restrictive temperature after 2–3 divisions of growth at permissive temperature).

• 4. Pellet cells by centrifugation in a 15 mL Falcon tube (  450 × g, 3 min) and wash once with 10 mL dH₂O.

• 5. Pour off the supernatant and resuspend the cell pellet in remaining liquid.

• 6. Place 2 μL of cell suspension on a slide, press a coverslip over before inspection under a fluorescence microscope.

3.3.2 Reconstitution of Crm1-Nuclear Export Complexes In Vitro

In actively growing yeast cells , every minute ~25 pre-ribosomal particles are exported into the cytoplasm [2]. The exportin Crm1 (yeast Xpo1) plays an essential role in transporting pre-ribosomes into the cytoplasm. To initiate export, Crm1 , in the presence of RanGTP recognizes a nuclear export signal (NES) on adaptor proteins bound to pre-ribosomes, and cooperatively forms a Crm1-export complex [1, 23, 24, 76, 77]. In vitro binding assay with recombinant Crm1 and Ran^QL are used to analyze potential NES containing export adaptors of preribosomal subunits [58]. Here, using Ltv1 :Crm1 :Ran^QLGTP as an example to outline a protocol for assembly of a trimeric Crm1-export complex in vitro (Fig. 5a).

Fig. 5

In vitro binding assays for nuclear export. (A) Ltv1 NES-dependent export complex formation. Left panel: Workflow overview of the binding assay. POI = protein of interest. Right panel: GST-Ltv1 or GST-Ltv1 lacking NES sequence (450-463aa) was immobilized on the Glutathione Sepharose, washed with PBS-KMT and incubated with buffer alone (lanes 1 and 5), buffer with 2μM His6-Ran^QLGTP (lanes 2 and 6), 50nM Crm1-His6 (lanes 3 and 7) or 50nM Crm1-His6 and 2μM His6-Ran^QLGTP simultaneously (lane 4 and 8) for 1h at 4°C. Bound proteins were washed with PBS-KMT, eluted in 2XLDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining and Western analyses using α-RanGTP and α-Crm1 antibodies. L = input. Adapted from Fischer et al., 2015. (B) Heterodimeric transport receptor Mex67-Mtr2 interacts with FG repeat sequences of different nucleoporins. Left panel: Workflow overview of the binding assay. POI = protein of interest. Right panel: Indicated GST-Nucleoporin fusion proteins were immobilized on Glutathione Sepharose and washed with PBS-KMT before 1 h incubation with 3.6 μg His6-Mex67-Mtr2 at 4°C. Bound proteins were washed, eluted with 2X LDS buffer and analyzed by SDS-PAGE followed by Coomassie staining or Western blotting using α-Mex67/Mtr2 antibody. L = Load; Asterisks indicate Mex67 bands. Adapted from Altvater et al., 2012

3.3.2.1 Buffers and Solutions

PBS-KMT buffer (pH 7.3): 150 mM NaCl, 25 mM sodium phosphate, 3 mMKCl, 1 mM MgCl₂ Tween-20.

3.3.2.2 Method (See Also Notes 17–21)

• 1. Pipet 50 μL of GSH-Sepharose slurry (per reaction) into a 1.5 mL reaction tube and wash 3 times with 1 mL PBS-KMT buffer; spin down in between for 30 s at 850 × g. Wash GSH-Sepharose beads for all reactions together.

• 2. In meanwhile thaw GST-fusion proteins (GST-Ltv1 , GST-Ltv1 ^ΔNES ) and GST sample on ice.

• 3. Centrifuge the GST-Ltv1 and GST-alone or lysates for 10 min at 4 °C at 16,000 × g to pellet aggregated proteins and store the tubes on ice until use.

• 4. Add 12 μg GST-Ltv1 protein or GST-alone to washed GSH-Sepharose beads suspended in 500 μL PBS-KMT buffer.

• 5. Incubate on a rotating platform for 1 h at 4 °C to immobilize GST-Ltv1 and GST-alone on GSH-Sepharose beads.

• 6. Wash samples as indicated in step 1 to remove unbound protein. Keep samples on ice between each washing step.

• 7. The amount of GST-Ltv1 or GST-alone bound to the GST-Sepharose beads can be assessed at this stage. For this, remove supernatant completely, add 30 μL 2× LDS sample buffer, mix and heat up at 70 °C for 10 min to elute the bound proteins. Separate the eluted denatured proteins on a 12–15% SDS-PAGE and analyze by Coomassie Blue staining.

