Identification and characterization of a prevacuolar compartment in stigmas of Nicotiana alata

The stigmas of the ornamental tobacco plant Nicotiana alata accumulate larg e quantities of a series of 6-kD proteinase inhibitors (PIs) in the central vacuole that are derived from a 40-kD precursor protein, Na-PI. The sortin g information that directs Na-PI to the vacuole is likely to reside in a C- terminal propeptide domain of 25 amino acids that forms an amphipathic or h elix. Using cell fractionation techniques, we have examined transit of Na-P I through the endomembrane system and have identified a prevacuolar compart ment that contains Na-PI with an intact targeting signal. In contrast, the targeting signal is not present on the predominant form of Na-PI in the vac uole. The prevacuolar compartment is marked by the presence of homologs of both the t-SNARE, PEP12p, and the putative vacuolar sorting receptor BP-80. Cross-linking and affinity precipitation studies revealed that Na-PI assoc iates with BP-80 within this compartment, providing in vivo evidence for th e function of BP-80 as a sorting receptor for a protein with a C-terminal v acuolar targeting signal.