A gene encoding mitochondrial S13 is generally present in the mitochondrial genome of higher plants, but is lacking from the Arabidopsis thaliana mitochondrial genome. Previous research has failed to identify a nuclear gene capable of encoding a mitochondrial S13 protein or the protein itself. Doubts have even been raised as to whether a mitochondrial S13 exists in Arabidopsis. Here, we show that the nuclear gene encoding the plastid S13 has been partially duplicated in A. thaliana, such that the copy has lost the exon encoding the plastid transit peptide and acquired a sequence capable of encoding a mitochondrial targeting sequence. The two S13 sequences were fused to green fluorescent protein and shown to be targeted to plastids and mitochondria respectively.

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We prepared two derivatives of notexin, a phospholipase A2 from Motechis scutatus scutatus venom, by modifying the protein with 2-nitrophenylsulfenylchloride, a tryptophan-specific reagent. One derivative was modified at both tryptophans 20 and 110 whereas the other was modified at tryptophan 20. Evidence based on circular dichroic analysis and antigenicity towards a notexin-specific monoclonal antibody indicated that derivatization at both tryptophans did not affect the tertiary structure of notexin. Concomitant modification of tryptophans 20 and 110 induced a marked decrease in the capacity of notexin to kill mice and to block neuromuscular transmission in the chick biventer cervicis preparation, whereas selective modification at tryptophan 20 had no effect on the lethal properties of notexin. This implies that the decrease in the lethal properties of notexin after derivatization was due to modification at tryptophan 110. However, the diderivatized notexin retained full enzymatic activity, implying that neither tryptophan 20 and tryptophan 110 are involved in the catalytic function of the molecule. We conclude that notexin harbours two functional sites. One of them corresponds to the enzymatic site, whereas the other, which includes tryptophan 110, provides specific toxic characteristics to notexin. By reference to previous crystallographic studies, the relative spatial positions of elements involved in toxicity and the catalytic site, we propose a possible orientation of notexin with respect to its putative membrane-bound target.

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