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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

Ionic interactions are critical determinants of L10-N binding by Sqt1.(a) Representation of the mode of L10-N recognition by ScSqt1. The backbone and side chains of ScL10-N (residues 2–13) are shown as an elongated peptide. L10-N residues are labelled in grey (e.g.,: A2 for Ala2). The Sqt1 residues that form interactions, either via their side chains or main-chain carbonyls, with the L10-N peptide are indicated. Dotted lines indicate ionic interactions or hydrogen bonds and grey, curved lines hydrophobic interactions. The interaction representation was created with Accelrys Draw 4.1. (b) Y2H interaction between Sqt1 and Rpl10 variants harbouring mutations within the N-terminal residues. The residues mutated in Rpl10 (for example, R3E for Arg3 to glutamate), as well as the Sqt1 residues they are contacting (blue arrowheads, Sqt1*), are indicated. (c) Y2H interaction between Rpl10 and mutant Sqt1 variants. The residues mutated in Sqt1 (for example, E110K for Glu110 to lysine), as well as the L10-N residues they are contacting (blue arrowheads, Rpl10*), are indicated. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; D, Asp; E, Glu; I, Ile; K, Lys; R, Arg; T, Thr; and Y, Tyr.
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f4: Ionic interactions are critical determinants of L10-N binding by Sqt1.(a) Representation of the mode of L10-N recognition by ScSqt1. The backbone and side chains of ScL10-N (residues 2–13) are shown as an elongated peptide. L10-N residues are labelled in grey (e.g.,: A2 for Ala2). The Sqt1 residues that form interactions, either via their side chains or main-chain carbonyls, with the L10-N peptide are indicated. Dotted lines indicate ionic interactions or hydrogen bonds and grey, curved lines hydrophobic interactions. The interaction representation was created with Accelrys Draw 4.1. (b) Y2H interaction between Sqt1 and Rpl10 variants harbouring mutations within the N-terminal residues. The residues mutated in Rpl10 (for example, R3E for Arg3 to glutamate), as well as the Sqt1 residues they are contacting (blue arrowheads, Sqt1*), are indicated. (c) Y2H interaction between Rpl10 and mutant Sqt1 variants. The residues mutated in Sqt1 (for example, E110K for Glu110 to lysine), as well as the L10-N residues they are contacting (blue arrowheads, Rpl10*), are indicated. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; D, Asp; E, Glu; I, Ile; K, Lys; R, Arg; T, Thr; and Y, Tyr.

Mentions: Since the interaction between Sqt1 and the L10-N peptide involves many salt bridges and hydrogen bonds (Fig. 4a), we mainly focused on the arginine residues within the L10-N peptide (Arg3, 4, 7 and 10) and the four conserved negatively charged residues of Sqt1 (Glu110, Glu156, Glu315 and Asp420) in order to determine their contribution to the interaction by Y2H analyses. While mutation of Arg10 to glutamate or alanine abolished or already reduced the interaction with Rpl10, respectively, only combinations of simultaneous substitutions of Arg3, Arg4 and Arg7 abrogated or interfered with Sqt1 binding (Fig. 4b and Supplementary Fig. 7c). In agreement with this result, mutation of Sqt1 residue Glu315, which contacts Arg10, to lysine abolished the interaction between Sqt1 and Rpl10 (Fig. 4c). Moreover, similar reductions in Y2H interaction were observed for the E110K, E156K, D420K and E315A Sqt1 variants; and, as above, only combinations of Glu110, Glu156 and Asp420 substitutions eliminated or reduced the interaction with Rpl10 (Fig. 4c and Supplementary Fig. 7d). We conclude that the Arg10–Glu315 interaction is the main binding determinant and that the Arg3–Glu110/Glu156 and Arg4–Asp420 interactions are individually not strictly required for but clearly contribute to binding.


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)

Ionic interactions are critical determinants of L10-N binding by Sqt1.(a) Representation of the mode of L10-N recognition by ScSqt1. The backbone and side chains of ScL10-N (residues 2–13) are shown as an elongated peptide. L10-N residues are labelled in grey (e.g.,: A2 for Ala2). The Sqt1 residues that form interactions, either via their side chains or main-chain carbonyls, with the L10-N peptide are indicated. Dotted lines indicate ionic interactions or hydrogen bonds and grey, curved lines hydrophobic interactions. The interaction representation was created with Accelrys Draw 4.1. (b) Y2H interaction between Sqt1 and Rpl10 variants harbouring mutations within the N-terminal residues. The residues mutated in Rpl10 (for example, R3E for Arg3 to glutamate), as well as the Sqt1 residues they are contacting (blue arrowheads, Sqt1*), are indicated. (c) Y2H interaction between Rpl10 and mutant Sqt1 variants. The residues mutated in Sqt1 (for example, E110K for Glu110 to lysine), as well as the L10-N residues they are contacting (blue arrowheads, Rpl10*), are indicated. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; D, Asp; E, Glu; I, Ile; K, Lys; R, Arg; T, Thr; and Y, Tyr.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4491177&req=5

f4: Ionic interactions are critical determinants of L10-N binding by Sqt1.(a) Representation of the mode of L10-N recognition by ScSqt1. The backbone and side chains of ScL10-N (residues 2–13) are shown as an elongated peptide. L10-N residues are labelled in grey (e.g.,: A2 for Ala2). The Sqt1 residues that form interactions, either via their side chains or main-chain carbonyls, with the L10-N peptide are indicated. Dotted lines indicate ionic interactions or hydrogen bonds and grey, curved lines hydrophobic interactions. The interaction representation was created with Accelrys Draw 4.1. (b) Y2H interaction between Sqt1 and Rpl10 variants harbouring mutations within the N-terminal residues. The residues mutated in Rpl10 (for example, R3E for Arg3 to glutamate), as well as the Sqt1 residues they are contacting (blue arrowheads, Sqt1*), are indicated. (c) Y2H interaction between Rpl10 and mutant Sqt1 variants. The residues mutated in Sqt1 (for example, E110K for Glu110 to lysine), as well as the L10-N residues they are contacting (blue arrowheads, Rpl10*), are indicated. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; D, Asp; E, Glu; I, Ile; K, Lys; R, Arg; T, Thr; and Y, Tyr.
Mentions: Since the interaction between Sqt1 and the L10-N peptide involves many salt bridges and hydrogen bonds (Fig. 4a), we mainly focused on the arginine residues within the L10-N peptide (Arg3, 4, 7 and 10) and the four conserved negatively charged residues of Sqt1 (Glu110, Glu156, Glu315 and Asp420) in order to determine their contribution to the interaction by Y2H analyses. While mutation of Arg10 to glutamate or alanine abolished or already reduced the interaction with Rpl10, respectively, only combinations of simultaneous substitutions of Arg3, Arg4 and Arg7 abrogated or interfered with Sqt1 binding (Fig. 4b and Supplementary Fig. 7c). In agreement with this result, mutation of Sqt1 residue Glu315, which contacts Arg10, to lysine abolished the interaction between Sqt1 and Rpl10 (Fig. 4c). Moreover, similar reductions in Y2H interaction were observed for the E110K, E156K, D420K and E315A Sqt1 variants; and, as above, only combinations of Glu110, Glu156 and Asp420 substitutions eliminated or reduced the interaction with Rpl10 (Fig. 4c and Supplementary Fig. 7d). We conclude that the Arg10–Glu315 interaction is the main binding determinant and that the Arg3–Glu110/Glu156 and Arg4–Asp420 interactions are individually not strictly required for but clearly contribute to binding.

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