• 8. Thaw on ice and centrifuge substrate proteins (Ran^QLGTP and Crm1 ) at 10 min at 4 °C at 16,000 × g to pellet aggregated proteins.

• 9. Add simultaneously 2 μM Ran^QLGTP and 50 nM Crm1 to GSH-Sepharose beads immobilized GST-Ltv1 or GST-alone and suspended in 500 μL PBS-KMT buffer. In parallel, as controls, incubate immobilized GST-Ltv1 with Crm1 and Ran^QLGTP separately.

• 10. Incubate on a rotating platform for 1 h at 4 °C.

• 11. Wash samples as indicated in step 1 to remove unbound protein. Keep samples on ice between each washing step.

• 12. Remove supernatant completely. Add 30 μL 2× LDS sample buffer, mix, and heat up at 70 °C for 10 min to elute and denature the bound proteins.

• 13. Separate proteins on a 12–15% SDS-PAGE and subject them to Coomassie Blue staining and Western blot analysis using directed antibodies against Ran and Crm1 .

3.3.3 FG-Repeat Binding Assay

40S and 60S pre-ribosomes are transported through NPC into the cytoplasm by export factors that interact transiently with FG-repeat lining nucleoporins that line the transport channel. In addition to RanGTP-dependent exportins such as Crm1 and Exp5 [23, 24, 78] several shuttling factors such as Mex67-Mtr2 , Arx1 , Ecm1 , Bud20 , and Rrp12 promote nuclear export of pre-ribosomes by directly interacting with FG-rich meshwork of NPC [1, 57, 71, 79,80,81,82]. Here, we outline a protocol that demonstrates interactions between the heterodimeric transport receptor Mex67-Mtr2 with FG-rich nucleoporins (Fig. 5b).

3.3.3.1 Buffers and Solutions

Universal buffer (Künzler and Hurt, 1998 [83]): 20 mM HEPES–KOH, pH 7.0, 100 mM KOAc, 2 mM Mg(OAc, 0,1% Tween 20, 10% glycerol. Supplemented with (see Note 1 ) 1 complete protease inhibitor cocktail tablet (Roche) per 100 mL 5 mM ß-mercaptoethanol.

3.3.3.2 Method (See Also Notes 22–28)

• 1. Wash 80 μL of GSH-Sepharose slurry (per reaction) into a 1.5 mL reaction tube and wash 3 times with 1 mL universal buffer; spin down in between for 30 s at 3000 rpm at 4 °C. Wash GSH-Sepharose beads for all reactions together.

• 2. Meanwhile thaw lysates containing GST-nucleoporins (Nup1, Nup100, Nup116, Nup42) or GST alone on ice.

• 3. Centrifuge the lysates for 10 min at 4 °C at  16,000 × g to pellet aggregated proteins and store the tubes on ice until use.

• 4. Add 1 mL of each lysate to washed GSH-Sepharose beads (combine GSH-Sepharose beads for reactions with same GST-fusion protein) (see Note 22 ).

• 5. Incubate tubes on a rotating platform for 1 h at 4 °C to immobilize GST and GST fusion protein on GSH-Sepharose beads.

• 6. Wash samples 3× (850 × g, 30 s, 4 °C) with 1.5 mL universal buffer and resuspended in 1 mL buffer each.

• 7. Split beads, by pipetting 500 μL, into two new 1.5 mL eppendorf tubes.

• 8. Add 1–5 μg of recombinant affinity-purified His₆-tagged protein (3.6 μg for Mex67-Mtr2 ) or respective volume of buffer as negative control to the GST-/GST-nucleoporin-coated beads and fill up to 1 mL with universal buffer.

• 9. Incubate tubes on a rotating platform for 1 h at 4 °C.

• 10. Wash beads 3× (850 × g, 30s , 4 °C) with 1 mL universal buffer, remove supernatant completely. Elute the bound proteins by adding 80 μL 2× LDS buffer and heating at 70 °C for 10 min.

• 11. Spin down beads and analyze the supernatant by SDS-PAGE, Coomassie Blue staining and Western analysis using directed antibodies against Mex67-Mtr2 (see Note 28) .

