E1- and ubiquitin-like proteins provide a direct link between protein conjugation and sulfur transfer in archaea

Abstract

Based on our recent work with Haloferax volcanii, ubiquitin-like (Ubl) proteins (SAMP1 and SAMP2) are known to be covalently attached to proteins in archaea. Here, we investigated the enzymes required for the formation of these Ubl-protein conjugates (SAMPylation) and whether this system is linked to sulfur transfer. Markerless in-frame deletions were generated in H. volcanii target genes. The mutants were examined for: (i) the formation of Ubl protein conjugates, (ii) growth under various conditions, including those requiring the synthesis of the sulfur-containing molybdenum cofactor (MoCo), and (iii) the thiolation of tRNA. With this approach we found that UbaA of the E1/MoeB/ThiF superfamily was required for the formation of both SAMP1-and SAMP2-protein conjugates. In addition, UbaA, SAMP1, and MoaE (a homolog of the large subunit of molybdopterin synthase) were essential for MoCo-dependent dimethyl sulfoxide reductase activity, suggesting that these proteins function in MoCo-biosynthesis. UbaA and SAMP2 were also crucial for optimal growth at high temperature and the thiolation of tRNA. Based on these results, we propose a working model for archaea in which the E1-like UbaA can activate multiple Ubl SAMPs for protein conjugation as well as for sulfur transfer. In sulfur transfer, SAMP1 and SAMP2 appear specific for MoCo biosynthesis and the thiolation of tRNA, respectively. Overall, this study provides a fundamental insight into the diverse cellular functions of the Ubl system. posttranslational modification | tRNA modification | proteasomes

Biosynthesis of 4-Thiouridine in tRNA in the Methanogenic Archaeon Methanococcus maripaludis

“…The reaction was stopped by addition of an equal volume of the formamide loading dye. The tRNA thiolation was then analyzed with the [(N-acryloylamino)phenyl]mercuric chloride (APM)-retardation gel (58). The thiolated and unmodified tRNA Cys (2.5 g per lane) were separated by electrophoresis in 12% urea-polyacrylamide gels supplemented with 9 g/ml APM.…”

Identification of novel components of NAD-utilizing metabolic pathways and prediction of their biochemical functions

“…6 In a similar vein, multiple components of the peptide ligation and deubiquitination pathways in the eukaryotic ubiquitin system show evolutionary relationships with enzymes involved in diverse bacterial biosynthetic systems for cofactors (thiamine and molybdopterin), siderophores, antibiotics and the amino acid cysteine. [1][2][3][7][8][9][10] Enzymes catalyzing other major forms of peptide tagging of proteins in eukaryotes, e.g. protein polyglutamylation, polyglycination and tyrosinylation also display evolutionary connections to peptide ligases involved in diverse prokaryotic pathways for the biosynthesis of various antibiotics, the amino acid lysine and cofactors like peptidylated tetrahydrosarcinapterin (a folate-like pterin derivative) and Given these connections, we were interested in understanding the links between the biosynthetic and regulatory pathways centered on the ancient and ubiquitous metabolite, nicotinamide adenine dinucleotide (NAD) or its phosphorylated derivative NADP.…”

“…[1][2][3][4] For example, the origin of several eukaryotic enzymes that add or remove a methyl group on lysines and arginines in histones and other proteins can be directly traced to bacterial pathways involved in synthesizing peptide-derived antibiotics and siderophores. 5 A 2-oxoglutarate-dependent dioxygenase derived from similar bacterial systems has also spawned the wybutosine hydroxylase/ peroxidase, an enzyme that introduces a key modification in eukaryotic tRNAPhe.…”

Dual Role of the Molybdenum Cofactor Biosynthesis Protein MOCS3 in tRNA Thiolation and Molybdenum Cofactor Biosynthesis in Humans

“…The regulation is achieved by the covalent conjugation of the Ubls to target proteins via a lysine residue. Only recently, it was discovered that Ubls are not restricted to eukaryotes, with the identification of Pup in bacteria (1) and the characterization of SAMP proteins in archaea (2,3).…”

“…Ubiquitin (Ub) 3 and ubiquitin-like proteins (Ubls) are involved in a large number of diverse processes within the eukaryotic cell. The regulation is achieved by the covalent conjugation of the Ubls to target proteins via a lysine residue.…”