POTATO MITOCHONDRIAL MANGANESE SUPEROXIDE-DISMUTASE IS AN RNA-BINDINGPROTEIN

An RNA-binding protein present in potato mitochondrial lysates was pur ified and identified as manganese-containing superoxide dismutase (MnS OD). Using a gel mobility shift assay we found that proteins from mito chondrial lysates bind with high affinity to in vitro transcripts of m itochondrial orf206, encoding a subunit of the ABC-type heme transport er. By ammonium sulfate fractionation and two subsequent chromatograph ic steps on MonoQ columns we purified a 28 kDa protein to apparent hom ogeneity. Protein sequencing identified the purified polypeptide as ma nganese-containing superoxide dismutase, which is a specific enzymatic scavenger of superoxides in mitochondria. Using gel mobility shift an d competition assays, we show that RNA-binding of MnSOD of potato is n ot influenced by 400 mM KCl or heparin and is specific to heteropolyme ric RNAs. The labeled mitochondrial transcript could be competed with low amounts of unlabeled transcript while binding was stable to compet ition with large amounts of tRNA or high concentrations of NADH and NA DPH. The purified MnSOD of potato mitochondria was UV-cross-linked to the mitochondrial transcript. The Mn- and Fe-containing SODs from Esch erichia 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 pot ato mitochondria is probably not involved in modulating SOD enzyme act ivity and suggests a function different from superoxide degradation as ist biological role.