Mass Spectrometry of Ribosomes from Saccharomyces cerevisiae

Abstract

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 (P1␣-P2␤ and P1␤-P2␣) 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 P1␣ or P2␤ are deleted. This enables us to define novel post-translational modifications at very low levels, including a population of P2␣ molecules with both phosphorylation and trimethylation. The deletion mutants also reveal interactions within the heterodimers, specifically that the absence of P1␣ or P2␤ 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.Ribosomes are the universal translators of messenger RNA into proteins. X-ray analysis of the 30 S subunit from Thermus thermophilus, the 50 S subunit from Haloarcula marismortui, and the intact 70 S particle from T. thermophilus have revealed fascinating insights into the structures of these macromolecular machines (1-3). There are currently, however, no high resolution structures of eukaryotic ribosomes. These 80 S particles consist of two subunits, the 60 and 40 S subunits. Low resolution structures obtained by electron microscopy indicate an overall topology similar to that of the Escherichia coli 70 S ribosome but indicate that the eukaryotic particle is larger and more complex. In general, the core of the ribosome is highly conserved, but in S. cerevisiae there are large differences in the periphery of the particle. This is attributed to the presence of additional ribosomal proteins and ribosomal RNA (rRNA) regions of variable size, interrupting the universal core secondary structure of eukaryotic rRNA (4). The eukaryotic 60 S subunit is larger than the prokaryotic 50 S subunit, but the greatest differences are observed between the 30 S subunit and the eukaryotic 40 S subunit .A common feature of all ribosomes is ...

High heterogeneity within the ribosomal proteins of the Arabidopsis thaliana 80S ribosome

“…These proteins are known to be necessary for the correct functioning of most of the translational factors (reviewed by Liljas and Gudkov, 1987;Ballesta and Remacha, 1996), so much so that if exchanged between prokaryotes and eukaryotes, to function, the ribosome will require the corresponding elongation factor from the same species used for the stalk proteins (Uchiumi et al, 2002). Furthermore, the exact composition of the ribosomal stalk and the ratio of P-proteins within the stalk has been shown to be highly variable and developmentally regulated Hanson et al, 2004), leading to the suggestion that the composition of the stalk may be an important regulator of translation (Ballesta and Remacha, 1996).…”

Transcript profiling demonstrates absence of dosage compensation in Arabidopsis following loss of a single RPL23a paralog

“…For example, recent annotation suggests that there are approximately 259 (152 LSU and 107 SSU) and 228 (139 LSU and 89 SSU) r-protein genes for the 81 rproteins in Arabidopsis and rice (Oryza sativa), respectively (Barakat et al 2001;Chang et al 2005, and our unpublished observations). As the translating ribosome contains only a single molecule of each r-protein, with the exception of the acidic P-proteins (RPP1 to RPP3, Ban et al 2000;Wimberly et al 2000;Guarinos et al 2003;Hanson et al 2004;Schuwirth et al 2005), plants must possess mechanisms to govern both the coordinated production and equimolar accumulation of r-proteins. In budding yeast (Saccharomyces cerevisiae), regulation of r-protein paralogs occurs predominantly at the transcriptional level.…”

Arabidopsis Ribosomal Proteins RPL23aA and RPL23aB Are Differentially Targeted to the Nucleolus and Are Disparately Required for Normal Development

“…Plant cytoplasmic ribosomes synthesize the majority of cellular proteins (Bogorad, 1975;Bailey-Serres, 1998), and in Arabidopsis (Arabidopsis thaliana) are comprised of four ribosomal RNAs (rRNAs; small subunit [SSU] 18S and large subunit [LSU] 26S, 5.8S, and 5S) and 81 ribosomal proteins (r-proteins; 33 SSU, 48 LSU; Barakat et al, 2001;Chang et al, 2005). Ribosomes contain only a single copy of nearly all r-proteins Wimberly et al, 2000;Guarinos et al, 2003;Hanson et al, 2004;Schuwirth et al, 2005), yet in plants r-proteins are encoded by large multigene families containing more than one transcriptionally active member (Barakat et al, 2001;Popescu and Tumer, 2004;Hulm et al, 2005;McIntosh and Bonham-Smith, 2005;Ouyang et al, 2007). For example, the Arabidopsis genome contains 254 genes for the 81 r-proteins, with families of between two and five expressed members (Barakat et al, 2001;Chang et al, 2005).…”