Limits...
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

A thermophilic adaptation might sense the processing status of Rpl10's N-terminal methionine.(a) Close-up of the interaction between the L10-N residues and the WD-repeat β-propeller domain of CtSqt1 (left panel) and of ScSqt1 (right panel). Sqt1 is shown in its cartoon representation with superimposed electrostatic surface properties. The Sqt1 residues involved in the interaction are shown as sticks. L10-N residues (yellow) are shown in a mixed cartoon/stick representation. The relevant Sqt1 and L10-N residues are labelled in blue and black, respectively (for example, A2 for Ala2). N- and C termini of L10-N are indicated (N′ and C′). (b) Analysis of the L10-N interaction with the WD-repeat β-propeller domain of CtSqt1 and ScSqt1 by ITC. Shown are ITC measurements of CtSqt1.52C/ScSqt1.53C with CtL10-N/ScL10-N peptides either lacking (amino acids 2–20) or including Met1 (amino acids 1–20), as indicated in each panel. The upper part of each panel shows the raw injection heats (μcal s−1). The lower part of each panel displays the corresponding specific binding isotherms (Kcal mol−1 of injectant) plotted against the molar ratio. The measured interaction parameters are listed within the profiles and the approximate Kd is shown in blue.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4491177&req=5

f3: A thermophilic adaptation might sense the processing status of Rpl10's N-terminal methionine.(a) Close-up of the interaction between the L10-N residues and the WD-repeat β-propeller domain of CtSqt1 (left panel) and of ScSqt1 (right panel). Sqt1 is shown in its cartoon representation with superimposed electrostatic surface properties. The Sqt1 residues involved in the interaction are shown as sticks. L10-N residues (yellow) are shown in a mixed cartoon/stick representation. The relevant Sqt1 and L10-N residues are labelled in blue and black, respectively (for example, A2 for Ala2). N- and C termini of L10-N are indicated (N′ and C′). (b) Analysis of the L10-N interaction with the WD-repeat β-propeller domain of CtSqt1 and ScSqt1 by ITC. Shown are ITC measurements of CtSqt1.52C/ScSqt1.53C with CtL10-N/ScL10-N peptides either lacking (amino acids 2–20) or including Met1 (amino acids 1–20), as indicated in each panel. The upper part of each panel shows the raw injection heats (μcal s−1). The lower part of each panel displays the corresponding specific binding isotherms (Kcal mol−1 of injectant) plotted against the molar ratio. The measured interaction parameters are listed within the profiles and the approximate Kd is shown in blue.

Mentions: Interestingly, there are subtle differences between the binding surfaces formed by S. cerevisiae and C. thermophilum Sqt1, which mainly affect the recognition of the N-terminal residue Ala2 and the C-terminal part of the L10-N peptide. In the case of ctSqt1, the amino group of Ala2 is triangulated by hydrogen bonds involving the backbone carbonyls of Gly88, Ala90 and Ala93 (Fig. 3a). These residues are part of a thermophile-specific insertion within the surface loop connecting β-strands 1b and 1c, notably forming a narrow, cap-like binding pocket, which would not provide enough space for the accommodation of the N-terminal methionine (Met1). In the case of S. cerevisiae Sqt1, the amino group of Ala2 is held in place via interactions with the main-chain carbonyl of Gly85 and the side chains of Asn87, Glu110 and Ser111 (Fig. 3a). At the C-terminal end of the L10-N peptide, only Lys13 of S. cerevisiae engages in a contact, involving Asp311, with Sqt1. Since Met1 of Rpl10 is not present in the S. cerevisiae 60S structure1, we next addressed whether co-translational removal of the N-terminal methionine by the ribosome-associated methionine amino peptidase4546 is a pre-requisite for the recognition of L10-N by Sqt1. To this end, we quantified the binding of Sqt1 to L10-N peptides, either containing (amino acids 1–20) or lacking Met1 (amino acids 2–20), by isothermal titration calorimetry (ITC; Fig. 3b). Sqt1 from S. cerevisiae showed only a slight preference for the L10-N peptide lacking Met1, as indicated by the dissociation constants (Kd) of ∼21 and 43 nM, respectively. In the case of ctSqt1, however, the presence of Met1 reduced the affinity for the L10-N peptide by about tenfold (Kd of ∼35 and ∼442 nM), but did not abolish the interaction completely. We conclude that Sqt1 forms a remarkably stable interaction with the N-terminal residues of Rpl10, which is, at least as observed for ctSqt1, very sensitive to the presence of the N-terminal methionine.


