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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

Crystal structures of the eight-bladed WD-repeat β-propeller domain of Sqt1 with bound L10-N from S. cerevisiae and C. thermophilum.(a) Crystal structure of ctSqt1.52C (residues 52–533) with bound ctL10-N (residues 2–13). Cartoon representation showing ctSqt1.52C in rainbow colours from N- to C terminus and ctL10-N in yellow with side chains (left panel). The eight-bladed WD-repeat β-propeller is shown in its top view. Assignment of the top and bottom surface as well as numbering of the propeller blades (1–8) and labelling of the β-strands within each blade (a–d) is according to the conventional definition for WD-repeat β-propellers. N- and C termini are indicated. Electrostatic properties of the top surface of ctSqt1.52C with bound L10-N in yellow (right panel). (b) Crystal structure of ScSqt1.53C (residues 53–431) with bound ScL10-N (residues 2–15). Cartoon representation (left panel) and electrostatic properties (right panel) of ScSqt1.53C with bound ScL10-N in yellow. Labels and colouring is as in a. (c) Comparison of the interaction modes of ScL10-N with helix H89 of the 25S rRNA and with ScSqt1, respectively. Cartoon representation of Rpl10 bound to H38 and H89 of the 25S rRNA as observed in the mature 60S subunit (PDB 3U5I and 3U5H for Rpl10 and 25S rRNA, respectively)1 (left panel) and bound to ScSqt1.53C (right panel). The N-terminal residues of Rpl10 (amino acids 2–15) are shown in yellow with side chains, the remainder of Rpl10 in grey, and bases of H38 in turquoise and of H89 in purple (phosphate backbones of H38 and H89 are shown in orange). Sqt1 is shown in its surface representation with electrostatic properties. The upper right part shows a comparison of the L10-N peptide in the ribosome-bound (left) and Sqt1-bound (right) state.
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f2: Crystal structures of the eight-bladed WD-repeat β-propeller domain of Sqt1 with bound L10-N from S. cerevisiae and C. thermophilum.(a) Crystal structure of ctSqt1.52C (residues 52–533) with bound ctL10-N (residues 2–13). Cartoon representation showing ctSqt1.52C in rainbow colours from N- to C terminus and ctL10-N in yellow with side chains (left panel). The eight-bladed WD-repeat β-propeller is shown in its top view. Assignment of the top and bottom surface as well as numbering of the propeller blades (1–8) and labelling of the β-strands within each blade (a–d) is according to the conventional definition for WD-repeat β-propellers. N- and C termini are indicated. Electrostatic properties of the top surface of ctSqt1.52C with bound L10-N in yellow (right panel). (b) Crystal structure of ScSqt1.53C (residues 53–431) with bound ScL10-N (residues 2–15). Cartoon representation (left panel) and electrostatic properties (right panel) of ScSqt1.53C with bound ScL10-N in yellow. Labels and colouring is as in a. (c) Comparison of the interaction modes of ScL10-N with helix H89 of the 25S rRNA and with ScSqt1, respectively. Cartoon representation of Rpl10 bound to H38 and H89 of the 25S rRNA as observed in the mature 60S subunit (PDB 3U5I and 3U5H for Rpl10 and 25S rRNA, respectively)1 (left panel) and bound to ScSqt1.53C (right panel). The N-terminal residues of Rpl10 (amino acids 2–15) are shown in yellow with side chains, the remainder of Rpl10 in grey, and bases of H38 in turquoise and of H89 in purple (phosphate backbones of H38 and H89 are shown in orange). Sqt1 is shown in its surface representation with electrostatic properties. The upper right part shows a comparison of the L10-N peptide in the ribosome-bound (left) and Sqt1-bound (right) state.

Mentions: As a first step towards the elucidation of the recognition mode of the N-terminal residues of Rpl10 by the predicted β-propeller domain of Sqt1 at the atomic level, we independently determined the crystal structure of ctSqt1 at 1.94 Å by molecular replacement and of ctSqt1.52C, lacking the dispensable N-terminal extension, at 1.5 Å resolution by single-anomalous dispersion (SAD) followed by molecular replacement using the ctSqt1.52C Se-Met structure as the search model (Table 1). While the N-terminal extension could not be resolved, the crystal structures revealed that residues 52–533 of ctSqt1 form a typical eight-bladed WD-repeat β-propeller (Supplementary Fig. 5a); thus, being composed of eight blades that each contain four β-strands and showing the characteristic ‘velcro' closure owing to the presence of the N-terminal β-strand as the outermost β-strand of the eighth blade. Subsequently, we could solve the structure of the S. cerevisiae Sqt1 WD-repeat β-propeller domain at 2.0 Å resolution by molecular replacement using the native ctSqt1.52C structure as the search model (Supplementary Fig. 5a). Sqt1 and ctSqt1 share a high degree of overall structural conservation and mainly differ in three ctSqt1-specific insertions located in the loops connecting β-strands 1c-1d, 5c-5d and 7c-7d (Supplementary Figs 5b and 11). Analysis of the electrostatic properties revealed that both β-propeller structures notably contain a negatively charged top surface, whereas the bottom sides exhibit a charge-mixed surface (Fig. 2 and Supplementary Fig. 5a).


