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Co-translational capturing of nascent ribosomal proteins by their dedicated chaperones.

Pausch P, Singh U, Ahmed YL, Pillet B, Murat G, Altegoer F, Stier G, Thoms M, Hurt E, Sinning I, Bange G, Kressler D - Nat Commun (2015)

Bottom Line: Owing to their difficult physicochemical properties, the synthesis of assembly-competent ribosomal proteins represents a major challenge.Recent evidence highlights that dedicated chaperone proteins recognize the N-terminal regions of ribosomal proteins and promote their soluble expression and delivery to the assembly site.Co-translational capturing of nascent ribosomal proteins by dedicated chaperones constitutes an elegant mechanism to prevent unspecific interactions and aggregation of ribosomal proteins on their road to incorporation.

View Article: PubMed Central - PubMed

Affiliation: LOEWE Center for Synthetic Microbiology (SYNMIKRO) and Department of Chemistry, Philipps-University Marburg, Hans-Meerwein-Straße, Marburg D-35043, Germany.

ABSTRACT
Exponentially growing yeast cells produce every minute >160,000 ribosomal proteins. Owing to their difficult physicochemical properties, the synthesis of assembly-competent ribosomal proteins represents a major challenge. Recent evidence highlights that dedicated chaperone proteins recognize the N-terminal regions of ribosomal proteins and promote their soluble expression and delivery to the assembly site. Here we explore the intuitive possibility that ribosomal proteins are captured by dedicated chaperones in a co-translational manner. Affinity purification of four chaperones (Rrb1, Syo1, Sqt1 and Yar1) selectively enriched the mRNAs encoding their specific ribosomal protein clients (Rpl3, Rpl5, Rpl10 and Rps3). X-ray crystallography reveals how the N-terminal, rRNA-binding residues of Rpl10 are shielded by Sqt1's WD-repeat β-propeller, providing mechanistic insight into the incorporation of Rpl10 into pre-60S subunits. Co-translational capturing of nascent ribosomal proteins by dedicated chaperones constitutes an elegant mechanism to prevent unspecific interactions and aggregation of ribosomal proteins on their road to incorporation.

No MeSH data available.


Related in: MedlinePlus

The essential function of Sqt1 consists in Rpl10 binding.(a) Allele-specific synthetic lethality between interaction surface mutants of sqt1 and rpl10. The growth phenotypes of cells harbouring the SQT1 wild-type allele or the indicated sqt1 alleles in combination with the RPL10 wild-type allele or the rpl10.R3E and rpl10.R4A allele were scored on 5-FOA-containing plates, which were incubated for 4 days at 30 °C. The mutated Sqt1 residues, as well as the L10-N residues they are contacting (blue arrowheads, Rpl10*), are indicated. (b) Allele-specific abrogation of the interaction between the above interaction surface mutants of sqt1 and rpl10. Y2H interactions were assessed for combinations between Sqt1 or the indicated Sqt1 variants and Rpl10 or the Rpl10.R3E and Rpl10.R4A mutant variant.
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f6: The essential function of Sqt1 consists in Rpl10 binding.(a) Allele-specific synthetic lethality between interaction surface mutants of sqt1 and rpl10. The growth phenotypes of cells harbouring the SQT1 wild-type allele or the indicated sqt1 alleles in combination with the RPL10 wild-type allele or the rpl10.R3E and rpl10.R4A allele were scored on 5-FOA-containing plates, which were incubated for 4 days at 30 °C. The mutated Sqt1 residues, as well as the L10-N residues they are contacting (blue arrowheads, Rpl10*), are indicated. (b) Allele-specific abrogation of the interaction between the above interaction surface mutants of sqt1 and rpl10. Y2H interactions were assessed for combinations between Sqt1 or the indicated Sqt1 variants and Rpl10 or the Rpl10.R3E and Rpl10.R4A mutant variant.

