Mitochondria are multi-functional organelles that play crucial roles in the cell. They are widely known by the moniker 'powerhouse of the cell' because of their role in ATP production. However, they also synthesize metabolic intermediates, oxidize fatty acids, detoxify ammonia, regulate intracellular Ca2+, and participate in the pathway of programmed cell death. Consequently, mitochondrial dysfunction is a common source of inborn errors of metabolism and underlies many pathologies, including Parkinson's disease, cancer and Barth syndrome. The lipid composition of the mitochondrial outer and inner membranes is critical for the function of this organelle. Cardiolipin, the signature lipid of mitochondria acts as both a lubricant and a glue in supporting the machinery of the mitochondrial respiratory chain that produces ATP. The inability to synthesize the mature form of cardiolipin has devastating consequences. Barth syndrome (BTHS), an X-linked disease, is caused by defects in tafazzin an enzyme needed to synthesize mature cardiolipin. BTHS patients present with cardioskeletal myopathy and neutropenia, with heart failure being a major cause of death. Mitochondria import phospholipids and phospholipid precursors from the endoplasmic reticulum. Thus, cardiolipin is synthesized in the inner membrane of mitochondria from a phosphatidic acid precursor that is brought in from the outside. To reach internal mitochondrial compartments, phospholipids must first cross the outer membrane by flipping from the cytoplasmic side to the internal side. As phospholipid flip-flop across a membrane is a very slow process, occurring spontaneously at a rate of 1 flip per day, we hypothesize that the outer membrane must have dedicated molecular machinery to promote rapid flip-flop. Our preliminary data indicate strongly that a prominent protein of the outer membrane called VDAC is responsible for facilitating fast flip-flop of phospholipids. We will confirm this idea in the present proposal and also start to learn how VDAC works to achieve lipid flip-flop. We will purify VDAC protein and use different techniques to assay its ability to flip-flop lipids after reconstitution into a synthetic membrane environment. We will also study VDAC using computer methods to understand better how the protein works. These studies will address a long-standing question in mitochondrial biology, namely how mitochondria obtain the lipids they need, and specifically address the question of how precursor phospholipids necessary for cardiolipin synthesis reach their intramitochondrial destinations.