Autophagy is a self-cleaning mechanism that degrades cellular debris including damaged organelles, misfolded proteins, and invading microorganisms. Defects in autophagy lead to cancer, infection, neurodegeneration, aging, and heart disease. The rate-limiting factor required for formation of autophagosomes appears to be a lipid kinase, the class III PI3 kinase (PI3KC3), which catalyzes the conversion of phosphatidylinositol (PtdIns) to PtdIns3P. PI3KC3 functions as the initiating factor for autophagosome formation but very little is known about its regulation. We have recently purified and characterized a human PI3KC3 complex that is pivotal for autophagosome initiation and nucleation. In this complex, we have identified three novel regulatory components. One of them that we named Barkor clearly has a function in autophagy through specific direction of PI3KC3 to autophagosomes. We hypothesize that Barkor functions an apical factor to bind to autophagosome membrane and to ignite a signal cascade for autophagosome formation. Extended from Barkor, we aim to elucidate the protein and lipid requirement for autophagosome formation via biochemical approaches. We anticipate that biochemical reconstitution of autophagosome formation will lend insight into the mechanisms of human diseases implicated in autophagy dysfunction.