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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

Sqt1 and Rrb1 recognize the N-terminal residues of Rpl10 and Rpl3, respectively.(a) Sqt1 and Rrb1 are exclusively associated with Rpl10 and Rpl3. TAP of C-terminally TAP-tagged Sqt1 (Sqt1-TAP, lane 1) and N-terminally TAP-tagged Rrb1 (NTAP-Rrb1, lane 2) from yeast cell lysates. Final EGTA eluates were analysed by SDS–polyacrylamide gel electrophoresis (PAGE) and Coomassie staining. (b) Y2H interaction between Rpl10 and Sqt1. Note that the Sqt1.53C protein lacks amino acids 1–52 and thus essentially contains the WD-repeat β-propeller domain of Sqt1. Rpl10.12C corresponds to an Rpl10 variant starting with amino acid 12. (c) Y2H interaction between Rpl3 and Rrb1. Note that the Rrb1.60C protein lacks amino acids 2–59 and thus contains the WD-repeat β-propeller domain, including a predicted N-terminal α-helix, of Rrb1 (Supplementary Fig. 2d). Rpl3.8C corresponds to an Rpl3 variant starting with amino acid eight. (d) In vitro binding assay between Rpl10 and Sqt1. The indicated C-terminally (His)6-tagged Rpl10 and C-terminally Flag-tagged Sqt1 variants were co-expressed in E. coli and purified via Ni-affinity purification. Proteins were revealed by SDS–PAGE and Coomassie staining (top) or by western blot analysis using anti-Flag (Sqt1-Flag variants) and anti-His (Rpl10-(His)6 variants) antibodies (bottom). T, total extract (lane 1); P, pellet fraction (insoluble proteins, lane 2); S, soluble extract (lane 3); E, imidazole eluate (lane 4); M, molecular weight standard. The bands highlighted by blue arrowheads correspond to the different Rpl10 variants used as baits for the purifications. Black arrowheads indicate the position of Sqt1-Flag and Sqt1.53C-Flag. Note that the third panel can be considered as a reference for the background binding of Sqt1-Flag to the Ni-NTA agarose resin.
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f1: Sqt1 and Rrb1 recognize the N-terminal residues of Rpl10 and Rpl3, respectively.(a) Sqt1 and Rrb1 are exclusively associated with Rpl10 and Rpl3. TAP of C-terminally TAP-tagged Sqt1 (Sqt1-TAP, lane 1) and N-terminally TAP-tagged Rrb1 (NTAP-Rrb1, lane 2) from yeast cell lysates. Final EGTA eluates were analysed by SDS–polyacrylamide gel electrophoresis (PAGE) and Coomassie staining. (b) Y2H interaction between Rpl10 and Sqt1. Note that the Sqt1.53C protein lacks amino acids 1–52 and thus essentially contains the WD-repeat β-propeller domain of Sqt1. Rpl10.12C corresponds to an Rpl10 variant starting with amino acid 12. (c) Y2H interaction between Rpl3 and Rrb1. Note that the Rrb1.60C protein lacks amino acids 2–59 and thus contains the WD-repeat β-propeller domain, including a predicted N-terminal α-helix, of Rrb1 (Supplementary Fig. 2d). Rpl3.8C corresponds to an Rpl3 variant starting with amino acid eight. (d) In vitro binding assay between Rpl10 and Sqt1. The indicated C-terminally (His)6-tagged Rpl10 and C-terminally Flag-tagged Sqt1 variants were co-expressed in E. coli and purified via Ni-affinity purification. Proteins were revealed by SDS–PAGE and Coomassie staining (top) or by western blot analysis using anti-Flag (Sqt1-Flag variants) and anti-His (Rpl10-(His)6 variants) antibodies (bottom). T, total extract (lane 1); P, pellet fraction (insoluble proteins, lane 2); S, soluble extract (lane 3); E, imidazole eluate (lane 4); M, molecular weight standard. The bands highlighted by blue arrowheads correspond to the different Rpl10 variants used as baits for the purifications. Black arrowheads indicate the position of Sqt1-Flag and Sqt1.53C-Flag. Note that the third panel can be considered as a reference for the background binding of Sqt1-Flag to the Ni-NTA agarose resin.

Mentions: To address whether Rrb1 and Sqt1 are exclusively associated with Rpl3 and Rpl10, respectively, we performed tandem-affinity purification (TAP) of NTAP-Rrb1 (NTAP, proteinA-TEV-CBP-Flag), expressed from a monocopy plasmid under the control of its cognate promoter in an rrb1 strain, and genomically expressed Sqt1-TAP. Importantly, both SQT1-TAP and NTAP-RRB1, unlike the genomic RRB1-TAP strain (see also ref. 37), were completely functional as judged by their capacity to confer wild-type growth (Supplementary Fig. 2a,b). Notably, we observed that mild overexpression of Rpl3 weakly suppressed the slow-growth phenotype of rrb1-TAP mutant cells (Supplementary Fig. 2c). In agreement with previous Rrb1 purifications (Rrb1-HA and Rrb1-TAP)3637, NTAP-Rrb1 showed robust co-purification of Rpl3 (Fig. 1a). In affirmation of the proposed role of Sqt1 as a specific chaperone of Rpl10, we obtained good co-enrichment of Rpl10 when purifying the Sqt1-TAP bait (Fig. 1a). Unlike the dedicated transport adaptor Syo1, which binds simultaneously to the ribosomal proteins Rpl5 and Rpl11 (ref. 11), we did not observe any association of additional ribosomal proteins, besides Rpl3 or Rpl10, or biogenesis factors with purified Rrb1 or Sqt1.


