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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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HvJAMM1 (desampylase) collapses SAMP2-NcsA conjugates.NcsA-StrepII fractions were purified from ΔncsA and ΔubaA strains, incubated with HvJAMM1 in the presence and absence of EDTA, and analyzed by IB as indicated. 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-g005: HvJAMM1 (desampylase) collapses SAMP2-NcsA conjugates.NcsA-StrepII fractions were purified from ΔncsA and ΔubaA strains, incubated with HvJAMM1 in the presence and absence of EDTA, and analyzed by IB as indicated. 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: To further investigate the SAMP2-modified forms of NcsA, NcsA-StrepII pull-down fractions from an ΔncsA mutant co-expressing Flag-SAMP2 were treated with the desampylating enzyme, HvJAMM1, and analyzed by IB (Figure 5). HvJAMM1 is a Zn2+-dependent metalloprotease of the JAB1/MPN+/MOV34 superfamily that hydrolyzes isopeptide and linear linkages of SAMP1-3 to target proteins and is inhibited by the metal chelator EDTA [24], [25]. Thus, samples incubated in the presence of EDTA (lanes 2 and 5) or in the absence of HvJAMM1 (lanes 1, 4 and 8) served as controls. By this approach, HvJAMM1 was found to hydrolyze the SAMP2-modified forms of NcsA but not the unmodified form of NcsA detected at 36 kDa by anti-StrepII IB (lane 3). Likewise, HvJAMM1 hydrolyzed the majority of SAMP2-conjugates detected by anti-Flag IB in NcsA pull down fractions of the ΔncsA strain with exception of two Flag-SAMP2 specific bands of ∼60 and 75 kDa (lane 6). The SAMP2-specific bands of comparable migration (∼60 and 75 kDa) detected in NcsA pull down fractions of the ΔubaA mutant were also resistant to cleavage by HvJAMM1 (Figure 5, lanes 7–8) suggesting a small subset of proteins are linked to SAMP2 by a mechanism that is independent of UbaA and not reversed by HvJAMM1. However, the majority of NcsA isoforms were collapsed to a single species of 36 kDa by the desampylase HvJAMM1, providing further evidence that NcsA is covalently bound to SAMP2 and suggesting NcsA modification may, in part, be regulated by HvJAMM1.


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)

HvJAMM1 (desampylase) collapses SAMP2-NcsA conjugates.NcsA-StrepII fractions were purified from ΔncsA and ΔubaA strains, incubated with HvJAMM1 in the presence and absence of EDTA, and analyzed by IB as indicated. Molecular weight markers are indicated to the left of each blot. Pull down assays were from 1 L cultures. See Methods section for details.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0099104-g005: HvJAMM1 (desampylase) collapses SAMP2-NcsA conjugates.NcsA-StrepII fractions were purified from ΔncsA and ΔubaA strains, incubated with HvJAMM1 in the presence and absence of EDTA, and analyzed by IB as indicated. 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: To further investigate the SAMP2-modified forms of NcsA, NcsA-StrepII pull-down fractions from an ΔncsA mutant co-expressing Flag-SAMP2 were treated with the desampylating enzyme, HvJAMM1, and analyzed by IB (Figure 5). HvJAMM1 is a Zn2+-dependent metalloprotease of the JAB1/MPN+/MOV34 superfamily that hydrolyzes isopeptide and linear linkages of SAMP1-3 to target proteins and is inhibited by the metal chelator EDTA [24], [25]. Thus, samples incubated in the presence of EDTA (lanes 2 and 5) or in the absence of HvJAMM1 (lanes 1, 4 and 8) served as controls. By this approach, HvJAMM1 was found to hydrolyze the SAMP2-modified forms of NcsA but not the unmodified form of NcsA detected at 36 kDa by anti-StrepII IB (lane 3). Likewise, HvJAMM1 hydrolyzed the majority of SAMP2-conjugates detected by anti-Flag IB in NcsA pull down fractions of the ΔncsA strain with exception of two Flag-SAMP2 specific bands of ∼60 and 75 kDa (lane 6). The SAMP2-specific bands of comparable migration (∼60 and 75 kDa) detected in NcsA pull down fractions of the ΔubaA mutant were also resistant to cleavage by HvJAMM1 (Figure 5, lanes 7–8) suggesting a small subset of proteins are linked to SAMP2 by a mechanism that is independent of UbaA and not reversed by HvJAMM1. However, the majority of NcsA isoforms were collapsed to a single species of 36 kDa by the desampylase HvJAMM1, providing further evidence that NcsA is covalently bound to SAMP2 and suggesting NcsA modification may, in part, be regulated by HvJAMM1.

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
Related in: MedlinePlus