Dual location of a family of proteinase inhibitors within the stigmas of Nicotiana alata.

Reproductive and storage tissues of many plants produce large amounts of serine proteinase inhibitors (PIs). The ornamental tobacco, Nicotiana alata, produces a series of 6 kDa chymotrypsin and trypsin inhibitors that accumulate to up to 30% of soluble protein in the stigma. These inhibitors are derived by proteolytic processing of two closely related multidomain precursor proteins. Using immunogold electron microscopy, we find that the stigmatic PIs accumulate in both the central vacuole and in the extracellular mucilage. Labelling with antibodies specific for the C-terminal vacuolar targeting peptide (VTS) of each precursor confirms earlier biochemical data showing that the VTS is removed during passage through the secretory pathway. We have isolated and characterised the extracellular population of PIs, which are largely identical to PIs isolated from whole stigmas and are functional inhibitors of serine proteases. Subcellular fractionation of immature stigmas reveals that a sub-population of the PI precursor protein is proteolytically processed within the endoplasmic reticulum. This proteolysis results in the removal of the vacuolar sorting information, causing secretion of this PI population. We propose a novel mechanism whereby a single gene product may be simultaneously trafficked to two separate compartments mediated by proteolysis early in the secretory pathway.