title: Characterization and interaction studies of two isoforms of the dual localized 3-mercaptopyruvate sulfurtransferase TUM1 from humans.
abstract: The human tRNA thiouridine modification protein (TUM1), also designated as 3-mercaptopyruvate sulfurtransferase (MPST), has been implicated in a wide range of physiological processes in the cell. The roles range from an involvement in thiolation of cytosolic tRNAs to the generation of H2S as signaling molecule both in mitochondria and the cytosol. TUM1 is a member of the sulfurtransferase family and catalyzes the conversion of 3-mercaptopyruvate to pyruvate and protein-bound persulfide. Here, we purified and characterized two novel TUM1 splice variants, designated as TUM1-Iso1 and TUM1-Iso2. The purified proteins showed similar kinetic behavior and comparable pH and temperature dependence. Cellular localization studies, however, showed a different localization pattern between the isoforms. TUM1-Iso1 is exclusively localized in the cytosol, whereas TUM1-Iso2 showed a dual localization both in the cytosol and mitochondria. Interaction studies were performed with the isoforms both in vitro using the purified proteins and in vivo by fluorescence analysis in human cells, using the split-EGFP system. The studies showed that TUM1 interacts with the l-cysteine desulfurase NFS1 and the rhodanese-like protein MOCS3, suggesting a dual function of TUM1 both in sulfur transfer for the biosynthesis of the molybdenum cofactor, and for the thiolation of tRNA. Our studies point to distinct roles of each TUM1 isoform in the sulfur transfer processes in the cell, with different compartmentalization of the two splice variants of TUM1.
authors.affiliation: From the University of Potsdam, Institute of Biochemistry and Biology, D-14476 Potsdam, Germany.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Fräsdorf
authors.affiliation: From the University of Potsdam, Institute of Biochemistry and Biology, D-14476 Potsdam, Germany.
authors.forename: Christin
authors.initials: C
authors.lastname: Radon
authors.affiliation: From the University of Potsdam, Institute of Biochemistry and Biology, D-14476 Potsdam, Germany sleim@uni-potsdam.de.
authors.forename: Silke
authors.initials: S
authors.lastname: Leimkühler
mesh_terms: D000595:Amino Acid Sequence; D013437:Carbon-Sulfur Lyases / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D013455:Sulfur / Q000378:metabolism; D013466:Sulfurtransferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature
chemical_list: D007527:Isoenzymes; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D013455:Sulfur; C502790:MOCS3 protein, human; D009713:Nucleotidyltransferases; D013466:Sulfurtransferases; C021550:3-mercaptopyruvate sulphurtransferase; D013437:Carbon-Sulfur Lyases; C513492:NFS1 protein, human
keywords: Fluorescence; Mitochondria; Molybdenum; Sulfur; Transfer RNA (tRNA)