Mentions: In order to assess the functional relevance of the Y2H interaction data, we next determined the in vivo phenotypes of the sqt1 mutations that affect interaction with Rpl10 by growth assays. In agreement with the above binding studies, the Glu315 to lysine substitution was the only sqt1 single mutation that did not support growth, while combinations of alanine or lysine substitutions of Glu110, Glu156 and Asp420 were required to reduce or abolish growth (Fig. 5a and Supplementary Fig. 8a). In support of Rpl10 binding being the exclusive cellular role of Sqt1, overexpression of Rpl10 from a multicopy plasmid fully suppressed the slow-growth phenotypes of sqt1 mutants (Fig. 5b), while we observed partial growth restoration in case of the lethal sqt1 alleles (Supplementary Fig. 8b). Strikingly, Rpl10 overexpression even conferred very weak growth to cells lacking Sqt1 (Fig. 5b and Supplementary Fig. 8b). In agreement with a chaperone function of Sqt1, we observed that the solubility of newly synthesized Rpl10-2xHA, expressed for 20 min from a copper-inducible promoter, is strongly reduced in sqt1.E315A and sqt1.E110A/D420A mutant cells (Supplementary Fig. 9). Since the N-terminal residues of Rpl10 make extensive contacts with rRNA and have been implicated in coordination of tRNA movement (Supplementary Fig. 1; ref. 39), the effects of their mutation on growth cannot simply be correlated to their contribution to Sqt1 binding. Accordingly, the substitution of Arg4 to glutamate and the double substitution of Arg3/Arg4 to alanine, which only slightly reduced the interaction with Sqt1 (Fig. 4b), resulted in a lethal phenotype (Fig. 5c). Nevertheless, it was possible to obtain slow-growing rpl10 mutants, for example, rpl10.R3E and rpl10.R4A (Fig. 5c and Supplementary Fig. 8c), which were suitable to be exploited for the determination of synthetic lethal interactions with sqt1 alleles. In validation of the co-crystal structure and the above Y2H data, we only observed synthetic lethal phenotypes when the combined sqt1 and rpl10 mutations affected different interaction pairs (Fig. 6a). While the sqt1.E156K mutation, which interferes with Arg3 interaction, was selectively synthetically lethal with the Arg4 to alanine substitution within L10-N, there was no synthetic growth defect when this sqt1 allele was combined with the Arg3 to glutamate substitution. Likewise, only the combination of the sqt1.D420K allele, which abrogates Arg4 binding, with the Arg3 to glutamate, but not the Arg4 to alanine, substitution resulted in lethality. Finally, the sqt1.E315A allele, which abolishes interaction with Arg10, was synthetically lethal with both rpl10 mutations. These allele-specific effects were even more striking at the level of the Y2H interaction (Fig. 6b). As expected, the R3E/D420K, R3E/E315A, R4A/E156K and R4A/E315A combinations abolished the Rpl10–Sqt1 interaction. However, reversion or elimination of the charge repulsion in the case of the R3E/E156K and R4A/D420K pairs resulted in a substantially improved interaction compared with the Y2H binding of wild-type Rpl10 to the E156K and D420K variants.


Co-translational capturing of nascent ribosomal proteins by their dedicated chaperones.

Pausch P, Singh U, Ahmed YL, Pillet B, Murat G, Altegoer F, Stier G, Thoms M, Hurt E, Sinning I, Bange G, Kressler D - Nat Commun (2015)

The essential function of Sqt1 consists in Rpl10 binding.(a) Allele-specific synthetic lethality between interaction surface mutants of sqt1 and rpl10. The growth phenotypes of cells harbouring the SQT1 wild-type allele or the indicated sqt1 alleles in combination with the RPL10 wild-type allele or the rpl10.R3E and rpl10.R4A allele were scored on 5-FOA-containing plates, which were incubated for 4 days at 30 °C. The mutated Sqt1 residues, as well as the L10-N residues they are contacting (blue arrowheads, Rpl10*), are indicated. (b) Allele-specific abrogation of the interaction between the above interaction surface mutants of sqt1 and rpl10. Y2H interactions were assessed for combinations between Sqt1 or the indicated Sqt1 variants and Rpl10 or the Rpl10.R3E and Rpl10.R4A mutant variant.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4491177&req=5

