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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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SAMP2 is covalently linked in apparent poly-SAMP2 chains to NcsA.NcsA-StrepII fractions purified from H26 (wt, parent), Δsamp2, and ΔubaA strains expressing NcsA-StrepII, Flag-SAMP2 and/or Flag-SAMP2 K>R variant were analyzed by IB [α-Flag (left panel) and α-StrepII (right panel)] with molecular weight markers indicated on left and right of each panel, respectively. Coomassie blue stain (CB stain) was used to confirm equal protein loading. Pull down assays were from 50 ml cultures. See Methods section for details.
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pone-0099104-g006: SAMP2 is covalently linked in apparent poly-SAMP2 chains to NcsA.NcsA-StrepII fractions purified from H26 (wt, parent), Δsamp2, and ΔubaA strains expressing NcsA-StrepII, Flag-SAMP2 and/or Flag-SAMP2 K>R variant were analyzed by IB [α-Flag (left panel) and α-StrepII (right panel)] with molecular weight markers indicated on left and right of each panel, respectively. Coomassie blue stain (CB stain) was used to confirm equal protein loading. Pull down assays were from 50 ml cultures. See Methods section for details.

Mentions: Ub/Ubl proteins can form polymeric chains on target substrates, such as the Lys48-linked Ub chains that serve as signals for degradation of proteins by proteasomes in eukaryotic cells [26]. Lys58-linked SAMP2 chains have been identified in Hfx. volcanii[13]. However, it is not known whether these chains are anchored to protein targets or whether the second lysine residue (SAMP2 Lys64) can serve as a site for chain formation [13]. In an effort to determine whether NcsA is modified by SAMP2 chains, NcsA-StrepII was co-expressed with a Flag-SAMP2 variant devoid of lysine residues (K58R and K64R, named K>R) in wild-type, Δsamp2, and ΔubaA backgrounds. NcsA was subjected to pull down assays and probed with anti-Flag and anti-StrepII antibodies (Figure 6A and B, respectively). With this approach, SAMP2 and its K>R variant were found functional in formation of robust levels of protein conjugates in wild type and Δsamp2 strains (Figure 6A, lanes 1–2 and 4–5) vs. the ΔubaA mutant strain (lanes 3 and 6). However, only a single modified form of NcsA (∼50 kDa) was detected when SAMP2 K>R was expressed in the Δsamp2 background (Figure 6B, lane 5) compared to wild type cells (lane 4) or cells expressing SAMP2 (lane 1–2). While the NcsA that is linked to the single moiety of SAMP2 K>R is at low levels, the conjugate is detected and may be limited due to editing by a desampylase similarly to deubiquitylases of eukaryotic cells [27]. The physiological role of poly-SAMP2 chain formation on NcsA is unclear. However, our results provide evidence that poly-SAMP2 chains are anchored on NcsA and that these chains do not form on NcsA when SAMP2 lysines are modified to arginine residues.


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)

SAMP2 is covalently linked in apparent poly-SAMP2 chains to NcsA.NcsA-StrepII fractions purified from H26 (wt, parent), Δsamp2, and ΔubaA strains expressing NcsA-StrepII, Flag-SAMP2 and/or Flag-SAMP2 K>R variant were analyzed by IB [α-Flag (left panel) and α-StrepII (right panel)] with molecular weight markers indicated on left and right of each panel, respectively. Coomassie blue stain (CB stain) was used to confirm equal protein loading. Pull down assays were from 50 ml cultures. See Methods section for details.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4048286&req=5

pone-0099104-g006: SAMP2 is covalently linked in apparent poly-SAMP2 chains to NcsA.NcsA-StrepII fractions purified from H26 (wt, parent), Δsamp2, and ΔubaA strains expressing NcsA-StrepII, Flag-SAMP2 and/or Flag-SAMP2 K>R variant were analyzed by IB [α-Flag (left panel) and α-StrepII (right panel)] with molecular weight markers indicated on left and right of each panel, respectively. Coomassie blue stain (CB stain) was used to confirm equal protein loading. Pull down assays were from 50 ml cultures. See Methods section for details.
Mentions: Ub/Ubl proteins can form polymeric chains on target substrates, such as the Lys48-linked Ub chains that serve as signals for degradation of proteins by proteasomes in eukaryotic cells [26]. Lys58-linked SAMP2 chains have been identified in Hfx. volcanii[13]. However, it is not known whether these chains are anchored to protein targets or whether the second lysine residue (SAMP2 Lys64) can serve as a site for chain formation [13]. In an effort to determine whether NcsA is modified by SAMP2 chains, NcsA-StrepII was co-expressed with a Flag-SAMP2 variant devoid of lysine residues (K58R and K64R, named K>R) in wild-type, Δsamp2, and ΔubaA backgrounds. NcsA was subjected to pull down assays and probed with anti-Flag and anti-StrepII antibodies (Figure 6A and B, respectively). With this approach, SAMP2 and its K>R variant were found functional in formation of robust levels of protein conjugates in wild type and Δsamp2 strains (Figure 6A, lanes 1–2 and 4–5) vs. the ΔubaA mutant strain (lanes 3 and 6). However, only a single modified form of NcsA (∼50 kDa) was detected when SAMP2 K>R was expressed in the Δsamp2 background (Figure 6B, lane 5) compared to wild type cells (lane 4) or cells expressing SAMP2 (lane 1–2). While the NcsA that is linked to the single moiety of SAMP2 K>R is at low levels, the conjugate is detected and may be limited due to editing by a desampylase similarly to deubiquitylases of eukaryotic cells [27]. The physiological role of poly-SAMP2 chain formation on NcsA is unclear. However, our results provide evidence that poly-SAMP2 chains are anchored on NcsA and that these chains do not form on NcsA when SAMP2 lysines are modified to arginine residues.

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