Figure 4. The Rsa4–Nsa2 interaction is... | Rockefeller University Press

Figure 4.

$Figure 4. The Rsa4–Nsa2 interaction is essential for ribosome biogenesis. (A) In vitro reconstitution of the Nsa2–Rsa4 interaction. Purified MBP-Nsa2 wild-type (wt; lanes 3 and 4), Nsa2 Y90A (lanes 5 and 6), Nsa2 Y90F (lanes 7 and 8), and MBP alone (lanes 9 and 10) were immobilized on amylose beads and incubated with E. coli lysate with or without HIS-TEV-Rsa4 (for input, see lanes 1 and 2). Eluates were analyzed by 4–12% SDS-PAGE and Coomassie staining, and the positions of the bands are indicated. (B) The highly conserved tyrosine 90 in Nsa2 is essential for yeast cell growth. Nsa2 shuffle strain was transformed with plasmids carrying the NSA2 or the indicated nsa2 mutant alleles. Complementation was analyzed by incubation of transformants on SDC+FOA plates at 30°C for 2 d. (C) Overexpression of nsa2 Y90A is dominant lethal. A yeast wild-type strain with endogenous NSA2 was transformed with 2μ plasmids carrying NSA2 or nsa2 Y90A alleles under the control of the galactose-inducible GAL promoter. The dominant-negative phenotype by NSA2 overexpression was tested on galactose-containing plates after incubation for 3 d at 30°C. (D) Dominant-lethal nsa2 Y90A induction arrests 60S biogenesis. Pulse-chase analysis of HA-Rpl25-Flag-ProtA (uL23) isolated from cells expressing dominant-negative nsa2 Y90A and wild-type Nsa2. HA-Rpl25-Flag-ProtA was pulsed for 7 min with Ome-Tyr after a 60-min galactose induction, and subsequently chased for 20 min with glucose/tetracycline in cells expressing GAL::NSA2 (WT) or GAL::nsa2 Y90A (Y90A). Affinity-purified HA-Rpl25-Flag-ProtA was analyzed by SDS-PAGE and Coomassie staining, and bands were identified by mass spectrometry. (E) Mutant Nsa2 Y90A is efficiently assembled into preribosomes. Whole-cell lysates derived from wild-type cells expressing plasmid-borne NSA2-FTpA and nsa2 Y90A-FTpA, respectively, were fractionated on a 10–50% sucrose gradient, and fractions were analyzed by Western blotting using anti-ProtA antibodies to detect wild-type and mutant Nsa2 proteins. (F) Dominant-lethal nsa2 Y90A induction causes a defect in 60S subunit export. Wild-type yeast cells were transformed with plasmids harboring the 60S reporter Rpl25-EGFP (uL23) and mRFP-Nop1, and 2μ plasmids harboring GAL::NSA2 or GAL::nsa2 Y90A alleles, respectively. Cells were shifted to galactose-containing medium for 6 h before intracellular localization of Rpl25-EGFP, and mRFP-Nop1 was analyzed by fluorescence microscopy.$

The Rsa4–Nsa2 interaction is essential for ribosome biogenesis. (A) In vitro reconstitution of the Nsa2–Rsa4 interaction. Purified MBP-Nsa2 wild-type (wt; lanes 3 and 4), Nsa2 Y90A (lanes 5 and 6), Nsa2 Y90F (lanes 7 and 8), and MBP alone (lanes 9 and 10) were immobilized on amylose beads and incubated with E. coli lysate with or without HIS-TEV-Rsa4 (for input, see lanes 1 and 2). Eluates were analyzed by 4–12% SDS-PAGE and Coomassie staining, and the positions of the bands are indicated. (B) The highly conserved tyrosine 90 in Nsa2 is essential for yeast cell growth. Nsa2 shuffle strain was transformed with plasmids carrying the NSA2 or the indicated nsa2 mutant alleles. Complementation was analyzed by incubation of transformants on SDC+FOA plates at 30°C for 2 d. (C) Overexpression of nsa2 Y90A is dominant lethal. A yeast wild-type strain with endogenous NSA2 was transformed with 2μ plasmids carrying NSA2 or nsa2 Y90A alleles under the control of the galactose-inducible GAL promoter. The dominant-negative phenotype by NSA2 overexpression was tested on galactose-containing plates after incubation for 3 d at 30°C. (D) Dominant-lethal nsa2 Y90A induction arrests 60S biogenesis. Pulse-chase analysis of HA-Rpl25-Flag-ProtA (uL23) isolated from cells expressing dominant-negative nsa2 Y90A and wild-type Nsa2. HA-Rpl25-Flag-ProtA was pulsed for 7 min with Ome-Tyr after a 60-min galactose induction, and subsequently chased for 20 min with glucose/tetracycline in cells expressing GAL::NSA2 (WT) or GAL::nsa2 Y90A (Y90A). Affinity-purified HA-Rpl25-Flag-ProtA was analyzed by SDS-PAGE and Coomassie staining, and bands were identified by mass spectrometry. (E) Mutant Nsa2 Y90A is efficiently assembled into preribosomes. Whole-cell lysates derived from wild-type cells expressing plasmid-borne NSA2-FTpA and nsa2 Y90A-FTpA, respectively, were fractionated on a 10–50% sucrose gradient, and fractions were analyzed by Western blotting using anti-ProtA antibodies to detect wild-type and mutant Nsa2 proteins. (F) Dominant-lethal nsa2 Y90A induction causes a defect in 60S subunit export. Wild-type yeast cells were transformed with plasmids harboring the 60S reporter Rpl25-EGFP (uL23) and mRFP-Nop1, and 2μ plasmids harboring GAL::NSA2 or GAL::nsa2 Y90A alleles, respectively. Cells were shifted to galactose-containing medium for 6 h before intracellular localization of Rpl25-EGFP, and mRFP-Nop1 was analyzed by fluorescence microscopy.