Plant cells store a diverse variety of molecules in vacuoles. Storage is the central reason why plants are so important to humankind. The wars against cocaine and heroin result from the harvest of products in storage vacuoles, the morning coffee is brewed to release products stored in vacuoles, the flowers in our garden are there because of pigments stored in vacuoles, and the nutrition of domestic animals and humans ultimately depends upon proteins stored in plant vacuoles. Many molecules that are targets in plant biotechnology are stored in vacuoles. Only by understanding the different compartments, their internal contents and environment, and how proteins and membrane are directed to each, will we be able to program a cell to make and accumulate a desired product. The need to separate a digestive compartment similar to the yeast vacuole or mammalian lysosome from the storage compartments has resulted in a complex plant vacuolar system. Of the storage compartments, protein storage vacuoles have been best studied because proteins are relatively easy to track in a cell. In many plant cells, separate protein storage and lytic vacuoles coexist. Proteins are delivered to each from the Golgi complex by separate vesicular trafficking pathways, clathrin coated vesicles for the lytic pathway, and dense vesicles (or their equivalents)for the protein storage vacuole pathway. However,in developing seeds and certain other cells, the two pathways converge on the same vacuole. In this instance, for example in protein storage vacuoles in seeds, the resultant organelle is a multivesicular body, where the storage products are partitioned in the "soup " and lytic functions are partitioned into the internal vesicles. Thus the seed protein storage vacuole is a compound organelle,where two functionally distinct compartments exist within the limiting membrane. How proteins are delivered by the two separate vesicular pathways to the two compartments within the organelle is an important unsolved question in cell biology. This project focuses on mechanisms by which proteins are sorted into the protein storage vacuole pathway in the Golgi complex. Results from recent ligand binding experiments indicate that the lumenal domain of a plant RMR protein specifically interacts with the targeting determinants that sort proteins into the protein storage vacuole pathway. RMR proteins are integral membrane proteins that traffic from Golgi to the protein storage vacuole where they are incorporated into a membrane-containing crystalloid within the storage compartment.Thus it is likely they serve as a unique type of sorting receptor.RMR proteins are also expressed in avian and mammalian cells. The association and dissociation constants, and stoichiometry, for binding of RMR protein lumenal domains for a model ligand will be determined. The function of RMR proteins in plants will be assessed by generating antisense knockouts in tobacco and by identifying transposon/T DNA insertions in individual RMR protein genes in Arabidopsis. Motifs in the RMR proteins' cytoplasmic tails responsible for traffic from Golgi to the storage compartment will be identified. A separate experimental strategy will address mechanisms by which the storage compartment crystalloid is formed. Dr. Rogers' laboratory has purified protein storage vacuole crystalloids away from other membranes in the vacuole. Using a proteomics approach, the integral membrane proteins specifically incorporated into PSV crystalloids, and then tonoplast and globoids in B. napus seeds will be identified. The mechanisms by which a tomato storage protein related to 11S globulins that appears to have transmembrane helices is incorporated into crystalloid membranes will be defined. Although protein storage compartments were thought initially to be unique to plant cells, evidence now emerging indicates that animal cells also have a dense vesicle pathway, and that multivesicular body endosomes in animal cells may partition two separate functions within the same organelle. Thus,an understanding of fundamental processes of compartmentation in plant cells may have broader impact in cell biology. The ability to visualize the two compartments easily in protein storage vacuoles and to track proteins in each of the vesicular pathways provides great advantages for use of a plant system in these studies.