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Archaeal Tuc1/Ncs6 homolog required for wobble uridine tRNA thiolation is associated with ubiquitin-proteasome, translation, and RNA processing system homologs.

Chavarria NE, Hwang S, Cao S, Fu X, Holman M, Elbanna D, Rodriguez S, Arrington D, Englert M, Uthandi S, Söll D, Maupin-Furlow JA - PLoS ONE (2014)

Bottom Line: When purified from Hfx. volcanii, NcsA was found to be modified at Lys204 by isopeptide linkage to polymeric chains of the ubiquitin-fold protein SAMP2.Non-covalent protein partners that specifically associated with NcsA were also identified including UbaA, SAMP2, proteasome activating nucleotidase (PAN)-A/1, translation elongation factor aEF-1α and a β-CASP ribonuclease homolog of the archaeal cleavage and polyadenylation specificity factor 1 family (aCPSF1).Together, our study reveals that NcsA is essential for growth at high temperature, required for formation of thiolated tRNA(Lys)UUU and intimately linked to homologs of ubiquitin-proteasome, translation and RNA processing systems.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida, United States of America.

ABSTRACT
While cytoplasmic tRNA 2-thiolation protein 1 (Tuc1/Ncs6) and ubiquitin-related modifier-1 (Urm1) are important in the 2-thiolation of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) at wobble uridines of tRNAs in eukaryotes, the biocatalytic roles and properties of Ncs6/Tuc1 and its homologs are poorly understood. Here we present the first report of an Ncs6 homolog of archaea (NcsA of Haloferax volcanii) that is essential for maintaining cellular pools of thiolated tRNA(Lys)UUU and for growth at high temperature. When purified from Hfx. volcanii, NcsA was found to be modified at Lys204 by isopeptide linkage to polymeric chains of the ubiquitin-fold protein SAMP2. The ubiquitin-activating E1 enzyme homolog of archaea (UbaA) was required for this covalent modification. Non-covalent protein partners that specifically associated with NcsA were also identified including UbaA, SAMP2, proteasome activating nucleotidase (PAN)-A/1, translation elongation factor aEF-1α and a β-CASP ribonuclease homolog of the archaeal cleavage and polyadenylation specificity factor 1 family (aCPSF1). Together, our study reveals that NcsA is essential for growth at high temperature, required for formation of thiolated tRNA(Lys)UUU and intimately linked to homologs of ubiquitin-proteasome, translation and RNA processing systems.

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NcsA is covalently associated with SAMP2 through a UbaA-dependent mechanism.A) NcsA StrepII-affinity purified fractions purified from ΔncsA strains expressing NscA-StrepII with and without Flag-SAMP1/2 proteins were separated by reducing SDS-PAGE and analyzed by Coomassie blue stain (upper panel) and α-StrepII and α-Flag IB (middle and bottom panels, respectively) as indicated. B) α-StrepII immunoblot of NcsA-StrepII purified from H26 (wt, parent), ΔubaA, and Δsamp2. C) α-Flag immunoblot of NcsA-StrepII purified from H26 (wt, parent) and ΔubaA strains co-expressing Flag-SAMP2. Molecular weight markers are indicated to the left of each blot. Pull down assays were from 1 L cultures. See Methods section for details.
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pone-0099104-g004: NcsA is covalently associated with SAMP2 through a UbaA-dependent mechanism.A) NcsA StrepII-affinity purified fractions purified from ΔncsA strains expressing NscA-StrepII with and without Flag-SAMP1/2 proteins were separated by reducing SDS-PAGE and analyzed by Coomassie blue stain (upper panel) and α-StrepII and α-Flag IB (middle and bottom panels, respectively) as indicated. B) α-StrepII immunoblot of NcsA-StrepII purified from H26 (wt, parent), ΔubaA, and Δsamp2. C) α-Flag immunoblot of NcsA-StrepII purified from H26 (wt, parent) and ΔubaA strains co-expressing Flag-SAMP2. Molecular weight markers are indicated to the left of each blot. Pull down assays were from 1 L cultures. See Methods section for details.

Mentions: Once purified, NcsA-pull down fractions were analyzed by reducing SDS-PAGE followed by total protein staining (Coomassie Blue, CB) and immunoblotting (IB) (Figure 4A). Based on total protein staining (Figure 4A, upper panel), no proteins were detected in the empty vector control (lane 1) revealing the NcsA-pull down assay was relatively specific for NcsA and NcsA-associated proteins. The NcsA-pull down fractions (lanes 2–4) were found to be primarily composed of a 36 kDa protein (of molecular mass consistent with the 37.4 kDa theoretical mass of NcsA-StrepII) with additional protein bands of varying abundance also detected. In particular, an ∼20 kDa protein was found to be relatively abundant in fractions purified from the strain expressing Flag-SAMP2 in trans (lane 3). Further examination of these samples by anti-StrepII IB to probe for NcsA-StrepII (Figure 4A, middle panel) revealed the majority of NcsA was in the 36 kDa form with high molecular mass bands of >50 kDa also detected. Use of anti-Flag IB to probe for the Flag-SAMPs (Figure 4A, lower panel) revealed SAMP2 to be associated with NcsA in both free (∼20 kDa) and high molecular mass (>50 kDa) forms. This association appeared specific for SAMP2, as SAMP1 was not detected in NcsA-pull down fractions from strains co-expressing Flag-SAMP1 with NcsA-StrepII (Figure 4A, lower panel, lane 4). Based on these results, NcsA is covalently modified and co-purifies with a variety of protein partners including the Ub-fold SAMP2, which is linked to the NcsA protein network by covalent and non-covalent bonds.


