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Hrr25/CK1δ-directed release of Ltv1 from pre-40S ribosomes is necessary for ribosome assembly and cell growth.

Ghalei H, Schaub FX, Doherty JR, Noguchi Y, Roush WR, Cleveland JL, Stroupe ME, Karbstein K - J. Cell Biol. (2015)

Bottom Line: Conversely, phosphomimetic Ltv1 variants rescued viability after Hrr25 depletion.Finally, Ltv1 knockdown in human breast cancer cells impaired apoptosis induced by CK1δ/ε inhibitors, establishing that the antiproliferative activity of these inhibitors is due, at least in part, to disruption of ribosome assembly.These findings validate the ribosome assembly pathway as a novel target for the development of anticancer therapeutics.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cancer Biology and Department of Chemistry, The Scripps Research Institute, Jupiter, FL 33458.

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Ltv1 release is the essential function of Hrr25. (A) Growth of wild-type yeast (BY4741), and those with galactose-inducible/glucose-repressible Hrr25 (GAL::Hrr25) or lacking Ltv1 (ΔLtv1), and of double mutant GAL::Hrr25;ΔLtv1 and ΔHrr25;ΔLtv1 cells, was compared. Fig. S2 B has a close-up to compare colony size. (B) Growth rates of wild-type, GAL::Hrr25,ΔLtv1, and GAL::Hrr25;ΔLtv1 yeast cells, as determined by their doubling times in YPD. All measurements were done in triplicate, and error bars show the standard deviation of these data. (C) Northern blot analysis of the 18S rRNA precursor (20S) and 25S rRNA. The quantitation shown below the data is the mean and standard deviation from four independent experiments. (D) Dominant-negative effects from Ltv1-S/A. Wild-type yeast (BY4741) were transformed with plasmids encoding wild-type Ltv1, Ltv1-S/A, or Ltv1-S/D under a galactose-inducible promoter. Induction of Ltv1 is weakly dominant-negative for wild-type Ltv1 and Ltv1-S/D, and strongly dominant-negative for Ltv1-S/A. (E) Growth of GAL::Hrr25;ΔLtv1 cells carrying plasmids of wild-type or phosphomutant Ltv1, with and without Hrr25, is compared on glucose (no endogenous Hrr25) and galactose-containing (endogenous Hrr25) plates. (F) Alanine mutations in the Ltv1 phosphosite block Enp1/Ltv1 release in vitro. Shown are Western blots of gradient fractions from ΔLtv1;Rio2TAP ribosomes, purified from cells containing Hrr25_I82G, and reconstituted on the calmodulin beads with Enp1–Yar1–Rps3 and wild-type Ltv1 or Ltv1-S/A. The positions of 40S ribosomes and free proteins are indicated.
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fig4: Ltv1 release is the essential function of Hrr25. (A) Growth of wild-type yeast (BY4741), and those with galactose-inducible/glucose-repressible Hrr25 (GAL::Hrr25) or lacking Ltv1 (ΔLtv1), and of double mutant GAL::Hrr25;ΔLtv1 and ΔHrr25;ΔLtv1 cells, was compared. Fig. S2 B has a close-up to compare colony size. (B) Growth rates of wild-type, GAL::Hrr25,ΔLtv1, and GAL::Hrr25;ΔLtv1 yeast cells, as determined by their doubling times in YPD. All measurements were done in triplicate, and error bars show the standard deviation of these data. (C) Northern blot analysis of the 18S rRNA precursor (20S) and 25S rRNA. The quantitation shown below the data is the mean and standard deviation from four independent experiments. (D) Dominant-negative effects from Ltv1-S/A. Wild-type yeast (BY4741) were transformed with plasmids encoding wild-type Ltv1, Ltv1-S/A, or Ltv1-S/D under a galactose-inducible promoter. Induction of Ltv1 is weakly dominant-negative for wild-type Ltv1 and Ltv1-S/D, and strongly dominant-negative for Ltv1-S/A. (E) Growth of GAL::Hrr25;ΔLtv1 cells carrying plasmids of wild-type or phosphomutant Ltv1, with and without Hrr25, is compared on glucose (no endogenous Hrr25) and galactose-containing (endogenous Hrr25) plates. (F) Alanine mutations in the Ltv1 phosphosite block Enp1/Ltv1 release in vitro. Shown are Western blots of gradient fractions from ΔLtv1;Rio2TAP ribosomes, purified from cells containing Hrr25_I82G, and reconstituted on the calmodulin beads with Enp1–Yar1–Rps3 and wild-type Ltv1 or Ltv1-S/A. The positions of 40S ribosomes and free proteins are indicated.

