The major objective of this work is to identify the protein domain that allows vacuolar proteins to be targeted to the vacuoles of plant cells, and to identify the receptor(s) with which they interact. The work will concentrate on three vacuolar proteins present in bean (Phaseolus vulgaris) seeds, phytohemagglutinin, phaseolin, and cx-amylase inhibitor and will use two different transgenic systems, the yeast Saccharomyces cerevisiae and tobacco (Nicotiana tabacum). In addition, we will attempt to transform bean with Agrobacterium tumefaciens to be in a position to study transport to the vacuole. The work will utilize translational fusions between portions of these three proteins and the yeast invertase gene (a secreted protein) to delineate the targeting domain. Mutagenesis will be used together with computer-based sequence comparisons to identify the critical residues. Identification of the receptor will rely on affinity studies (either columns or gel/blot binding procedures) using the targeting domains of these proteins and solubilized membrane fractions. Once the receptor has been isolated, we will attempt to clone the gene and carry out immunocytochemical work to study its subcellular distribution. This project will provide important basic information relevant to our understanding of how cellular proteins get to their specific functional sites within the cell. It will also provide important information concerning how seed storage proteins get to the sites of storage within the cell, which is of practical value in terms of genetic engineering of nutritionally "improved" seeds and grains.