Archaeal Tuc1/Ncs6 homolog required for wobble uridine tRNA thiolation is associated with ubiquitin-proteasome, translation, and RNA processing system homologs.

Chavarria NE, Hwang S, Cao S, Fu X, Holman M, Elbanna D, Rodriguez S, Arrington D, Englert M, Uthandi S, Söll D, Maupin-Furlow JA - PLoS ONE (2014)

NcsA is covalently associated with SAMP2 through a UbaA-dependent mechanism.A) NcsA StrepII-affinity purified fractions purified from ΔncsA strains expressing NscA-StrepII with and without Flag-SAMP1/2 proteins were separated by reducing SDS-PAGE and analyzed by Coomassie blue stain (upper panel) and α-StrepII and α-Flag IB (middle and bottom panels, respectively) as indicated. B) α-StrepII immunoblot of NcsA-StrepII purified from H26 (wt, parent), ΔubaA, and Δsamp2. C) α-Flag immunoblot of NcsA-StrepII purified from H26 (wt, parent) and ΔubaA strains co-expressing Flag-SAMP2. Molecular weight markers are indicated to the left of each blot. Pull down assays were from 1 L cultures. See Methods section for details.
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Related In: Results  -  Collection

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pone-0099104-g004: NcsA is covalently associated with SAMP2 through a UbaA-dependent mechanism.A) NcsA StrepII-affinity purified fractions purified from ΔncsA strains expressing NscA-StrepII with and without Flag-SAMP1/2 proteins were separated by reducing SDS-PAGE and analyzed by Coomassie blue stain (upper panel) and α-StrepII and α-Flag IB (middle and bottom panels, respectively) as indicated. B) α-StrepII immunoblot of NcsA-StrepII purified from H26 (wt, parent), ΔubaA, and Δsamp2. C) α-Flag immunoblot of NcsA-StrepII purified from H26 (wt, parent) and ΔubaA strains co-expressing Flag-SAMP2. Molecular weight markers are indicated to the left of each blot. Pull down assays were from 1 L cultures. See Methods section for details.
Mentions: Once purified, NcsA-pull down fractions were analyzed by reducing SDS-PAGE followed by total protein staining (Coomassie Blue, CB) and immunoblotting (IB) (Figure 4A). Based on total protein staining (Figure 4A, upper panel), no proteins were detected in the empty vector control (lane 1) revealing the NcsA-pull down assay was relatively specific for NcsA and NcsA-associated proteins. The NcsA-pull down fractions (lanes 2–4) were found to be primarily composed of a 36 kDa protein (of molecular mass consistent with the 37.4 kDa theoretical mass of NcsA-StrepII) with additional protein bands of varying abundance also detected. In particular, an ∼20 kDa protein was found to be relatively abundant in fractions purified from the strain expressing Flag-SAMP2 in trans (lane 3). Further examination of these samples by anti-StrepII IB to probe for NcsA-StrepII (Figure 4A, middle panel) revealed the majority of NcsA was in the 36 kDa form with high molecular mass bands of >50 kDa also detected. Use of anti-Flag IB to probe for the Flag-SAMPs (Figure 4A, lower panel) revealed SAMP2 to be associated with NcsA in both free (∼20 kDa) and high molecular mass (>50 kDa) forms. This association appeared specific for SAMP2, as SAMP1 was not detected in NcsA-pull down fractions from strains co-expressing Flag-SAMP1 with NcsA-StrepII (Figure 4A, lower panel, lane 4). Based on these results, NcsA is covalently modified and co-purifies with a variety of protein partners including the Ub-fold SAMP2, which is linked to the NcsA protein network by covalent and non-covalent bonds.

Bottom Line: When purified from Hfx. volcanii, NcsA was found to be modified at Lys204 by isopeptide linkage to polymeric chains of the ubiquitin-fold protein SAMP2.Non-covalent protein partners that specifically associated with NcsA were also identified including UbaA, SAMP2, proteasome activating nucleotidase (PAN)-A/1, translation elongation factor aEF-1α and a β-CASP ribonuclease homolog of the archaeal cleavage and polyadenylation specificity factor 1 family (aCPSF1).Together, our study reveals that NcsA is essential for growth at high temperature, required for formation of thiolated tRNA(Lys)UUU and intimately linked to homologs of ubiquitin-proteasome, translation and RNA processing systems.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida, United States of America.

ABSTRACT
While cytoplasmic tRNA 2-thiolation protein 1 (Tuc1/Ncs6) and ubiquitin-related modifier-1 (Urm1) are important in the 2-thiolation of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) at wobble uridines of tRNAs in eukaryotes, the biocatalytic roles and properties of Ncs6/Tuc1 and its homologs are poorly understood. Here we present the first report of an Ncs6 homolog of archaea (NcsA of Haloferax volcanii) that is essential for maintaining cellular pools of thiolated tRNA(Lys)UUU and for growth at high temperature. When purified from Hfx. volcanii, NcsA was found to be modified at Lys204 by isopeptide linkage to polymeric chains of the ubiquitin-fold protein SAMP2. The ubiquitin-activating E1 enzyme homolog of archaea (UbaA) was required for this covalent modification. Non-covalent protein partners that specifically associated with NcsA were also identified including UbaA, SAMP2, proteasome activating nucleotidase (PAN)-A/1, translation elongation factor aEF-1α and a β-CASP ribonuclease homolog of the archaeal cleavage and polyadenylation specificity factor 1 family (aCPSF1). Together, our study reveals that NcsA is essential for growth at high temperature, required for formation of thiolated tRNA(Lys)UUU and intimately linked to homologs of ubiquitin-proteasome, translation and RNA processing systems.

Show MeSH