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Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification.

Perrochia L, Guetta D, Hecker A, Forterre P, Basta T - Nucleic Acids Res. (2013)

Bottom Line: We confirmed that Pcc1 promotes dimerization of the KEOPS/EKC complex and uncovered that together with Kae1, it forms the tRNA binding core of the complex.Kae1 binds l-threonyl-carbamoyl-AMP intermediate in a metal-dependent fashion and transfers the l-threonyl-carbamoyl moiety to substrate tRNA.Overall, our data support a mechanistic model in which the final step in the biosynthesis of t(6)A relies on a strictly catalytic component, Kae1, and three partner proteins necessary for dimerization, tRNA binding and regulation.

View Article: PubMed Central - PubMed

Affiliation: Institut de Génétique et Microbiologie, Université Paris-Sud, IFR115, UMR8621-CNRS, 91405 Orsay, France and Université de Lorraine, UMR 1136 INRA/Université de Lorraine Interactions Arbres-Microorganismes, Labex ARBRE, FR EFABA, Faculté des Sciences, 54500 Vandoeuvre, France.

ABSTRACT
N(6)-threonylcarbamoyladenosine (t(6)A) is a universal tRNA modification essential for normal cell growth and accurate translation. In Archaea and Eukarya, the universal protein Sua5 and the conserved KEOPS/EKC complex together catalyze t(6)A biosynthesis. The KEOPS/EKC complex is composed of Kae1, a universal metalloprotein belonging to the ASHKA superfamily of ATPases; Bud32, an atypical protein kinase and two small proteins, Cgi121 and Pcc1. In this study, we investigated the requirement and functional role of KEOPS/EKC subunits for biosynthesis of t(6)A. We demonstrated that Pcc1, Kae1 and Bud32 form a minimal functional unit, whereas Cgi121 acts as an allosteric regulator. We confirmed that Pcc1 promotes dimerization of the KEOPS/EKC complex and uncovered that together with Kae1, it forms the tRNA binding core of the complex. Kae1 binds l-threonyl-carbamoyl-AMP intermediate in a metal-dependent fashion and transfers the l-threonyl-carbamoyl moiety to substrate tRNA. Surprisingly, we found that Bud32 is regulated by Kae1 and does not function as a protein kinase but as a P-loop ATPase possibly involved in tRNA dissociation. Overall, our data support a mechanistic model in which the final step in the biosynthesis of t(6)A relies on a strictly catalytic component, Kae1, and three partner proteins necessary for dimerization, tRNA binding and regulation.

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Analysis of ATPase and protein kinase activity of Bud32. (A) As a part of the KEOPS complex, Bud32 does not exhibit a significant protein kinase activity. Sua5 and the KEOPS complex were incubated under standard t6A assay conditions in presence of γ-P32 ATP (see Materials and Methods). The reaction mixture was analyzed by SDS-PAGE (part a), and the radioactivity retained by the proteins was recorded by phosphorimaging (part b). The same reaction mixture (part c) and a negative control lacking proteins (part -) was analyzed by TLC. The radioactive spots correspond to different nucleotides and free inorganic phosphate, as indicated. (B) Bud32 exhibits autophosphorylation activity in presence of Cgi121. The Bud32–Cgi121 binary complex was incubated in presence of γ-P32 ATP under standard t6A assay conditions, except that tRNA was omitted in the assay. The reaction mixture was analyzed by SDS-PAGE (part a), and the radioactivity retained by the proteins was recorded by phosphorimaging (part b). An aliquot of the reaction mixture (part c) and a negative control lacking proteins (part -) was analyzed by TLC. (C) Bud32 phosphotransferase activity is switched off in presence of Kae1. The Bud32–Cgi121 complex was incubated in presence of γ-P32 ATP under standard t6A assay conditions, except that tRNA was omitted in the assay. Reaction mixtures contained Bud32–Cgi121 (BC) alone (leftmost sample) or BC mixed with increasing concentrations of Pcc1–Kae1 (PK). In the rightmost sample, PK and BC subcomplexes were present in the reaction mixture in equimolar amounts. The reaction mixtures were analyzed by SDS-PAGE, and the radioactivity retained by the proteins was recorded by phosphorimaging (upper panel). The production of free inorganic phosphate for each reaction mixture was monitored by TLC analysis (lower panel).
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gkt720-F5: Analysis of ATPase and protein kinase activity of Bud32. (A) As a part of the KEOPS complex, Bud32 does not exhibit a significant protein kinase activity. Sua5 and the KEOPS complex were incubated under standard t6A assay conditions in presence of γ-P32 ATP (see Materials and Methods). The reaction mixture was analyzed by SDS-PAGE (part a), and the radioactivity retained by the proteins was recorded by phosphorimaging (part b). The same reaction mixture (part c) and a negative control lacking proteins (part -) was analyzed by TLC. The radioactive spots correspond to different nucleotides and free inorganic phosphate, as indicated. (B) Bud32 exhibits autophosphorylation activity in presence of Cgi121. The Bud32–Cgi121 binary complex was incubated in presence of γ-P32 ATP under standard t6A assay conditions, except that tRNA was omitted in the assay. The reaction mixture was analyzed by SDS-PAGE (part a), and the radioactivity retained by the proteins was recorded by phosphorimaging (part b). An aliquot of the reaction mixture (part c) and a negative control lacking proteins (part -) was analyzed by TLC. (C) Bud32 phosphotransferase activity is switched off in presence of Kae1. The Bud32–Cgi121 complex was incubated in presence of γ-P32 ATP under standard t6A assay conditions, except that tRNA was omitted in the assay. Reaction mixtures contained Bud32–Cgi121 (BC) alone (leftmost sample) or BC mixed with increasing concentrations of Pcc1–Kae1 (PK). In the rightmost sample, PK and BC subcomplexes were present in the reaction mixture in equimolar amounts. The reaction mixtures were analyzed by SDS-PAGE, and the radioactivity retained by the proteins was recorded by phosphorimaging (upper panel). The production of free inorganic phosphate for each reaction mixture was monitored by TLC analysis (lower panel).