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)

Sqt1 and Rrb1 recognize the N-terminal residues of Rpl10 and Rpl3, respectively.(a) Sqt1 and Rrb1 are exclusively associated with Rpl10 and Rpl3. TAP of C-terminally TAP-tagged Sqt1 (Sqt1-TAP, lane 1) and N-terminally TAP-tagged Rrb1 (NTAP-Rrb1, lane 2) from yeast cell lysates. Final EGTA eluates were analysed by SDS–polyacrylamide gel electrophoresis (PAGE) and Coomassie staining. (b) Y2H interaction between Rpl10 and Sqt1. Note that the Sqt1.53C protein lacks amino acids 1–52 and thus essentially contains the WD-repeat β-propeller domain of Sqt1. Rpl10.12C corresponds to an Rpl10 variant starting with amino acid 12. (c) Y2H interaction between Rpl3 and Rrb1. Note that the Rrb1.60C protein lacks amino acids 2–59 and thus contains the WD-repeat β-propeller domain, including a predicted N-terminal α-helix, of Rrb1 (Supplementary Fig. 2d). Rpl3.8C corresponds to an Rpl3 variant starting with amino acid eight. (d) In vitro binding assay between Rpl10 and Sqt1. The indicated C-terminally (His)6-tagged Rpl10 and C-terminally Flag-tagged Sqt1 variants were co-expressed in E. coli and purified via Ni-affinity purification. Proteins were revealed by SDS–PAGE and Coomassie staining (top) or by western blot analysis using anti-Flag (Sqt1-Flag variants) and anti-His (Rpl10-(His)6 variants) antibodies (bottom). T, total extract (lane 1); P, pellet fraction (insoluble proteins, lane 2); S, soluble extract (lane 3); E, imidazole eluate (lane 4); M, molecular weight standard. The bands highlighted by blue arrowheads correspond to the different Rpl10 variants used as baits for the purifications. Black arrowheads indicate the position of Sqt1-Flag and Sqt1.53C-Flag. Note that the third panel can be considered as a reference for the background binding of Sqt1-Flag to the Ni-NTA agarose resin.
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f1: Sqt1 and Rrb1 recognize the N-terminal residues of Rpl10 and Rpl3, respectively.(a) Sqt1 and Rrb1 are exclusively associated with Rpl10 and Rpl3. TAP of C-terminally TAP-tagged Sqt1 (Sqt1-TAP, lane 1) and N-terminally TAP-tagged Rrb1 (NTAP-Rrb1, lane 2) from yeast cell lysates. Final EGTA eluates were analysed by SDS–polyacrylamide gel electrophoresis (PAGE) and Coomassie staining. (b) Y2H interaction between Rpl10 and Sqt1. Note that the Sqt1.53C protein lacks amino acids 1–52 and thus essentially contains the WD-repeat β-propeller domain of Sqt1. Rpl10.12C corresponds to an Rpl10 variant starting with amino acid 12. (c) Y2H interaction between Rpl3 and Rrb1. Note that the Rrb1.60C protein lacks amino acids 2–59 and thus contains the WD-repeat β-propeller domain, including a predicted N-terminal α-helix, of Rrb1 (Supplementary Fig. 2d). Rpl3.8C corresponds to an Rpl3 variant starting with amino acid eight. (d) In vitro binding assay between Rpl10 and Sqt1. The indicated C-terminally (His)6-tagged Rpl10 and C-terminally Flag-tagged Sqt1 variants were co-expressed in E. coli and purified via Ni-affinity purification. Proteins were revealed by SDS–PAGE and Coomassie staining (top) or by western blot analysis using anti-Flag (Sqt1-Flag variants) and anti-His (Rpl10-(His)6 variants) antibodies (bottom). T, total extract (lane 1); P, pellet fraction (insoluble proteins, lane 2); S, soluble extract (lane 3); E, imidazole eluate (lane 4); M, molecular weight standard. The bands highlighted by blue arrowheads correspond to the different Rpl10 variants used as baits for the purifications. Black arrowheads indicate the position of Sqt1-Flag and Sqt1.53C-Flag. Note that the third panel can be considered as a reference for the background binding of Sqt1-Flag to the Ni-NTA agarose resin.
Mentions: To address whether Rrb1 and Sqt1 are exclusively associated with Rpl3 and Rpl10, respectively, we performed tandem-affinity purification (TAP) of NTAP-Rrb1 (NTAP, proteinA-TEV-CBP-Flag), expressed from a monocopy plasmid under the control of its cognate promoter in an rrb1 strain, and genomically expressed Sqt1-TAP. Importantly, both SQT1-TAP and NTAP-RRB1, unlike the genomic RRB1-TAP strain (see also ref. 37), were completely functional as judged by their capacity to confer wild-type growth (Supplementary Fig. 2a,b). Notably, we observed that mild overexpression of Rpl3 weakly suppressed the slow-growth phenotype of rrb1-TAP mutant cells (Supplementary Fig. 2c). In agreement with previous Rrb1 purifications (Rrb1-HA and Rrb1-TAP)3637, NTAP-Rrb1 showed robust co-purification of Rpl3 (Fig. 1a). In affirmation of the proposed role of Sqt1 as a specific chaperone of Rpl10, we obtained good co-enrichment of Rpl10 when purifying the Sqt1-TAP bait (Fig. 1a). Unlike the dedicated transport adaptor Syo1, which binds simultaneously to the ribosomal proteins Rpl5 and Rpl11 (ref. 11), we did not observe any association of additional ribosomal proteins, besides Rpl3 or Rpl10, or biogenesis factors with purified Rrb1 or Sqt1.

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