3.4 Genetic Analyses of Nuclear Export

Pre-ribosomal particles are among the largest and most abundant cargoes that need to cross the permeability barrier of the NPC. Rapid transit through the NPC is especially important, as any delay within the channel may hinder transport of other cargoes. Therefore, pre-ribosomal particles deploy a set of multiple redundant export receptors for their transport to the cytoplasm [1, 21]. High-copy suppressor screens and synthetic lethal interactions and in budding yeast have uncovered ancillary factors that also mediate efficient pre-ribosome nuclear export . These approaches have proved to be powerful tools to either discover new components of the ribosome export machinery or to directly test functional overlap of a factor of interest with the process of nuclear export .

The essential export adaptor Nmd3 was identified as a high copy suppressor of the bud20 Δ mutant slow growth and 60S pre-ribosome export defect [72]. Further, the bud20 Δ strain exhibits synthetic lethal or synthetic enhanced growth defects when combined with mutants of established 60S pre-ribosome export factors (nmd3ΔNES1 , xpo1-1 , arx1 Δ , gle2-1, and ecm1 Δ ), included mex67 and mtr2 mutant alleles ( mex67kraa and mtr2-33 ) that are specifically impaired in 60S subunit [72]. These analyses culminated in the discovery of new FG-repeat binding proteins that functions directly to the process of nuclear export of 60S pre-ribosomes.

Similarly, a high-copy suppressor screen aimed at identifying genes that suppress the impaired growth of the slx9Δ mutant, led to the discovery of the mRNA and 60S export receptor Mex67-Mtr2 as an auxiliary export receptor for in 40S pre-ribosome export [57]. Mex67-Mtr2 mutants that fail to bind to the 40S pre-ribosome were synthetically lethal when combined with the slx9Δ mutant [57]. Further, the slx9Δ mutant displayed a synthetic growth defect when combined with a strain expressing Rrp12-GFP. Rrp12 is a 40S pre-ribosome export factor that directly interacts with FG-rich nucleoporins [79]. Finally, Yrb2 that stimulate the assembly of Crm1-export complexes on certain NES-containing cargos by cooperatively binding Crm1 and RanGTP also showed a strong genetic interaction with Slx9 [57]. This genetic link culminated in the identification of a new type of RanGTP binding protein that binds to a specific NES-containing cargo and RanGTP and promotes Crm1 recruitment.

Tables 2 and 3 provide a comprehensive list of mutants and their sources to investigate 60S and 40S preribosome export using standard yeast genetic approaches.

Table 2 Yeast strains used for ribosomal nuclear export studies

Table 3 Plasmids for genetic studies of nucleo-cytoplasmic transport

4 Conclusions

Diverse approaches in budding yeast have greatly advanced our understanding of ribosome assembly and transport. Genetic perturbation of energy-consuming enzymes can now be combined with biochemical affinity purifications to obtain high-resolution snapshots of the ribosome assembly pathway. We also anticipate that traditional genetic, biochemical, and cell-biological approaches will provide eagerly awaited integration of the ribosome pathway with other biological pathways including nucleocytoplasmic transport , cellular quality control and signaling.

5 Notes

• 1. Add just before use.

• 2. Outlined below is a step-by-step protocol to lyse frozen yeast noodles using a planetary ball mill (Pulverisette 6, Fritsch).

• 3. You MUST hear the balls rattling around in the jar!

  If there is no rattling: add or remove balls to the jar until you hear it rattle. It is not considered a grinding cycle unless there is rattling.

• 4. Typically, ~90% of yeast cells can be disrupted in such procedure.

• 5. Use cryoprotective gloves for harvesting cells.

• 6. Work on ice or cold room as much as possible.

• 7. Prepare powder stock from big culture, store at −80 °C and weight only necessary amount for an experiment.

• 8. The M-IgG beads are prepared by conjugation of Dynabeads (Invitrogen) with rabbit IgG (Sigma) according to the standard protocol.

• 9. Plasmids encoding GST-Kap123 and GST-Pse1 [86], Ran^QLGTP mutant [87] originate from Schlenstedt laboratory. Slx9 [58] and eS26^Flag (His₆-eS26^FLAG; [56]) can be requested from Panse laboratory.

• 10. GST recombinant proteins were expressed in E.coli and lysed in PBS-KMT (150 mM NaCl, 25 mM sodium phosphate, 3 mM KCl, 1 mM MgCl2, 0.1% Tween, pH 7.3) buffer. His-tagged proteins were purified in buffer containing 50 mM HEPES pH 7.5, 50 mM NaCl, 10% glycerol. It is possible to use directly E.coli lysates containing recombinant proteins without purification steps [56, 86]. His₆-tagged importins and Ran^QLGTP (His₆-Gsp1Q71L-GTP) were expressed and purified as previously described [86,87,88].

