Processing of thionin precursors in barley leaves by a vacuolar proteinase.

Thionins are synthesized as precursors with a signal peptide and a long C-terminal acidic peptide that is post-translationally processed. A fusion protein including the maltose-binding protein from Escherichia coli (MalE), thionin DG3 from barley leaves, and its acidic C-terminal peptide has been used to obtain antibodies that recognize both domains of the precursor. In barley leaf sections, mature thionins accumulated in the vacuolar content, while the acidic peptide was not detected in any cell fraction. Brefeldin A and monensin inhibited processing of the precursor but its export from the microsomal fraction was not inhibited. Both purified vacuoles and an acid (pH 5.5) extract from leaves processed the fusion protein into a MalE-thionin and an acidic peptide fragment. A 70-kDa proteinase that effected this cleavage was purified from the acid extract. Processing of the fusion protein by both lysed vacuoles and the purified proteinase was inhibited by Zn2+ and by Cu2+, but not by inhibitors of the previously described vacuolar processing thiol or aspartic proteinases. In vivo processing of the thionin precursor in leaf sections was also inhibited by Zn2+ and Cu2+. Variants of the fusion protein with altered processing sites that represented those of thionin precursors from different taxa were readily processed by the proteinase, whereas changing the polarity of either the C-terminal or N-terminal residues of the processing site prevented cleavage by the proteinase.