Potato mitochondrial manganese superoxide dismutase is an RNA-binding protein.
An RNA-binding protein present in potato mitochondrial lysates was purified and identified as manganese-containing superoxide dismutase (MnSOD). Using a gel mobility shift assay we found that proteins from mitochondrial lysates bind with high affinity to in vitro transcripts of mitochondrial orf206, encoding a subunit of the ABC-type heme transporter. By ammonium sulfate fractionation and two subsequent chromatographic steps on MonoQ columns we purified a 28 kDa protein to apparent homogeneity. Protein sequencing identified the purified polypeptide as manganese-containing superoxide dismutase, which is a specific enzymatic scavenger of superoxides in mitochondria. Using gel mobility shift and competition assays, we show that RNA-binding of MnSOD of potato is not influenced by 400 mM KCl or heparin and is specific to heteropolymeric RNAs. The labeled mitochondrial transcript could be competed with low amounts of unlabeled transcript while binding was stable to competition with large amounts of tRNA or high concentrations of NADH and NADPH. The purified MnSOD of potato mitochondria was UV-cross-linked to the mitochondrial transcript. The Mn- and Fe-containing SODs from Escherichia coli showed no binding to the RNA by either gel mobility shift or UV-cross-linking. Enzyme activity assays revealed that binding of RNA to the mitochondrial MnSOD does not significantly influence enzyme activity. This indicates that the RNA-binding feature of MnSOD of potato mitochondria is probably not involved in modulating SOD enzyme activity and suggests a function different from superoxide degradation as ist biological role.
Fester
T
T
Institut für Genbiologische Forschung Berlin GmbH, Berlin, Federal Republic of Germany.
Schuster
W
W
D000595:Amino Acid Sequence; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D006493:Heparin / Q000494:pharmacology; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D012313:RNA / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D012260:Ribonucleases / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D011198:Solanum tuberosum / Q000201:enzymology*; D013482:Superoxide Dismutase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic
D016601:RNA-Binding Proteins; D012313:RNA; D006493:Heparin; D012343:RNA, Transfer; D013482:Superoxide Dismutase; D012260:Ribonucleases