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)

Crystal structures of the eight-bladed WD-repeat β-propeller domain of Sqt1 with bound L10-N from S. cerevisiae and C. thermophilum.(a) Crystal structure of ctSqt1.52C (residues 52–533) with bound ctL10-N (residues 2–13). Cartoon representation showing ctSqt1.52C in rainbow colours from N- to C terminus and ctL10-N in yellow with side chains (left panel). The eight-bladed WD-repeat β-propeller is shown in its top view. Assignment of the top and bottom surface as well as numbering of the propeller blades (1–8) and labelling of the β-strands within each blade (a–d) is according to the conventional definition for WD-repeat β-propellers. N- and C termini are indicated. Electrostatic properties of the top surface of ctSqt1.52C with bound L10-N in yellow (right panel). (b) Crystal structure of ScSqt1.53C (residues 53–431) with bound ScL10-N (residues 2–15). Cartoon representation (left panel) and electrostatic properties (right panel) of ScSqt1.53C with bound ScL10-N in yellow. Labels and colouring is as in a. (c) Comparison of the interaction modes of ScL10-N with helix H89 of the 25S rRNA and with ScSqt1, respectively. Cartoon representation of Rpl10 bound to H38 and H89 of the 25S rRNA as observed in the mature 60S subunit (PDB 3U5I and 3U5H for Rpl10 and 25S rRNA, respectively)1 (left panel) and bound to ScSqt1.53C (right panel). The N-terminal residues of Rpl10 (amino acids 2–15) are shown in yellow with side chains, the remainder of Rpl10 in grey, and bases of H38 in turquoise and of H89 in purple (phosphate backbones of H38 and H89 are shown in orange). Sqt1 is shown in its surface representation with electrostatic properties. The upper right part shows a comparison of the L10-N peptide in the ribosome-bound (left) and Sqt1-bound (right) state.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Crystal structures of the eight-bladed WD-repeat β-propeller domain of Sqt1 with bound L10-N from S. cerevisiae and C. thermophilum.(a) Crystal structure of ctSqt1.52C (residues 52–533) with bound ctL10-N (residues 2–13). Cartoon representation showing ctSqt1.52C in rainbow colours from N- to C terminus and ctL10-N in yellow with side chains (left panel). The eight-bladed WD-repeat β-propeller is shown in its top view. Assignment of the top and bottom surface as well as numbering of the propeller blades (1–8) and labelling of the β-strands within each blade (a–d) is according to the conventional definition for WD-repeat β-propellers. N- and C termini are indicated. Electrostatic properties of the top surface of ctSqt1.52C with bound L10-N in yellow (right panel). (b) Crystal structure of ScSqt1.53C (residues 53–431) with bound ScL10-N (residues 2–15). Cartoon representation (left panel) and electrostatic properties (right panel) of ScSqt1.53C with bound ScL10-N in yellow. Labels and colouring is as in a. (c) Comparison of the interaction modes of ScL10-N with helix H89 of the 25S rRNA and with ScSqt1, respectively. Cartoon representation of Rpl10 bound to H38 and H89 of the 25S rRNA as observed in the mature 60S subunit (PDB 3U5I and 3U5H for Rpl10 and 25S rRNA, respectively)1 (left panel) and bound to ScSqt1.53C (right panel). The N-terminal residues of Rpl10 (amino acids 2–15) are shown in yellow with side chains, the remainder of Rpl10 in grey, and bases of H38 in turquoise and of H89 in purple (phosphate backbones of H38 and H89 are shown in orange). Sqt1 is shown in its surface representation with electrostatic properties. The upper right part shows a comparison of the L10-N peptide in the ribosome-bound (left) and Sqt1-bound (right) state.
Mentions: As a first step towards the elucidation of the recognition mode of the N-terminal residues of Rpl10 by the predicted β-propeller domain of Sqt1 at the atomic level, we independently determined the crystal structure of ctSqt1 at 1.94 Å by molecular replacement and of ctSqt1.52C, lacking the dispensable N-terminal extension, at 1.5 Å resolution by single-anomalous dispersion (SAD) followed by molecular replacement using the ctSqt1.52C Se-Met structure as the search model (Table 1). While the N-terminal extension could not be resolved, the crystal structures revealed that residues 52–533 of ctSqt1 form a typical eight-bladed WD-repeat β-propeller (Supplementary Fig. 5a); thus, being composed of eight blades that each contain four β-strands and showing the characteristic ‘velcro' closure owing to the presence of the N-terminal β-strand as the outermost β-strand of the eighth blade. Subsequently, we could solve the structure of the S. cerevisiae Sqt1 WD-repeat β-propeller domain at 2.0 Å resolution by molecular replacement using the native ctSqt1.52C structure as the search model (Supplementary Fig. 5a). Sqt1 and ctSqt1 share a high degree of overall structural conservation and mainly differ in three ctSqt1-specific insertions located in the loops connecting β-strands 1c-1d, 5c-5d and 7c-7d (Supplementary Figs 5b and 11). Analysis of the electrostatic properties revealed that both β-propeller structures notably contain a negatively charged top surface, whereas the bottom sides exhibit a charge-mixed surface (Fig. 2 and Supplementary Fig. 5a).

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