Mentions: To confirm the role of Hrr25 in releasing Ltv1 in vivo, we used suppression analysis. While Hrr25 is an essential protein, Ltv1 is not (Fig. 4 A; Hoekstra et al., 1991; Winzeler et al., 1999; Seiser et al., 2006). Thus, we reasoned that if displacement of Ltv1 from pre-40S ribosomes was the essential function of Hrr25, then deletion of Ltv1 would be predicted to rescue the lethal phenotype of Hrr25 depletion. Depletion of Hrr25 in yeast cells engineered to express a galactose-inducible/glucose-repressible Hrr25 transgene (Gal::Hrr25) essentially abolished growth in glucose, as expected because Hrr25 is an essential gene (Fig. 4, A and B). Strikingly, however, loss of Ltv1 rescued growth of this mutant strain when grown in glucose (Fig. 4, A and B), and the ΔLtv1ΔHrr25 strain is viable (Fig. 4 A). Thus, the essential function of Hrr25 is linked to Ltv1. The more profound growth defect in the ΔLtv1ΔHrr25 cells relative to ΔLtv1 cells likely reflects roles of Hrr25 in other cellular processes as discussed above (Knippschild et al., 2005a). ΔLtv1ΔHrr25 cells also do not grow well on galactose, which indicates a role for Hrr25 in the regulation of galactose metabolism, consistent with previously observed interactions between Hrr25 and Gal10, as well as Sgm1 (Ho et al., 2002; Fasolo et al., 2011).


Hrr25/CK1δ-directed release of Ltv1 from pre-40S ribosomes is necessary for ribosome assembly and cell growth.

Ghalei H, Schaub FX, Doherty JR, Noguchi Y, Roush WR, Cleveland JL, Stroupe ME, Karbstein K - J. Cell Biol. (2015)

