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

Abstract

Ribosome biogenesis takes place successively in the nucleolar, nucleoplasmic, and cytoplasmic compartments. Numerous nonribosomal factors transiently associate with the nascent ribosomes, but the mechanisms driving ribosome formation are mostly unknown. Here, we show that an energy-consuming enzyme, the AAA-type (ATPases associated with various cellular activities) ATPase Rix7, restructures a novel pre-60S particle at the transition from the nucleolus to nucleoplasm. Rix7 interacts genetically with Nsa1 and is targeted to the Nsa1-defined preribosomal particle. In vivo, Nsa1 cannot dissociate from pre-60S particles in rix7 mutants, causing nucleolar Nsa1 to escape to the cytoplasm, where it remains associated with aberrant 60S subunits. Altogether, our data suggest that Rix7 is required for the release of Nsa1 from a discrete preribosomal particle, thereby triggering the progression of 60S ribosome biogenesis.

Figure 1.

Rix7 is functionally linked to the WD40-repeat protein Nsa1. (A) Identification of Nsa1 in an sl screen with the rix7-31 mutant allele. Mutant sl30 was transformed with YCplac111 (empty vector), pHAC111-RIX7, pHAC111-rix7-31, and YCplac111-NSA1. Transformants were streaked out on SDC-Leu-Trp and SDC + 5-fluoroorotic acid (5-FOA) plates, which were incubated for 5 d at 30°C. (B) The rix7-31 or rix7Δ14N mutants synthetically enhance the growth phenotype of the nsa1-6 strain. The RIX7/NSA1 double shuffle strain was transformed with plasmids that carry the indicated wild-type or mutant alleles. After 5-FOA shuffling at 23°C, cells were spotted in 10-fold serial dilution steps onto yeast peptone dextrose plates, which were incubated for 4 d at 30°C.

Figure 2.

Rix7 is associated with the Nsa1 pre-60S particle. (A) Protein composition of the Nsa1 pre-60S ribosomal particle. Nsa1-TAP was affinity-purified via the TAP method, and the final EGTA eluate was analyzed by SDS-PAGE and Coomassie staining. The indicated proteins (1–27) were identified by mass spectrometry. (B) Nsa1 defines a distinct pre-60S ribosomal particle that specifically carries Rix7. The indicated TAP-tagged bait proteins were affinity-purified from cells expressing Rix7-GFP. The final EGTA eluates were analyzed by SDS-PAGE and Coomassie staining (top) and Western blotting using anti-GFP, anti-Noc1, anti-Noc3, anti-Nop7, anti-Nog1, anti-Nog2, anti-Nmd3, and anti-Rpl3 antibodies (bottom). The bands marked with asterisks correspond to the purified bait proteins. MW, molecular weight protein standard; Rpls, ribosomal L proteins.

Figure 3.

Nsa1-GFP mislocalizes to the cytoplasm in the rix7Δ14N mutant. The subcellular localization of the indicated GFP-tagged proteins was assessed by fluorescence microscopy in wild-type RIX7 and rix7Δ14N mutant cells that were first grown in yeast peptone dextrose at 23°C and then shifted for 3 h to 30°C. Location of Nsa1-GFP was also determined in RIX7 and rix7Δ14N cells that were grown at the permissive temperature of 23°C (top). Bar, 5 μm.

Figure 4.

Nsa1 is associated with mature 60S ribosomes in the rix7Δ14N mutant. (A) Affinity purification of Nsa1-TAP from wild-type RIX7 or rix7Δ14N mutant cells that were first grown at 23°C and then shifted for 3 h to 30°C. The final EGTA eluates were analyzed by SDS-PAGE and Coomassie staining (top) and Western blotting using anti-Nop7, anti-Nog1, anti-CBP, anti-Rpl3, anti-Rpp0, anti-Rpl10, and anti-Rps8 antibodies (bottom). The band marked with an asterisk corresponds to Rpp0. The bands corresponding to ribosomal proteins are indicated. (B) rRNA composition of Nsa1-TAP affinity-purified from wild-type RIX7 or rix7Δ14N mutant cells that were first grown at 23°C and then shifted for 3 h to 30°C. Aliquots of total RNA and of the final EGTA eluates were resolved on a 1.5% agarose-formaldehyde gel, transferred to a nylon membrane, and analyzed by Northern blotting using the indicated probes to detect pre-rRNA and mature rRNA species. (C) Affinity purification of mature 60S ribosomal subunits via Rpl24A-TAP from wild-type RIX7 or rix7Δ14N mutant cells expressing Nsa1-GFP. Cells were first grown at 23°C and then shifted for 3 h to 30°C. The final EGTA eluates were analyzed by SDS-PAGE and Coomassie staining (top) and Western blotting using anti-GFP, anti-CBP, anti-Rpl3, anti-Rpp0, and anti-Rps8 antibodies (bottom). The bands marked with asterisks contain Stm1, Rpl5, Rpp0/Asc1, and Rpl2/Rps1/Rps3/Rps4/Rps0/Rpl8/Rps6, respectively. MW, molecular weight protein standard.

Figure 5.

Nsa1 is associated with translating ribosomes in the rix7Δ14N mutant. Whole cell lysates derived from wild-type RIX7 and rix7Δ14N mutant strains (both expressing Nsa1-TAP), which were first grown at 23°C and then shifted for 3 h to 30°C, were analyzed by sucrose gradient centrifugation under polysome-conserving conditions (A) or polysome runoff conditions (B). The profiles were recorded at A₂₅₄. The peaks of free 40S and 60S subunits, 80S ribosomes, and polysomes are indicated. Half-mers are indicated by arrowheads. Aliquots of the total cell lysate (T) and the fractions from the gradient were analyzed by Western blotting using anti-ProtA and anti-Rpl3 antibodies. The fractions boxed with broken lines indicate the 80S fractions into which the polysomes are redistributed under polysome runoff conditions.

Figure 6.

Changes in the composition of “upstream” and “downstream” pre-60S ribosomal particles isolated from the rix7Δ14N mutant. Nop7-TAP, Rix1-TAP, and Kre35-TAP were affinity-purified from wild-type RIX7 or rix7Δ14N mutant cells expressing Nsa1-GFP. Cells were first grown at 23°C and then shifted for 3 h to 30°C. The final EGTA eluates were analyzed by SDS-PAGE and Coomassie staining (top) and Western blotting using anti-GFP and anti-Rpl3 antibodies (bottom). The indicated proteins (1–13) were identified by mass spectrometry. White lines indicate that intervening lanes have been spliced out. MW, molecular weight protein standard.