A CARBOXY-TERMINAL PLANT VACUOLAR TARGETING SIGNAL IS NOT RECOGNIZED BY YEAST

Three different classes of signals for plant vacuolar targeting have been defined. Previous work has demonstrated that the carboxyl-terminal propeptide (CTPP) of barley lectin (BL) is a vacuolar targeting signal in tobacco plants. When a mutant BL protein lacking the CTPP is expressed in tobacco, the protein is secreted. In an effort to determine the universality of this signal, the CTPP was tested for its ability to target proteins to the vacuole of Saccharomyces cerevisiae. Genes encoding fusion proteins between the yeast secreted protein invertase and BL domains were synthesized and transformed into an invertase deletion mutant of yeast. Invertase assays on intact and detergent-solubilized cells demonstrated that invertase+CTPP was secreted, while nearly 90% of the invertase::BL+CTPP (fusion protein between invertase and BL containing the CTPP) and invertase::BL-CTPP proteins (fusion between invertase and BL lacking the CTPP) were retained intracellularly. These fusions were secreted in a mutant of yeast that normally secretes proteins targeted to the vacuole. With this and previous work, proteins representing all three classes of plant vacuolar targeting signals have now been tested in yeast, and in all cases, the experiments indicate that the plant proteins are directed to the yeast vacuole using signals other than those recognized by plants.