Abstract
Posttranslational modification of proteins with ubiquitin and ubiquitin-like proteins plays important regulatory roles in eukaryotes. Although a homologous conjugation system has recently been reported in Archaea, there is no similar report in Bacteria. This report describes the identification of a ubiquitin-like conjugation system in the bacterium Thermus thermophilus. A series of in vivo analyses revealed that TtuB, a bacterial ubiquitin-like protein that functions as a sulfur carrier in tRNA thiouridine synthesis, was covalently attached to target proteins, most likely via its C-terminal glycine. The involvement of the ubiquitin-activating enzyme-like protein TtuC in conjugate formation and the attachments of TtuB to TtuC and TtuA, which are proteins required for tRNA thiouridine synthesis, were demonstrated. Mass spectrometry analysis revealed that lysine residues (Lys-137/Lys-226/Lys-229) of TtuA were covalently modified by the C-terminal carboxylate of TtuB. Intriguingly, a deletion mutant of a JAMM (JAB1/MPN/Mov34 metalloenzyme) ubiquitin isopeptidase homolog showed aberrant TtuB conjugates of TtuC and TtuA and an ∼50% decrease in thiouridine amounts in tRNA. These results would support the hypothesis that thiouridine synthesis is regulated by TtuB-conjugation.