Mass spectrometry of ribosomes from Saccharomyces cerevisiae: implications for assembly of the stalk complex.
The acidic ribosomal P proteins form a distinct protuberance on the 60 S subunit of eukaryotic ribosomes. In yeast this structure is composed of two heterodimers (P1alpha-P2beta and P1beta-P2alpha) attached to the ribosome via P0. Although for prokaryotic ribosomes the isolation of a pentameric stalk complex comprising the analogous proteins is well established, its observation has not been reported for eukaryotic ribosomes. We used mass spectrometry to examine the composition of the stalk proteins on ribosomes from Saccharomyces cerevisiae. The resulting mass spectra reveal a noncovalent complex of mass 77,291 +/- 7 Da assigned to the pentameric stalk. Tandem mass spectrometry confirms this assignment and is consistent with the location of the P2 proteins on the periphery of the stalk complex, shielding the P1 proteins, which in turn interact with P0. No other oligomers are observed, confirming the specificity of the pentameric complex. At lower m/z values the spectra are dominated by individual proteins, largely from the stalk complex, giving rise to many overlapping peaks. To define the composition of the stalk proteins in detail we compared spectra of ribosomes from strains in which genes encoding either or both of the interacting stalk proteins P1alpha or P2beta are deleted. This enables us to define novel post-translational modifications at very low levels, including a population of P2alpha molecules with both phosphorylation and trimethylation. The deletion mutants also reveal interactions within the heterodimers, specifically that the absence of P1alpha or P2beta destabilizes binding of the partner protein on the ribosome. This implies that assembly of the stalk complex is not governed solely by interactions with P0 but is a cooperative process involving binding to partner proteins for additional stability on the ribosome.
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom.
Charlotte L
CL
Hanson
Hortense
H
Videler
Cruz
C
Santos
Juan P G
JP
Ballesta
Carol V
CV
Robinson
D015153:Blotting, Western; D017353:Gene Deletion; D007477:Ions; D007525:Isoelectric Focusing; D013058:Mass Spectrometry / Q000379:methods*; D008745:Methylation; D009154:Mutation; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry; D010766:Phosphorylation; D012269:Ribosomal Proteins / Q000737:chemistry*; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization
D007477:Ions; D012269:Ribosomal Proteins; D010744:Phosphoric Monoester Hydrolases