Ltv1 release is the essential function of Hrr25. (A) Growth of wild-type yeast (BY4741), and those with galactose-inducible/glucose-repressible Hrr25 (GAL::Hrr25) or lacking Ltv1 (ΔLtv1), and of double mutant GAL::Hrr25;ΔLtv1 and ΔHrr25;ΔLtv1 cells, was compared. Fig. S2 B has a close-up to compare colony size. (B) Growth rates of wild-type, GAL::Hrr25,ΔLtv1, and GAL::Hrr25;ΔLtv1 yeast cells, as determined by their doubling times in YPD. All measurements were done in triplicate, and error bars show the standard deviation of these data. (C) Northern blot analysis of the 18S rRNA precursor (20S) and 25S rRNA. The quantitation shown below the data is the mean and standard deviation from four independent experiments. (D) Dominant-negative effects from Ltv1-S/A. Wild-type yeast (BY4741) were transformed with plasmids encoding wild-type Ltv1, Ltv1-S/A, or Ltv1-S/D under a galactose-inducible promoter. Induction of Ltv1 is weakly dominant-negative for wild-type Ltv1 and Ltv1-S/D, and strongly dominant-negative for Ltv1-S/A. (E) Growth of GAL::Hrr25;ΔLtv1 cells carrying plasmids of wild-type or phosphomutant Ltv1, with and without Hrr25, is compared on glucose (no endogenous Hrr25) and galactose-containing (endogenous Hrr25) plates. (F) Alanine mutations in the Ltv1 phosphosite block Enp1/Ltv1 release in vitro. Shown are Western blots of gradient fractions from ΔLtv1;Rio2TAP ribosomes, purified from cells containing Hrr25_I82G, and reconstituted on the calmodulin beads with Enp1–Yar1–Rps3 and wild-type Ltv1 or Ltv1-S/A. The positions of 40S ribosomes and free proteins are indicated.
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fig4: Ltv1 release is the essential function of Hrr25. (A) Growth of wild-type yeast (BY4741), and those with galactose-inducible/glucose-repressible Hrr25 (GAL::Hrr25) or lacking Ltv1 (ΔLtv1), and of double mutant GAL::Hrr25;ΔLtv1 and ΔHrr25;ΔLtv1 cells, was compared. Fig. S2 B has a close-up to compare colony size. (B) Growth rates of wild-type, GAL::Hrr25,ΔLtv1, and GAL::Hrr25;ΔLtv1 yeast cells, as determined by their doubling times in YPD. All measurements were done in triplicate, and error bars show the standard deviation of these data. (C) Northern blot analysis of the 18S rRNA precursor (20S) and 25S rRNA. The quantitation shown below the data is the mean and standard deviation from four independent experiments. (D) Dominant-negative effects from Ltv1-S/A. Wild-type yeast (BY4741) were transformed with plasmids encoding wild-type Ltv1, Ltv1-S/A, or Ltv1-S/D under a galactose-inducible promoter. Induction of Ltv1 is weakly dominant-negative for wild-type Ltv1 and Ltv1-S/D, and strongly dominant-negative for Ltv1-S/A. (E) Growth of GAL::Hrr25;ΔLtv1 cells carrying plasmids of wild-type or phosphomutant Ltv1, with and without Hrr25, is compared on glucose (no endogenous Hrr25) and galactose-containing (endogenous Hrr25) plates. (F) Alanine mutations in the Ltv1 phosphosite block Enp1/Ltv1 release in vitro. Shown are Western blots of gradient fractions from ΔLtv1;Rio2TAP ribosomes, purified from cells containing Hrr25_I82G, and reconstituted on the calmodulin beads with Enp1–Yar1–Rps3 and wild-type Ltv1 or Ltv1-S/A. The positions of 40S ribosomes and free proteins are indicated.
Mentions: To confirm the role of Hrr25 in releasing Ltv1 in vivo, we used suppression analysis. While Hrr25 is an essential protein, Ltv1 is not (Fig. 4 A; Hoekstra et al., 1991; Winzeler et al., 1999; Seiser et al., 2006). Thus, we reasoned that if displacement of Ltv1 from pre-40S ribosomes was the essential function of Hrr25, then deletion of Ltv1 would be predicted to rescue the lethal phenotype of Hrr25 depletion. Depletion of Hrr25 in yeast cells engineered to express a galactose-inducible/glucose-repressible Hrr25 transgene (Gal::Hrr25) essentially abolished growth in glucose, as expected because Hrr25 is an essential gene (Fig. 4, A and B). Strikingly, however, loss of Ltv1 rescued growth of this mutant strain when grown in glucose (Fig. 4, A and B), and the ΔLtv1ΔHrr25 strain is viable (Fig. 4 A). Thus, the essential function of Hrr25 is linked to Ltv1. The more profound growth defect in the ΔLtv1ΔHrr25 cells relative to ΔLtv1 cells likely reflects roles of Hrr25 in other cellular processes as discussed above (Knippschild et al., 2005a). ΔLtv1ΔHrr25 cells also do not grow well on galactose, which indicates a role for Hrr25 in the regulation of galactose metabolism, consistent with previously observed interactions between Hrr25 and Gal10, as well as Sgm1 (Ho et al., 2002; Fasolo et al., 2011).

Bottom Line: Conversely, phosphomimetic Ltv1 variants rescued viability after Hrr25 depletion.Finally, Ltv1 knockdown in human breast cancer cells impaired apoptosis induced by CK1δ/ε inhibitors, establishing that the antiproliferative activity of these inhibitors is due, at least in part, to disruption of ribosome assembly.These findings validate the ribosome assembly pathway as a novel target for the development of anticancer therapeutics.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cancer Biology and Department of Chemistry, The Scripps Research Institute, Jupiter, FL 33458.

Show MeSH
Related in: MedlinePlus