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)

A thermophilic adaptation might sense the processing status of Rpl10's N-terminal methionine.(a) Close-up of the interaction between the L10-N residues and the WD-repeat β-propeller domain of CtSqt1 (left panel) and of ScSqt1 (right panel). Sqt1 is shown in its cartoon representation with superimposed electrostatic surface properties. The Sqt1 residues involved in the interaction are shown as sticks. L10-N residues (yellow) are shown in a mixed cartoon/stick representation. The relevant Sqt1 and L10-N residues are labelled in blue and black, respectively (for example, A2 for Ala2). N- and C termini of L10-N are indicated (N′ and C′). (b) Analysis of the L10-N interaction with the WD-repeat β-propeller domain of CtSqt1 and ScSqt1 by ITC. Shown are ITC measurements of CtSqt1.52C/ScSqt1.53C with CtL10-N/ScL10-N peptides either lacking (amino acids 2–20) or including Met1 (amino acids 1–20), as indicated in each panel. The upper part of each panel shows the raw injection heats (μcal s−1). The lower part of each panel displays the corresponding specific binding isotherms (Kcal mol−1 of injectant) plotted against the molar ratio. The measured interaction parameters are listed within the profiles and the approximate Kd is shown in blue.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: A thermophilic adaptation might sense the processing status of Rpl10's N-terminal methionine.(a) Close-up of the interaction between the L10-N residues and the WD-repeat β-propeller domain of CtSqt1 (left panel) and of ScSqt1 (right panel). Sqt1 is shown in its cartoon representation with superimposed electrostatic surface properties. The Sqt1 residues involved in the interaction are shown as sticks. L10-N residues (yellow) are shown in a mixed cartoon/stick representation. The relevant Sqt1 and L10-N residues are labelled in blue and black, respectively (for example, A2 for Ala2). N- and C termini of L10-N are indicated (N′ and C′). (b) Analysis of the L10-N interaction with the WD-repeat β-propeller domain of CtSqt1 and ScSqt1 by ITC. Shown are ITC measurements of CtSqt1.52C/ScSqt1.53C with CtL10-N/ScL10-N peptides either lacking (amino acids 2–20) or including Met1 (amino acids 1–20), as indicated in each panel. The upper part of each panel shows the raw injection heats (μcal s−1). The lower part of each panel displays the corresponding specific binding isotherms (Kcal mol−1 of injectant) plotted against the molar ratio. The measured interaction parameters are listed within the profiles and the approximate Kd is shown in blue.
Mentions: Interestingly, there are subtle differences between the binding surfaces formed by S. cerevisiae and C. thermophilum Sqt1, which mainly affect the recognition of the N-terminal residue Ala2 and the C-terminal part of the L10-N peptide. In the case of ctSqt1, the amino group of Ala2 is triangulated by hydrogen bonds involving the backbone carbonyls of Gly88, Ala90 and Ala93 (Fig. 3a). These residues are part of a thermophile-specific insertion within the surface loop connecting β-strands 1b and 1c, notably forming a narrow, cap-like binding pocket, which would not provide enough space for the accommodation of the N-terminal methionine (Met1). In the case of S. cerevisiae Sqt1, the amino group of Ala2 is held in place via interactions with the main-chain carbonyl of Gly85 and the side chains of Asn87, Glu110 and Ser111 (Fig. 3a). At the C-terminal end of the L10-N peptide, only Lys13 of S. cerevisiae engages in a contact, involving Asp311, with Sqt1. Since Met1 of Rpl10 is not present in the S. cerevisiae 60S structure1, we next addressed whether co-translational removal of the N-terminal methionine by the ribosome-associated methionine amino peptidase4546 is a pre-requisite for the recognition of L10-N by Sqt1. To this end, we quantified the binding of Sqt1 to L10-N peptides, either containing (amino acids 1–20) or lacking Met1 (amino acids 2–20), by isothermal titration calorimetry (ITC; Fig. 3b). Sqt1 from S. cerevisiae showed only a slight preference for the L10-N peptide lacking Met1, as indicated by the dissociation constants (Kd) of ∼21 and 43 nM, respectively. In the case of ctSqt1, however, the presence of Met1 reduced the affinity for the L10-N peptide by about tenfold (Kd of ∼35 and ∼442 nM), but did not abolish the interaction completely. We conclude that Sqt1 forms a remarkably stable interaction with the N-terminal residues of Rpl10, which is, at least as observed for ctSqt1, very sensitive to the presence of the N-terminal methionine.

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