Autophagy is an important cellular process that is triggered by nutrient stress and also plays significant roles in normal turnover of damaged proteins and organelles, tissue remodeling in development, and innate immunity. In addition, decreased autophagic activity has been implicated in aging and in aging related diseases such as neurodegenerative disorders. The mammalian Class III PI 3-kinase, hVps34, is required for both vesicular trafficking and autophagy. hVps34 and its regulatory subunit, hVps15, are known to form complexes with a number of endocytic and autophagic regulatory proteins, including beclin-1, Atg14, UVRAG, Rab5/7, and calmodulin. hVps34 is also a substrate for chaperone-mediated autophagy, and undergoes enhanced degradation during serum starvation. The exact modes of hVps34 regulation by these different interactions, and their relative roles in different hVps34-dependent processes, have not yet been elucidated. In this proposal, a structurally based approach will be used to study the functions of hVps34 and hVps15 in autophagy, using both cell culture and an in vivo animal model. Two novel mutants will be produced, each selectively defective for a major hVps34 regulatory interaction: hVps34 binding to beclin-1, and hVps15 binding to Rab5 and Rab7. Two other mutants have already been designed, each disrupting a specific functionality of hVps34: calmodulin binding, and the regulated degradation of hVps34 by chaperone mediated autophagy. These four mutants will be studied in a knockdown/rescue approach in cell culture experiments, testing their ability to rescue hVps34 functions in autophagy and vesicular trafficking assays. In addition, Vps34 signaling will be studied in zebrafish, a vertebrate animal model. Vps34- or Vps15- targeted morpholino oligos will be used to suppress Vps34 or Vps15 expression in the zebrafish. The mutants described above will then be studied by rescuing the morphant phenotype with wild type or mutant Vps34 or Vps15. Studies will focus on the effects of these mutants on autophagy and endocytic trafficking. These experiments should lead to important insights into the regulation of Vps34 in vivo, and its role as a major regulator of autophagy.