Mentions: Bud32 was initially described as an RIO-type serine/threonine protein kinase and its phosphotransferase activity was demonstrated in vitro and in vivo (30). We therefore tested if the Bud32 ATPase activity corresponded to the protein kinase activity under the conditions used for the synthesis of t6A in vitro. t6A assay was performed using γ-P32 ATP, and we measured the radioactivity retained by the different proteins separated on a SDS-PAGE gel (Figure 5) . When the reaction mixture contained only Bud32–Cgi121, a significant autophosphorylation activity of Bud32 was observed, indicating that it can function as a protein kinase under the t6A assay conditions. However, the analysis of the complete mixture containing PaSua5 and PaKEOPS showed that, surprisingly, none of the proteins carried a significant level of radioactive signal, indicating a lack of stable protein–phosphate complexes. In line with this observation, the analysis of the same reaction mixture by TLC revealed the release of free inorganic phosphate, indicative of an ATPase activity.Figure 5.


Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification.

Perrochia L, Guetta D, Hecker A, Forterre P, Basta T - Nucleic Acids Res. (2013)

Analysis of ATPase and protein kinase activity of Bud32. (A) As a part of the KEOPS complex, Bud32 does not exhibit a significant protein kinase activity. Sua5 and the KEOPS complex were incubated under standard t6A assay conditions in presence of γ-P32 ATP (see Materials and Methods). The reaction mixture was analyzed by SDS-PAGE (part a), and the radioactivity retained by the proteins was recorded by phosphorimaging (part b). The same reaction mixture (part c) and a negative control lacking proteins (part -) was analyzed by TLC. The radioactive spots correspond to different nucleotides and free inorganic phosphate, as indicated. (B) Bud32 exhibits autophosphorylation activity in presence of Cgi121. The Bud32–Cgi121 binary complex was incubated in presence of γ-P32 ATP under standard t6A assay conditions, except that tRNA was omitted in the assay. The reaction mixture was analyzed by SDS-PAGE (part a), and the radioactivity retained by the proteins was recorded by phosphorimaging (part b). An aliquot of the reaction mixture (part c) and a negative control lacking proteins (part -) was analyzed by TLC. (C) Bud32 phosphotransferase activity is switched off in presence of Kae1. The Bud32–Cgi121 complex was incubated in presence of γ-P32 ATP under standard t6A assay conditions, except that tRNA was omitted in the assay. Reaction mixtures contained Bud32–Cgi121 (BC) alone (leftmost sample) or BC mixed with increasing concentrations of Pcc1–Kae1 (PK). In the rightmost sample, PK and BC subcomplexes were present in the reaction mixture in equimolar amounts. The reaction mixtures were analyzed by SDS-PAGE, and the radioactivity retained by the proteins was recorded by phosphorimaging (upper panel). The production of free inorganic phosphate for each reaction mixture was monitored by TLC analysis (lower panel).
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gkt720-F5: Analysis of ATPase and protein kinase activity of Bud32. (A) As a part of the KEOPS complex, Bud32 does not exhibit a significant protein kinase activity. Sua5 and the KEOPS complex were incubated under standard t6A assay conditions in presence of γ-P32 ATP (see Materials and Methods). The reaction mixture was analyzed by SDS-PAGE (part a), and the radioactivity retained by the proteins was recorded by phosphorimaging (part b). The same reaction mixture (part c) and a negative control lacking proteins (part -) was analyzed by TLC. The radioactive spots correspond to different nucleotides and free inorganic phosphate, as indicated. (B) Bud32 exhibits autophosphorylation activity in presence of Cgi121. The Bud32–Cgi121 binary complex was incubated in presence of γ-P32 ATP under standard t6A assay conditions, except that tRNA was omitted in the assay. The reaction mixture was analyzed by SDS-PAGE (part a), and the radioactivity retained by the proteins was recorded by phosphorimaging (part b). An aliquot of the reaction mixture (part c) and a negative control lacking proteins (part -) was analyzed by TLC. (C) Bud32 phosphotransferase activity is switched off in presence of Kae1. The Bud32–Cgi121 complex was incubated in presence of γ-P32 ATP under standard t6A assay conditions, except that tRNA was omitted in the assay. Reaction mixtures contained Bud32–Cgi121 (BC) alone (leftmost sample) or BC mixed with increasing concentrations of Pcc1–Kae1 (PK). In the rightmost sample, PK and BC subcomplexes were present in the reaction mixture in equimolar amounts. The reaction mixtures were analyzed by SDS-PAGE, and the radioactivity retained by the proteins was recorded by phosphorimaging (upper panel). The production of free inorganic phosphate for each reaction mixture was monitored by TLC analysis (lower panel).
Mentions: Bud32 was initially described as an RIO-type serine/threonine protein kinase and its phosphotransferase activity was demonstrated in vitro and in vivo (30). We therefore tested if the Bud32 ATPase activity corresponded to the protein kinase activity under the conditions used for the synthesis of t6A in vitro. t6A assay was performed using γ-P32 ATP, and we measured the radioactivity retained by the different proteins separated on a SDS-PAGE gel (Figure 5) . When the reaction mixture contained only Bud32–Cgi121, a significant autophosphorylation activity of Bud32 was observed, indicating that it can function as a protein kinase under the t6A assay conditions. However, the analysis of the complete mixture containing PaSua5 and PaKEOPS showed that, surprisingly, none of the proteins carried a significant level of radioactive signal, indicating a lack of stable protein–phosphate complexes. In line with this observation, the analysis of the same reaction mixture by TLC revealed the release of free inorganic phosphate, indicative of an ATPase activity.Figure 5.

Bottom Line: We confirmed that Pcc1 promotes dimerization of the KEOPS/EKC complex and uncovered that together with Kae1, it forms the tRNA binding core of the complex.Kae1 binds l-threonyl-carbamoyl-AMP intermediate in a metal-dependent fashion and transfers the l-threonyl-carbamoyl moiety to substrate tRNA.Overall, our data support a mechanistic model in which the final step in the biosynthesis of t(6)A relies on a strictly catalytic component, Kae1, and three partner proteins necessary for dimerization, tRNA binding and regulation.

View Article: PubMed Central - PubMed

Affiliation: Institut de Génétique et Microbiologie, Université Paris-Sud, IFR115, UMR8621-CNRS, 91405 Orsay, France and Université de Lorraine, UMR 1136 INRA/Université de Lorraine Interactions Arbres-Microorganismes, Labex ARBRE, FR EFABA, Faculté des Sciences, 54500 Vandoeuvre, France.

ABSTRACT
N(6)-threonylcarbamoyladenosine (t(6)A) is a universal tRNA modification essential for normal cell growth and accurate translation. In Archaea and Eukarya, the universal protein Sua5 and the conserved KEOPS/EKC complex together catalyze t(6)A biosynthesis. The KEOPS/EKC complex is composed of Kae1, a universal metalloprotein belonging to the ASHKA superfamily of ATPases; Bud32, an atypical protein kinase and two small proteins, Cgi121 and Pcc1. In this study, we investigated the requirement and functional role of KEOPS/EKC subunits for biosynthesis of t(6)A. We demonstrated that Pcc1, Kae1 and Bud32 form a minimal functional unit, whereas Cgi121 acts as an allosteric regulator. We confirmed that Pcc1 promotes dimerization of the KEOPS/EKC complex and uncovered that together with Kae1, it forms the tRNA binding core of the complex. Kae1 binds l-threonyl-carbamoyl-AMP intermediate in a metal-dependent fashion and transfers the l-threonyl-carbamoyl moiety to substrate tRNA. Surprisingly, we found that Bud32 is regulated by Kae1 and does not function as a protein kinase but as a P-loop ATPase possibly involved in tRNA dissociation. Overall, our data support a mechanistic model in which the final step in the biosynthesis of t(6)A relies on a strictly catalytic component, Kae1, and three partner proteins necessary for dimerization, tRNA binding and regulation.

Show MeSH
Related in: MedlinePlus