RESUMO
We studied two pathways that involve the transfer of persulfide sulfur in humans, molybdenum cofactor biosynthesis and tRNA thiolation. Investigations using human cells showed that the two-domain protein MOCS3 is shared between both pathways. MOCS3 has an N-terminal adenylation domain and a C-terminal rhodanese-like domain. We showed that MOCS3 activates both MOCS2A and URM1 by adenylation and a subsequent sulfur transfer step for the formation of the thiocarboxylate group at the C terminus of each protein. MOCS2A and URM1 are ß-grasp fold proteins that contain a highly conserved C-terminal double glycine motif. The role of the terminal glycine of MOCS2A and URM1 was examined for the interaction and the cellular localization with MOCS3. Deletion of the C-terminal glycine of either MOCS2A or URM1 resulted in a loss of interaction with MOCS3. Enhanced cyan fluorescent protein and enhanced yellow fluorescent protein fusions of the proteins were constructed, and the fluorescence resonance energy transfer efficiency was determined by the decrease in the donor lifetime. The cellular localization results showed that extension of the C terminus with an additional glycine of MOCS2A and URM1 altered the localization of MOCS3 from the cytosol to the nucleus.
Assuntos
Núcleo Celular/metabolismo , Coenzimas/biossíntese , Citosol/metabolismo , Metaloproteínas/biossíntese , Nucleotidiltransferases/metabolismo , RNA de Transferência/metabolismo , Sulfurtransferases/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Motivos de Aminoácidos , Animais , Núcleo Celular/genética , Coenzimas/genética , Células HEK293 , Células HeLa , Humanos , Metaloproteínas/genética , Cofatores de Molibdênio , Nucleotidiltransferases/genética , Dobramento de Proteína , Pteridinas , RNA de Transferência/genética , Spodoptera , Sulfurtransferases/genética , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMO
Because of mechanistic parallels in the activation of ubiquitin and the biosynthesis of several sulfur-containing cofactors, we have characterized the human Urm1 and Saccharomyces cerevisiae Uba4 proteins, which are very similar in sequence to MOCS2A and MOCS3, respectively, two proteins essential for the biosynthesis of the molybdenum cofactor (Moco) in humans. Phylogenetic analyses of MOCS3 homologues showed that Uba4 is the MOCS3 homologue in yeast and thus the only remaining protein of the Moco biosynthetic pathway in this organism. Because of the high levels of sequence identity of human MOCS3 and yeast Uba4, we purified Uba4 and characterized the catalytic activity of the protein in detail. We demonstrate that the C-terminal domain of Uba4, like MOCS3, has rhodanese activity and is able to transfer the sulfur from thiosulfate to cyanide in vitro. In addition, we were able to copurify stable heterotetrameric complexes of Uba4 with both human Urm1 and MOCS2A. The N-terminal domain of Uba4 catalyzes the activation of either MOCS2A or Urm1 by formation of an acyl-adenylate bond. After adenylation, persulfurated Uba4 was able to form a thiocarboxylate group at the C-terminal glycine of either Urm1 or MOCS2A. The formation of a thioester intermediate between Uba4 and Urm1 or MOCS2A was not observed. The functional similarities between Uba4 and MOCS3 further demonstrate the evolutionary link between ATP-dependent protein conjugation and ATP-dependent cofactor sulfuration.