• 11. To prepare E. coli lysate BL21 cells was grown overnight in 100 mL LB media at 37 °C and 220 rpm. Diluted in 1 l of LB media to OD₆₀₀ 0.2 and again incubated at the same condition. Cells were harvest at logarithmic growth phase and resuspended in PBS-KMT. After cell lysis and centrifugation supernatant is used for the experiment.

• 12. To prevent unspecific interactions with GST-tagged proteins or GST it is possible to add E. coli lysate to the incubation reaction (100 μL is usually sufficient).

• 13. The Ran protein mutant used in this assay, Ran^QL (Gsp1Q71L), is a GTPase deficient mutant originating from Schlenstedt laboratory. His₆-Ran^QL is stored in KPi buffer (24.8 mM KH₂PO₄, 25 mM, K₂HPO₄, 20 mM KCl, 5 mM MgCl₂, 2 mM imidazole, 5 mM β-mercaptoethanol, pH 6.8).

• 14. Antibodies against FLAG tag are commercially available reagent from Sigma (F1804).

• 15. This assay can be performed with strains containing the reporters growing on solid selective media. In this case, strains typically in stationary phase, are spread on fresh YPD plates to induce ribosome production. After growing the cells for 4–8 h at the appropriate temperature, cells can be scraped from plate using an inoculation loop, mixed with 2 μL H₂O placed on a glass slide and analyzed under a fluorescence microscope.

• 16. The uL23-GFP and uS5-GFP reporter plasmids with different selection markers (LEU2, URA3, TRP1, and HIS3) originate from the Hurt laboratory [73, 74], the uL5-GFP reporter (LEU2) originates from the Silver laboratory [75], and the uL18-GFP reporter plasmids (LEU2, URA3, TRP1 and HIS3) can be requested from the Panse laboratory [72]. All the above reporters are expressed under the endogenous promoters. The ribosomal protein uL18 can be C-terminally tagged with GFP at the genomic locus for monitoring the nuclear export of the large pre-ribosomal subunit.

• 17. Plasmids for yeast Crm1 (Xpo1-His₆) and Ran^QL mutant (His₆-Gsp1Q71L) originate from Schlenstedt lab [87], GST-Ltv1 and GST-Ltv1 ^ΔNES can be requested from Panse lab [58].

• 18. Recombinant proteins were expressed in E. coli and either purified in PBS-KMT or used as lysate.

• 19. GST-tagged proteins and His₆-Gsp1Q71L was expressed in E. coli as described in [87, 88]. Crm1-His₆ was expressed in E. coli as described in [58]. GST, GST-Ltv1 and GST-Ltv1 ^ΔNES , and Crm1-His₆ proteins are stored in PBS-KMT buffer. His₆-Gsp1Q71L is stored in KPi buffer (24.8 mM KH₂PO₄, 25 mM, K₂HPO₄, 20 mM KCl, 5 mM MgCl₂, 2 mM imidazole, 5 mM β-mercaptoethanol, pH 6.8).

• 20. To prevent unspecific interactions with GST-tagged proteins or GST it is possible to add E.coli lysate to the incubation reaction (100 μL is usually sufficient).

• 21. Antibodies against Ran and Crm1 can be requested in Panse laboratory.

• 22. For the load: Respective amount of affinity-purified His₆-tagged Mex67-Mtr2 is added to final 100 μL 2× LDS, heated at 70 °C for 10 min.

• 23. All the steps should be carried out at 4 °C or on ice.

• 24. Cut pipette tip when pipetting beads.

• 25. Recombinant His₆-tagged protein (Mex67-Mtr2 ) was expressed in BL21 E. coli strain, lysed by sonication in lysis buffer (500 mM NaCl, 20 mM Tris–HCl pH 8.0, 0.1% Triton X-100, 1 mM PMSF) and purified using Ni-Sepharose (GE Healthcare) following a standard protocol.

• 26. The lysates containing GST-tagged nucleoporins (Nup1, Nup100, Nup116, Nup42) or GST alone were expressed in BL21 E. coli strain lysed by sonication in universal buffer.

• 27. Due to different expression levels, different volumes of GST-NUPs lysates may need to be used to uniformly load GSH-Sepharose beads.

• 28. The antibody directed against Mex67-Mtr2 can be requested from the Panse laboratory [72].