The Saccharomyces cerevisiae 60 S Ribosome Biogenesis Factor Tif6p Is Regulated by Hrr25p-mediated Phosphorylation

Abstract

The biosynthesis of 60 S ribosomal subunits in Saccharomyces cerevisiae requires Tif6p, the yeast homologue of mammalian eIF6. This protein is necessary for the formation of 60 S ribosomal subunits because it is essential for the processing of 35 S pre-rRNA to the mature 25 S and 5.8 S rRNAs. In the present work, using molecular genetic and biochemical analyses, we show that Hrr25p, an isoform of yeast casein kinase I, phosphorylates Tif6p both in vitro and in vivo. Tryptic phosphopeptide mapping of in vitro phosphorylated Tif6p by Hrr25p and 32 Plabeled Tif6p isolated from yeast cells followed by mass spectrometric analysis revealed that phosphorylation occurred on a single tryptic peptide at Ser-174. Sucrose gradient fractionation and coimmunoprecipitation experiments demonstrate that a small but significant fraction of Hrr25p is bound to 66 S preribosomal particles that also contain bound Tif6p. Depletion of Hrr25p from a conditional yeast mutant that fails to phosphorylate Tif6p was unable to process pre-rRNAs efficiently, resulting in significant reduction in the formation of 25 S rRNA. These results along with our previous observations that phosphorylatable Ser-174 is required for yeast cell growth and viability, suggest that Hrr25p-mediated phosphorylation of Tif6p plays a critical role in the biogenesis of 60 S ribosomal subunits in yeast cells. Eukaryotic translation initiation factor 6 (eIF6)3 was initially purified as a protein that can bind the 60 S ribosomal subunit and prevent its association with the 40 S ribosomal subunit (1-4). Based on this ribosomal subunit anti-association property, the protein was originally thought to be an initiation factor that functions to provide a pool of free ribosomal subunits required for initiation of protein synthesis (5). The protein was named eIF6, although a role in translation was not demonstrated in these earlier studies. To understand the function of this protein in translation, Si et al. (6) first cloned the human cDNA and then the yeast Saccharomyces cerevisiae gene (7) encoding functionally active eIF6, each of 245 amino acids. The two proteins are 72% identical. The yeast gene, designated TIF6, is a single copy gene that is essential for cell growth and viability (7). These properties of TIF6 allowed the construction of a conditional null allele by placing its expression under the control of the regulatable GAL10 promoter. Depletion of Tif6p in this yeast mutant strain inhibited the rate of in vivo protein synthesis (7). However, a more detailed analysis of the protein synthesis parameters in Tif6p-depleted cells showed that the reduced rate of protein synthesis was not due to a direct inhibition in initiation (7). Rather, the biogenesis of 60 S ribosomal subunits was severely inhibited. Similar observations were also reported by Sanvito et al. (8), who identified eIF6 from mammalian cells as a ␤4 integrin-interacting protein. Specifically, lack of Tif6p in yeast cells prevented the processing of pre-ribosomal RNA (pre-rRNA) to the mature 25 S and 5.8 S...

The Activity-Dependent Regulation of Protein Kinase Stability by the Localization to P-Bodies

“…Hrr25 belongs to the casein kinase 1 (CK1) family of protein kinases and is the yeast ortholog of the mammalian CK1d enzyme (DeMaggio et al 1992). The Hrr25/CK1d proteins have conserved roles in ribosome maturation, vesicle trafficking, DNA repair, and chromosome segregation during meiosis (Hoekstra et al 1991;Petronczki et al 2006;Schafer et al 2006;Ray et al 2008;Grozav et al 2009;Biswas et al 2011;Isoda et al 2011;Lord et al 2011). We report here that this critical regulator of cell proliferation is efficiently recruited to P-bodies in both yeast and mammalian cells under all conditions examined.…”

“…To reduce Hrr25 protein levels in cells, we used a previously described strain, KKY387, that expresses an unstable, degrontagged version of Hrr25 (Hrr25 degron ) under the control of a galactose-inducible promoter Ray et al 2008). Briefly, this strain was grown to midlog phase in SCGal medium and then transferred to SCD for 8 hr to repress Hrr25 degron expression and arrest growth.…”

“…Expression from the CUP1 promoter was induced by the addition of 100 mM CuSO 4 to the growth medium. For the indicated stress treatments, yeast strains were grown to midlog phase in SCD medium and then transferred for 20 min to SC medium lacking glucose (2Glc), H 2 O, SC containing 2% galactose (SCGal), or SCD medium containing 3 mM H 2 O 2 , 0.5% NaN 3 , or 0.4% HCl.To reduce Hrr25 protein levels in cells, we used a previously described strain, KKY387, that expresses an unstable, degrontagged version of Hrr25 (Hrr25 degron ) under the control of a galactose-inducible promoter Ray et al 2008). Briefly, this strain was grown to midlog phase in SCGal medium and then transferred to SCD for 8 hr to repress Hrr25 degron expression and arrest growth.…”

Powering through ribosome assembly

“…Hrr25 binds to 90S and 40S, as well as to 66S pre-ribosomes, and its depletion leads to 18S and 25S processing defects (Schafer et al 2006;Ray et al 2008). Recent data from the Maitra and Tschocher laboratories suggest that cycles of phosphorylation and desphosphorylation, mediated by Hrr25, are required for export of both ribosomal subunits (Basu et al 2003;Schafer et al 2006;Ray et al 2008). Hrr25 phosphorylates Tif6 at serine 174 in vitro and in vivo (Basu et al 2003;Ray et al 2008).…”

“…Recent data from the Maitra and Tschocher laboratories suggest that cycles of phosphorylation and desphosphorylation, mediated by Hrr25, are required for export of both ribosomal subunits (Basu et al 2003;Schafer et al 2006;Ray et al 2008). Hrr25 phosphorylates Tif6 at serine 174 in vitro and in vivo (Basu et al 2003;Ray et al 2008). Mutation of this residue to alanine prevents phosphorylation, traps Tif6 in the nucleus, and leads to defective 60S assembly (Basu et al 2003).…”