IMMUNOCYTOCHEMICAL LOCALIZATION OF 22-KILODALTON POTATO PROTEINASE INHIBITORS

Abstract

Twenty-two kilodalton potato proteinase inhibitor (22-kD PPI) is synthesized as a pre-protein with a hydrophobic signal sequence of 40 amino acids. Using immunogoldcytochemistry we determined the subcellular localization of the 22-kD PPI. Using 22-kD PPI specific antibodies and GAR-IgG colloidal gold for electron-microscopical immunocytochemistry, the 22-kD PPI was found to be localized mainly in the vacuole and cytoplasm of both tubers and wounded leaves of potatoes (Solanum tuberosum L). Within the vacuole, label was found predominantly over the protein aggregates and protein cluster-like structure. Neither cell wall nor the intercellular space contained detectable levels of the 22-kD PPI. We found one basic residue with a hydrophobic core followed by a cleavage site for signal peptidase in the deduced amino acid sequence of the 22-kD PPI cDNA clone, p34021. A conserved glutamine, which is thought to be required for correct sorting in all vacuolar proteins sequenced to date, was also found in amino acid sequence derived from the 22-kD PPI cDNA clone, p34021.