Posttranslational Modification of Cellular Proteins by a Ubiquitin-like Protein in Bacteria

Naoki Shigi

doi:10.1074/jbc.m112.359844

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.

Highlights

Modification of proteins by ubiquitin (Ub) and ubiquitin-like proteins (Ubls) is essential in eukaryotes
This study focused on investigating whether a bacterial Ubl can function as a protein modifier in the bacterium Thermus thermophilus
TtuC has a Cys-192 that corresponds to the “catalytic Cys” of E1, and the TtuB-Gly65 ϳ TtuC-Cys-192 thioester that was formed in vitro (Fig. 1A) [29] was similar to the Ub/UblϳE1 thioester formed in the first step of Ub/Ubl conjugation [1]. These findings suggest that there exist Ub/Ubl homologous conjugation systems in Bacteria

Summary

Background

Modification of proteins by ubiquitin (Ub) and ubiquitin-like proteins (Ubls) is essential in eukaryotes. 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. Prior work from our group identified proteins required for tRNA thiolation in T. thermophilus, including cysteine desulfurases, TtuA, TtuB, and TtuC, and the reaction was reconstituted in vitro (Fig. 1A) [27,28,29]. In vivo analyses were used to examine the mechanism of TtuB conjugation and its functions, and the evolutionary implications for the eukaryotic Ub/Ubl system are discussed

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION