9315940 Chrispeels, Marten J. The goal of the proposal is to understand how proteins are targeted to and accumulated in vacuoles of plant cells. Although plant vacuoles are related to yeast vacuoles and lysosomes of mammalian cells, they contain a much greater variety of proteins, including storage proteins, lectins, enzyme inhibitors, defense enzymes, as well as many of the acidic hydrolases commonly found in all vacuoles and lysosomes. Much progress has been made in the last years in understanding the sorting signals that occur on the vacuolar proteins themselves, but there is at present no information on the cellular machinery that allows these proteins to be correctly targeted to their destination. Correct vacuolar targeting of Golgi-derived vesicles involves proteins on the outside of the vesicles that mediate vesicle budding, transport and fusion of the vesicles with the target membrane in a highly specific manner. These processes are thought to require coating and uncoating with proteins that bind to the membranes and can be recruited from the cytosol. The identification of these proteins is the major goal of this proposal. The Pi will exploit the recent isolation of the gene that encodes a phosphatidyl-inositol 3-kinase from Arabidopsis and investigate whether it has a role in vacuolar protein sorting. The function of this protein will be examined in stably transformed and transient systems, or protoplasts treated with antisense oligodeoxyribonucleotides. The Pi will transform a yeast vps34 mutant with the plant phosphatidylinositol 3-kinase gene to find interacting proteins, and then rescue this strain with a plant cDNA library. The two-hybrid method is also going to be used to identify this gene. The Pi will also continue work on the targeting of proteins to the vacuolar membrane or tonoplast. He will make constructs that involve a reporter protein against which he has antiserum, and different membrane spanning domains. %%% The significance of this work lies in a better understanding of vacuole biogenesis in plant cells. The research proposed here will contribute to our understanding of the mechanisms by which the vacuolar proteins reach their destinations. As with other aspects of the secretory system, some of the findings will be applicable to all eukaryotic cells, while others will be specific for plant cells. ***