f6: The essential function of Sqt1 consists in Rpl10 binding.(a) Allele-specific synthetic lethality between interaction surface mutants of sqt1 and rpl10. The growth phenotypes of cells harbouring the SQT1 wild-type allele or the indicated sqt1 alleles in combination with the RPL10 wild-type allele or the rpl10.R3E and rpl10.R4A allele were scored on 5-FOA-containing plates, which were incubated for 4 days at 30 °C. The mutated Sqt1 residues, as well as the L10-N residues they are contacting (blue arrowheads, Rpl10*), are indicated. (b) Allele-specific abrogation of the interaction between the above interaction surface mutants of sqt1 and rpl10. Y2H interactions were assessed for combinations between Sqt1 or the indicated Sqt1 variants and Rpl10 or the Rpl10.R3E and Rpl10.R4A mutant variant.
Mentions: In order to assess the functional relevance of the Y2H interaction data, we next determined the in vivo phenotypes of the sqt1 mutations that affect interaction with Rpl10 by growth assays. In agreement with the above binding studies, the Glu315 to lysine substitution was the only sqt1 single mutation that did not support growth, while combinations of alanine or lysine substitutions of Glu110, Glu156 and Asp420 were required to reduce or abolish growth (Fig. 5a and Supplementary Fig. 8a). In support of Rpl10 binding being the exclusive cellular role of Sqt1, overexpression of Rpl10 from a multicopy plasmid fully suppressed the slow-growth phenotypes of sqt1 mutants (Fig. 5b), while we observed partial growth restoration in case of the lethal sqt1 alleles (Supplementary Fig. 8b). Strikingly, Rpl10 overexpression even conferred very weak growth to cells lacking Sqt1 (Fig. 5b and Supplementary Fig. 8b). In agreement with a chaperone function of Sqt1, we observed that the solubility of newly synthesized Rpl10-2xHA, expressed for 20 min from a copper-inducible promoter, is strongly reduced in sqt1.E315A and sqt1.E110A/D420A mutant cells (Supplementary Fig. 9). Since the N-terminal residues of Rpl10 make extensive contacts with rRNA and have been implicated in coordination of tRNA movement (Supplementary Fig. 1; ref. 39), the effects of their mutation on growth cannot simply be correlated to their contribution to Sqt1 binding. Accordingly, the substitution of Arg4 to glutamate and the double substitution of Arg3/Arg4 to alanine, which only slightly reduced the interaction with Sqt1 (Fig. 4b), resulted in a lethal phenotype (Fig. 5c). Nevertheless, it was possible to obtain slow-growing rpl10 mutants, for example, rpl10.R3E and rpl10.R4A (Fig. 5c and Supplementary Fig. 8c), which were suitable to be exploited for the determination of synthetic lethal interactions with sqt1 alleles. In validation of the co-crystal structure and the above Y2H data, we only observed synthetic lethal phenotypes when the combined sqt1 and rpl10 mutations affected different interaction pairs (Fig. 6a). While the sqt1.E156K mutation, which interferes with Arg3 interaction, was selectively synthetically lethal with the Arg4 to alanine substitution within L10-N, there was no synthetic growth defect when this sqt1 allele was combined with the Arg3 to glutamate substitution. Likewise, only the combination of the sqt1.D420K allele, which abrogates Arg4 binding, with the Arg3 to glutamate, but not the Arg4 to alanine, substitution resulted in lethality. Finally, the sqt1.E315A allele, which abolishes interaction with Arg10, was synthetically lethal with both rpl10 mutations. These allele-specific effects were even more striking at the level of the Y2H interaction (Fig. 6b). As expected, the R3E/D420K, R3E/E315A, R4A/E156K and R4A/E315A combinations abolished the Rpl10–Sqt1 interaction. However, reversion or elimination of the charge repulsion in the case of the R3E/E156K and R4A/D420K pairs resulted in a substantially improved interaction compared with the Y2H binding of wild-type Rpl10 to the E156K and D420K variants.

Bottom Line: Owing to their difficult physicochemical properties, the synthesis of assembly-competent ribosomal proteins represents a major challenge.Recent evidence highlights that dedicated chaperone proteins recognize the N-terminal regions of ribosomal proteins and promote their soluble expression and delivery to the assembly site.Co-translational capturing of nascent ribosomal proteins by dedicated chaperones constitutes an elegant mechanism to prevent unspecific interactions and aggregation of ribosomal proteins on their road to incorporation.

View Article: PubMed Central - PubMed

Affiliation: LOEWE Center for Synthetic Microbiology (SYNMIKRO) and Department of Chemistry, Philipps-University Marburg, Hans-Meerwein-Straße, Marburg D-35043, Germany.

ABSTRACT
Exponentially growing yeast cells produce every minute >160,000 ribosomal proteins. Owing to their difficult physicochemical properties, the synthesis of assembly-competent ribosomal proteins represents a major challenge. Recent evidence highlights that dedicated chaperone proteins recognize the N-terminal regions of ribosomal proteins and promote their soluble expression and delivery to the assembly site. Here we explore the intuitive possibility that ribosomal proteins are captured by dedicated chaperones in a co-translational manner. Affinity purification of four chaperones (Rrb1, Syo1, Sqt1 and Yar1) selectively enriched the mRNAs encoding their specific ribosomal protein clients (Rpl3, Rpl5, Rpl10 and Rps3). X-ray crystallography reveals how the N-terminal, rRNA-binding residues of Rpl10 are shielded by Sqt1's WD-repeat β-propeller, providing mechanistic insight into the incorporation of Rpl10 into pre-60S subunits. Co-translational capturing of nascent ribosomal proteins by dedicated chaperones constitutes an elegant mechanism to prevent unspecific interactions and aggregation of ribosomal proteins on their road to incorporation.

No MeSH data available.


Related in: MedlinePlus