The Saccharomyces cerevisiae 60 S ribosome biogenesis factor Tif6p is regulated by Hrr25p-mediated phosphorylation.
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)P-labeled 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.
Ray
Partha
P
Department of Developmental and Molecular Biology, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY 10461, USA.
Basu
Uttiya
U
Ray
Anirban
A
Majumdar
Romit
R
Deng
Haiteng
H
Maitra
Umadas
U
D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D047389:Casein Kinase I / Q000235:genetics / Q000378:metabolism*; D039642:Eukaryotic Initiation Factors / Q000235:genetics; D007381:Intermediate Filament Proteins / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D009154:Mutation; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding / Q000502:physiology; D012323:RNA Processing, Post-Transcriptional / Q000502:physiology*; D012335:RNA, Ribosomal / Q000235:genetics / Q000378:metabolism*; D012269:Ribosomal Proteins; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D002352:Carrier Proteins; C109284:EIF6 protein, human; D039642:Eukaryotic Initiation Factors; D007381:Intermediate Filament Proteins; D010750:Phosphoproteins; D012335:RNA, Ribosomal; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins; C491609:TIF6 protein, S cerevisiae; D047389:Casein Kinase I; C070775:HRR25 protein, S cerevisiae